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Elastin as a kinetic elastomer : an analysis of its conformational, mechanical, and photoelastic properties 1980

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c l ELASTIN as A KINETIC ELASTOMER: An Analysis of Its Conformational, Mechanical, and Photoelastic Properties» by BEN-MEYER BENSON AARCN B.Sc., The University of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR A DEGREE OF MASTER OF SCIENCE in THE FACULTY CF GRADUATE STUDIES Department of Zcclogy We accept this thesis as conforming to the reguired standard THE UNIVERSITY OF ERITISH COLUMBIA August 1980 © BEN-MEYER BENSON AARON, 1980 / In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i ABSTRACT The e l a s t i c tissue composite i s made up of a number of materials that are characterized by d i f f e r e n t chemical and mechanical properties..The protein elastcmer e l a s t i n makes up almost 80% of bovine ligamgntum nachae with collagen and the matrix substances making up the other 20%. The analysis of the mechanical properties of the unpurified and p u r i f i e d tissue indicates that e l a s t i n i s the dominant mechanical component at low s t r a i n s with collagen contributing s i g n i f i c a n t l y at the higher extensions. The physical properties of s i n g l e , 5 to 8um diameter, water-swollen e l a s t i n f i b r e s were investigated on a micro-test apparatus attached to a polarizing microscope, and the r e s u l t s were analyzed by using the k i n e t i c theory relationships. The analysis of the mechanical properties at extensions below 100% indicate that e l a s t i c modulus, G = 4.1 X 10sNm-2, the average molecular weight of the chains between cross-links i s i n the range of 6000 to 7 100g/mol, and the s t r e s s - o p t i c a l coeffecient, C1 = 1 X 10-»m 2N-» at 24°C. Analysis of the temperature dependence of the s t r e s s - o p t i c a l coeffecient indicated that the p c l a r i z a b i l i t y of the random l i n k decreases with increasing temperature. The apparent ac t i v a t i o n energy for t h i s process i s i n the order of 1.6 kcal/mole. Analysis of the non-Gaussian mechanical and o p t i c a l properties at extensions above 100% suggest that the chain between cross- l i n k s contains approximately 10 ' e f f e c t i v e ' random l i n k s , with each l i n k consisting of 7 to 8 amino acid residues. i i The e x p l i c i t assumption of a random network that i s made by the k i n e t i c theory was tested by a number of techniques. 400 MHz pmr spectra cf the soluble alpha-elastin c l o s e l y resembled the spectra that were predicted for the random-coil conformation, and the spectra obtained for i t ' s amino acid hydrolysate. Polarized microscopy studies showed i n t a c t e l a s t i n f i b r e s to be devoid of any c r y s t a l l i n e structures. F i n a l l y , the parameters for the random chains in the e l a s t i n network were used to predict the dimensions of other random proteins. The close c o r r e l a t i o n of these predictions with published values for a series of proteins i n solution in 6M GuHCl provided an independent test cf the random conformation, validating the use of the k i n e t i c theory relationships to analyze the macroscopic properties of e l a s t i n . i i i TABLE OF CONTENTS ABSTRACT I TABLE OF CONTENTS i i i LIST OF TABLES X LIST OF FIGURES , .xi ACKNO WL E EG EM EN TS , . xi V I. .INTRODUCTION . 1 I I . GENERAL CHARACTERISTICS OF ELASTIC TISSOE 4 A. Introduction 4 B. . E l a s t i n Development • • (a) Embryology 4 (b) Morphogenesis Of E l a s t i n Fibres 5 (c) E l a s t i n Turnover 7 C. E l a s t i n Chemistry 7 (a) Amino Acid Composition 7 (b) E l a s t i n Cross-links 9 (c) Soluble E l a s t i n . . 1 0 D. Composition Of E l a s t i c Tissue 1 1 (a) Water Content . . 1 2 (b) E l a s t i n Content . . . 1 2 (c) Neutral Sugars . 1 2 (d) Mucopolysaccharides 1 2 (e) Collagen Content . . . 1 4 E. Organization Of E l a s t i c Tissue . 1 4 (a) Methodology . , . 1 4 i v (b) Organization Of ligament E l a s t i n 15 (c) Organization Of A r t e r i a l E l a s t i n 18 F. P u r i f i c a t i o n Techniques ......21 (a) Amino Acid Compositions 21 (b) Hexosamine And Neutral Sugar Content 24 (c) Evaluation Ey Scanning Electron Microscopy .....24 G. Mechanical Properties Of E l a s t i n Bundles 24 (a) Methods 23 (b) Results 28 H. Mechanical Properties Of Single E l a s t i n Fibres .....31 I . Discussion 34 (a) Variation In Elastin Biochemistry ? - 34 (b) The Composite Tissue .....35 (c) Evaluation Cf P u r i f i c a t i o n Techniques ......... 36 I I I . CONFORMATION OF ELASTIN: THE CONTEOVEESY. 38 A. . Introduction .....38 B. The Random Network Model . 33 (a) E l a s t i n Thermcelasticity .41 (b) D i f f e r e n t i a l Scanning Calorimetry 43 (c) E.M. And X-ray D i f f r a c t i o n Studies 43 (d) N.M.R. Evidence 44 C. .Liquid Crop Model .45 (a) Preliminary Evidence 45 (b) Thermodynamic Evidence 46 D. .Oiled C o i l Model .47 E. F i b r i l l a r Models .48 (a) Electron Microscope Evidence 49 V (b) Nuclear Magnetic Resonance Evidence . 5 0 F. D i s c u s s i o n .. • 54 (a) Two-phase Models . 54 (b) Evidence For Secondary S t r u c t u r e s 56 (c) Hydrogen Ecnded S t r u c t u r e s .....58 (d) F i b r i l l a r Models .. 60 G. C o n c l u s i o n s • 60 IV. CONFORMATION OF ELASTIN: COACERV ATE STRUCTURE. ........ 62 A. I n t r o d u c t i o n • • . 62 B. The Phenomenon Of Coacervation 62 C. P r e p a r a t i o n Of A l p h a - E l a s t i n • 6 3 (a) Enzyme H y d r o l y s i s .....64 (b) Removal Of SDS 65 (c) C h a r a c t e r i z a t i o n Of The A l p h a - e l a s t i n 65 Do V i s c o s i t y And Shape .66 (a) The Relevant Eguation 66 (b) E v a l u a t i o n Cf The Shape Parameter , .69 (c) A p p l i c a t i o n To S o l u b l e E l a s t i n s ..70 E. V i s c o s i t y Studies Of A l p h a - E l a s t i n 73 (a) V i s c o s i t y Measurements 73 (b) C a l c u l a t i o n Cf P a r t i a l S p e c i f i c Volume And Hydration 76 (c) R e s u l t s And D i s c u s s i o n . 7 6 F. Nuclear Magnetic Resonance .........80 (a) Theory .80 (b) The Chemical S h i f t . . . 8 4 (c) R e l a x a t i o n Processes •••• 85 v i G. N. Mo Ro Studies Of A l p h a - E l a s t i n ...87 (a) M a t e r i a l s And Method .87 (b) P r e d i c t i o n Cf Eandom-coil Spectra 9 1 (c) R e s u l t s And D i s c u s s i o n 92 H. Conclusi o n s .105 V. CON FO EM A HON OF ELASTIN: BIREFRINGENCE PEOPEBTIES. ...108 A. I n t r o d u c t i o n ..108 B. Phenomenological E x p l a n a t i o n Of Double R e f r a c t i o n .109 (a) R e t a r d a t i o n Of P o l a r i z e d L i g h t .110 (b) Q u a n t i t a t i n g The R e t a r d a t i o n ,, ,115 C. . Q u a l i f y i n g The Types Of B i r e f r i n g e n c e .....116 (a) I n t r i n s i c B i r e f r i n g e n c e 116 (b) Form E i r e f r i n g e n c e .........124 {c) S t r a i n B i r e f r i n g e n c e ....125 D. M a t e r i a l s And Methods - 126 E. B i r e f r i n g e n c e P r o p e r t i e s Of S i n g l e E l a s t i n F i b r e s .127 (a) Form B i r e f r i n g e n c e .127 (b) I n t r i n s i c B i r e f r i n g e n c e 132 (c) E x p l a n a t i o n For The Apparent B i r e f r i n g e n c e ....136 . F. D i s c u s s i o n ..146 (a) Previous S t u d i e s ...146 (b) The F i b r i l l a r Models .148 G. . Conclus i o n s .150 VI. , CONFORMATION OF ELASTIN: SCANNING ELECTRON MICROSCOPY .152 A. I n t r o d u c t i o n 152 B. Methods 152 v i i C. Results . ..153 D. Discussion 156 VII. .ELASTIN AS A KINETIC ELASTOMER. .....163 A. Introduction .163 B. .Entropy Elastomers: The Kinetic Theory Of Rubber E l a s t i c i t y 164 (a) Gaussian Chain S t a t i s t i c s And Entropy 164 (b) The E l a s t i c Network .168 (c) Mechanical Properties Of Kinetic Rubbers ...... 169 (d) Photoelasticity 171 (e) Non-Gaussian Effects And The Evaluation Of S ..172 (f) Reduction Cf E l a s t i n Data To The Unswollen Form 173 C. Materials And Method .174 (a) P u r i f i c a t i o n Of E l a s t i n .174 i (b) The Experimental Stage .175 i (c) Preparation Of Experimental Specimen 175 (d) Measurement Of Strain 178 (e) Measurement Of Force .178 (f) Calculation Of Cross-sectional Area .....179 (g) Measurement Of Birefringence ...180 (h) Errors 180 D. Physical Properties Of Single E l a s t i n Fibres 181 (a) General Characteristics r ^ 8 1 (b) Mechanical Properties And The Derivation Of Mc 18 1 (c) Photoelasticity . 185 (d) Temperature Dependence Of The Optical v i i i Anisotropy 188 (e) Non-Gaussian Properties Of The E l a s t i n Network 192 E. Conclusions 195 VIII. .A PREDICTIVE TEST FOR ELASTIN CONFORMATION 197 A. . Introduction 197 B. Characterization Of Random Proteins .197 (a) The Measurable Dimension 197 (b) Accounting For The Non-ideality .20 1 C. R.M.S. Values From Viscosity 202 (a) Viscosity Of Random Co i l s 202 (b) Correction Of Viscosity Values 203 D. Predictions From The E l a s t i n Network .204 (a) Calculation Of S .204 (b) Calculation Of L ..206 E. Discussion And Conclusions 207 IX. CONCLUSIONS 211 APPENDIX.I: Ihermoelasticity .216 (A) Thermodynamic Relationships .216 (B) The Thermodynamic Experiment 217 (C) Thermoelasticity Of Kinetic Elastomers ...........218 APPENDIX.II: PREPARATION OF SOLUBLE ELASTIN BY PHOTOLYSIS ....... 220 A. Introduction 220 B. Methodology .220 (a) Rationale .220 (b) Procedure • .224 C. Results And Discussion ......225 ix (a) Yield ..225 (b) Characterization Of The Soluble Peptides ......226 Appendix.Ill: PREDICTIONS FOR TROPOELASTIN VISCOSITY ...,231 A. The Relevant Equation 231 5. Application To Tropoelastin 231 Appendix.IV: EVALUATION OF PROTEIN CONFORMATION .236 Appendix.V: DETERMINATION OF SOLVENT EFFECTS ON RANDOM COILS .........242 Appendix.VI: AMINO ACID COMPOSITION AND ELASTIN EVOLUTION 245 LITERATURE CITED ..249 X LIST OF TABLES Table.2.1: Amino Acid Composition Of E l a s t i c Proteins. ., 8 Table.2.2: Chemical Composition Of E l a s t i c Tissue 13 Table.2.3: Amino Acid Composition Of E l a s t i n Preparations. ........................................ 25 Table. 4.1: Viscosity Of Alpha-elastin 77 Table.4.2: Spectral Parameters Used For Bandom-coil Predictions .90 Table.4.3: Peak Assignments For Alpha-elastin At 400 MHz. 9 5 Table.5.1: Form Birefringence Of Single E l a s t i n Fibres. .128 Table.7.1: Kinetic Theory Parameters For E l a s t i n . ..186 Table.8.1: Predictions For Random-coil Proteins. ..205 Taile.A.3.1: Prediction For Tropoelastin V i s c o s i t y . .....234 Table.A.6.1: Difference Index For E l a s t i n Composition. .,246 x i LIST OF FIGURES Figure.2.1: Organization Of Ligament E l a s t i n . . . .16 Figure.2.2: Organization Of A r t e r i a l E l a s t i n . .19 Figure.2.3: The E l a s t i c Tissue Composite.. 22 Figure.2.4: Evaluation Of P u r i f i c a t i o n Technigues. .26 Figure.2.5: Mechanical Properties Of E l a s t i n Bundles. ... 29 Figure.2.6: Mechanical Properties Of Single E l a s t i n Fibres. 3 2 Figure.3.1: Proposals For The Conformation Of E l a s t i n ....39 Figure.3.2: Eeta-turns. 51 Figure.4.1: Ccacervation P r o f i l e Of Alpha-elastin. ..67 Figure.4.2: Dependence Of The Simha Factor On Axial Ratio 71 Figure.4.3: Dependence Of E l a s t i n Water Content On Temperature .74 Figure.4. 4: Viscosity Of Alpha-elastin 78 Figure.4.5: Precession Of A Proton In A Magnetic F i e l d . . 81 Figure.4.6: The Lorentzian Line Shape ..88 Figure.4.7: 4C0 MHz Nmr Spectrum Of Alpha-elastin. .......93 Figure.4.8: Predicted Nmr Spectra Fcr Alpha-elastin 97 Figure.4.9: Nmr Spectrum Of Alpha-elastin Hydrolysate. ..100 Figure.4. 10: Nmr Spectra Of Albumin. 103 Figure.5.1: Propagation Of Polarized Light Through Isotropic Material. .....111 Figure.5.2: Propagation Of Polarized Light Through Anisotropic Material .113 x i i FIGUfiE . 5 . 3 : The Birefringence Experiment 1 1 7 F i g u r e . 5 . 4 : The Sign Of The Birefringence. . . . . . . . . 1 2 0 F i g u r e . 5 . 5 : Form Birefringence 1 2 2 F i g u r e . 5 . 6 : Form Birefringence Of E l a s t i n Fibres , 1 2 9 F i g u r e . 5 . 7 : Form Birefringence Of Collagen... 1 3 3 F i g u r e . 5 . 8 : Expected Relationship For I n t r i n s i c Birefringence. 1 3 7 F i g u r e . 5 . 9 : Expected Birefringence For An Anisotropic Coating 1 40 Figure. 5 . 10: Birefringence Pattern Of Single El a s t i n Fibres . . 1 44 F i g u r e . 6 . 1 : S.E.M. Of Unpurified Ligament E l a s t i n Fibre. 1 5 4 F i g u r e . 6 . 2 : Surface Texture Of Autoclaved E l a s t i n Fibres. 1 5 7 F i g u r e . 6 . 3 : Fracture Surfaces Of E l a s t i n Fibres. , 1 5 9 F i g u r e . 7 . 1 : The Random-coiled Chain 1 6 6 F i g u r e . 7 . 2 : The Experimental Stage.. . 1 7 6 F i g u r e . 7 . 3 : Physical Properties Of Single E l a s t i n Fibres. 1 8 3 F i g u r e . 7 . 4 : Temperature Dependence Of Photoelasticity. . . 1 8 9 F i g u r e . 7 . 5 : Non-Gaussian Properties Of The E l a s t i n Network. 1 9 3 F i g u r e . 8 . 1 : The Randcm Walk.. . . . . 1 9 9 F i g u r e . 8 . 2 : Predictions For The Dimensions Of Random-coil Protein 2 0 8 F i g u r e . 9 . 1 : Summary Figure For The Thesis. 213 Figure. A. 2 . 1: Photolysis Of E l a s t i n 2 2 2 x i i i Figure.A.2.2: Molecular Weight Of Photolysis Peptides. ..228 Figure.A.3. 1 : Viscosity Of Eandom-coil Proteins.. 232 Figure.A.4. 1 : Badius Of Gyration For Various Shapes. ..... 237 Figure.A.4.2: Evaluation Of Protein Conformation 239 Figure.A.5. 1 : Evaluation Of Solvent Effects On Random C o i l s . 24 3 Figure. A. 6. 1 : E l a s t i n Evcluticn. 247 xiv AKNOWLEDGEMENTS This research was made possible by the patience and support cf many people. I would esp e c i a l l y l i k e to thank Dr. J.M. Gosline who suffered through my everpresent bouts of incompetence and procrastination without losing interest in the d i r e c t i n g cf t h i s research. I would also l i k e to thank Karen Martin for her help with the preparation of thi s thesis and for giving me the emotional support over these past years. The occupants of the lab and my other associates, Mark Denny, Tony Harmon, Bob Shadwick, and Kevin Bush have a l l contributed to t h i s thesis through t h e i r many c r i t i c i s m s and discussions, and t h e i r input i s gr a t e f u l l y acknowledged. La s t l y , I would l i k e to thank Dr. P.D. Burns from the Department of Chemistry, without whose co-operation and many hours of experimental work the nmr studies would not have been possible, and Tina Duke from the Department of Computer Science who helped with the documentation of t h i s thesis. This research was supported by grants from the B.C. Heart Foundation and the Canadian Natural Sciences and Engineering Council to Dr. J.M. Gosline. XV This thesis i s dedicated to my family: my parents Benson and Seemah Aaron, and my brother Solomon Aaron, whose support and advice provided the ca t a l y s t for t h i s work. 1 Chapter.I. INTRODUCTION,, We have a l l experienced instances where having boasted about the knowledge of an objects function have subsequently been embarassed by our ignorance of the 'mode of functioning',. Proteins, which serve a vast number of functions i n nature, present the same dilema. As most of us know from watching detergent commercials on t e l e v i s i o n , enzymes break down certain compounds. This t e l l s us about t h e i r 'function' but does not i n any way inform us about the manner in which i t performs. If one i s to comment on t h i s aspect of the enzyme's character i t becomes necessary to obtain some s t r u c t u r a l information for the protein i n question. The same argument applies to e l a s t i n . Although one can claim that e l a s t i n i s a rubbery protein, the basis of t h i s e l a s t i c i t y , which i s the topic of i n t e r e s t , cannot be elucidated without knowledge of the protein's conformational state. Different theories of e l a s t i c i t y assume di f f e r e n t conformations at the molecular l e v e l for the material i n question. Hence, i t seems reasonable that one should investigate the molecular conformation of t h i s protein in an attempt to shed some l i g h t on the v a l i d i t y of the wide spectrum of theories that claim tc explain the basis of e l a s t i n e l a s t i c i t y . Hence the major focus of this thesis was to investigate the structure of the e l a s t i n protein and, then, to interpret i t ' s physical properties i n terms of a t h e o r e t i c a l framework for rubber e l a s t i c i t y . The.techniques of conformational analysis used i n t h i s i nvestigation were selected on their value as sensitive probes 2 of structure with the minimum amount of disturbance to the native conformation i n terms of experimental techniques. The studies themselves were developed at d i f f e r e n t l e v e l s of organization st a r t i n g with the investigation of soluble peptide properties and building upto the intact e l a s t i c f i b r e . Nmr and vi s c o s i t y experiments were used to study the structure of soluble proteins from e l a s t i n , with polarized microscopy and scanning electron microscopy providing the tools for the analysis of intact e l a s t i n f i b r e s . A l l of these studies showed e l a s t i n to be a random network elastomer, and on the basis of t h i s conclusion I then proceeded to use the t h e o r e t i c a l framework provided by the kinetic theory of rubber e l a s t i c i t y to characterize the macroscopic mechanical and photoelastic properties of t h i s protein. F i n a l l y , a test f o r the random network network conformation was conducted by predicting the dimensions of randcm-coil proteins, and comparing these to published values. The thesis i t s e l f i s organized i n the following manner, I have started by discussing the properties of the intact t i s s u e composite (chapter II) to show the exact relationship of the e l a s t i c f i b r e to the other components present i n the tissue. The following four chapters deal with the question of e l a s t i n conformation and involves the presentation of the current controversy (chapter I I I ) , the nmr and v i s c o s i t y investigations (chapter IV), the polarized microscopy studies (chapter V) , and the scanning electron microscopy result-s (chapter VI). The next chapter (chapter VIII) deals with the 3 evaluation of the e l a s t i n network properties i n terms of the k i n e t i c theory of rubber e l a s t i c i t y . The predictive test for e l a s t i n conformation i s presented i n chapter IX.. 4 Cha_)ter__II__ GENERAL CHARACTERISTICS OF ELASTIC TISSUE,, A. . Introdaction Given a mechanically functioning material, the macroscopic properties of t h i s tissue w i l l depend on, (a) the chemical composition of the tissue, (b) the mechanical properties of the i n d i v i d u a l components, (c) the a r c h i t e c t u r a l organization of these components, and (d) the ef f e c t of the chemical properties of one component on the mechanical properties of i t s e l f and the other components. As w i l l be shown i n t h i s chapter the e l a s t i n protein forms a major mechanical component of e l a s t i c tissue, and i t i s probably j u s t i f i a b l e to discuss the chemistry of t h i s p a r t i c u l a r protein which, along with i t ' s organization, w i l l eventually determine i t ' s functional properties..Since e l a s t i n i s implicated in the pathology of the vascular system, there has been considerable a c t i v i t y in t h i s f i e l d , so for the sake of convenience (and lack of breath) I have just outlined the major aspects of i t ' s biochemistry. A detailed discussion of the chemistry of e l a s t i n has been presented by Sandberg (1976) and Franzblau (1971). 1 E l a s t i n Development (a) Embryology A l l e l a s t i n , l i k e collagen and other connective t i s s u e , arises from the t h i r d germ layer commonly referred to as 5 mesoderm. The exact type of mesoderm that gives r i s e to any p a r t i c u l a r e l a s t i n i s dependent on where i t occurs (the mesodermal source of a r t e r i a l e l a s t i n i s d i f f e r e n t from that of ligament or s k i n ) . This could account for the v a r i a b i l i t y of the composition that i s observed between d i f f e r e n t types of e l a s t i c tissue (Kieth et^a1_._. 1S79). A review of e l a s t i n embryology i s given by Hass (1939) and i s summarized i n the following paragraph. According to Hass, the vascular system i s the f i r s t part of the body to be supplied with e l a s t i n , and can be demonstrated to be present i n four day old chick embryos. In humans the e l a s t i n i s f i r s t found in the third or fourth week. In the embryo, the majority of the e l a s t i n i s concentrated i n the aorta with the exact d i s t r i b u t i o n changing aft e r p a r t u r i t i o n . After b i r t h , the r e l a t i v e amount of e l a s t i n i n the artery decreases while the r e l a t i v e amounts i n the veins increases. A s i m i l a r time course can be found for the development of e l a s t i n in the lungs, with the development of e l a s t i n i n the skin lagging by about three months. The alimentary tr a c t i s thought to be one of the l a s t organs to recieve e l a s t i c tissue. Jbj_ Morphogenesis of e l a s t i n f i b r e s Although i t has been known fo r a long time that the e l a s t i n f i b r e i s a two component system, i«.e.. an inner amorphous core with a surrounding f i b r i l l a r coat, the exact relationship between the two has only recently been 6 elucidated. Ross and Eornstein (1969) demonstrated that these components of the e l a s t i n f i b r e are very d i s t i n c t chemically, the external f i b r i l l a r coat being a polar glycoprotein with the amorphous core being an extremely hydrophobic protein. The chemistry cf the m i c r o f i b r i l l a r component has been studied and i t has been shown to be composed of f i b r i l s , ranging from 10 to 40 nm in diameter, which could be extracted with agents that reduced di-sulphide bonds (Robert e t . a l . 1971, Anderson 1976) . In order to assign a functional r o l e to these glycoprotein f i b r i l s i t has been proposed that they are involved with the aligning of the e l a s t i n protein during i t ' s secretion. It has been shown that e l a s t i c ligaments i n the embryo are almost devoid of e l a s t i n i n t h e i r early stages of development, but have a high amount of glycoprotein present. In the l a t e r stages of development the e l a s t i n protein can be seen to be interspersed between these m i c r o f i b r i l s which, due to t h e i r negative charge, may tend to aggregate the e l a s t i n (which has a net positive charge) around themselves (Ross et_.al o_ 1 97 7). Further evidence for t h i s type of a r o l e f o r the m i c r o f i b r i l s has been obtained by Cotta-Pereira et.al..(1977), who h i s t o l o g i c a l l y demonstrated the presence of two types of developing e l a s t i n f i b r e s . Oxytalan f i b r e s , composed mainly of glycoproteins, and elaunin f i b r e s which consisted of m i c r o f i b r i l s and amorphous e l a s t i n . On the basis of t h e i r observations they proposed a oxytalan-- e l a u n i n — mature 7 e l a s t i c f i b r e hierarchy for the development of the e l a s t i n protein, which i s synthesized in vivo by f i b r o b l a s t and smooth muscle c e l l s (Boucek 1959, and Pathrapamkel e t . a l . 1977). Jcj_ E l a s t i n turnover The turnover of e l a s t i n in normal e l a s t i c tissue i s characterized by a h a l f - l i f e that i s approximately egual to the l i f e span of the animal (Ayer 1969). .It has been proposed by some workers that the diseased states represent an a l t e r a t i o n i n the turnover rate of the e l a s t i c t i s s u e , increasing with age and pathology. This increase in turnover i s l a r g e l y due to the presence of degradative processes (Robert 1 977) . . E l a s t i n Chemistry _[a}_ Amino acid composition In order to characterize the amino acid composition of a protein one has to f i r s t decide on the guestion of what i s to be considered as being the 'pure' protein..Fortunately the case f o r e l a s t i n i s guite clear cut, i n that investigators have been able to treat e l a s t i c tissue guite d r a s t i c a l l y and s t i l l arrive at a protein of constant composition which can account for the e l a s t i c i t y of the i n t a c t tissue. This residue that remains after treatment i s termed e l a s t i n , and w i l l be defined as such for the rest of t h i s thesis. Amino acid analysis of t h i s protein shows e l a s t i n to be 8 'Table, 2. 1: Ajinc acid composit ion of e l a s t i c proteins, AMINO ACID COMPOSITION OF ELASTIC PROTEINS. * ELASTIN 1 RESILIN 2 ABDUCTIN 3 CONNECTIN4 OCTUPUS FIBRES 5 asx 6.4 102 69.9 92 90.6 th r 8.9 28 7.4 59 64.4 ser 9.9 80 36.4 60 71.8 9lx 15 47 19.4 128 121 pro 120 77 7.4 65 54.9 hyp 10.7 - — 12 - g i y 324 385 620 104 85 a l a 232 111 26.5 84 71 c y 5 4.1 - - 4 7.4 val 135 28 3.5 60 62 met — - 117 23 21 1le 25.5 17 4 52 60 leu - 61.1 23 3 70 73.5 t y r 7.1 27 10.7 28 36.2 phe 30 26 51.3 29 42.3 h i s 0.6 9 0.3 15 21 l y s 7.4 5 12.4 61 68.3 arg 5.4 35 9.8 56 45.8 * 1n residues/1000. i ^ o s s and Bornstein 1969. We1s-Fogh 1961. 3 K e l l y and Rice 1967. 4Maruyama e t . a l . 1976. 5Shadwick 1980. 9 one of the most hydrophobic proteins yet discovered, with nearly 60% of the residues being non-polar. As w i l l be presented l a t e r , t h i s c h a r a c t e r i s t i c , more than any other, has been responsible for the mis-interpretation of the physical properties of t h i s elastomer. E l a s t i n also has an unusually large content of glycine, valine, and proline residues. The amino acid composition of e l a s t i n (Eoss and Bornstein 1969) and i t ' s comparison to the other known elastomers, R e s i l i n (Anderson 1971) , Abductin (Kelly and Rice 1967) , Connectin (Maruyama ______ 1976), and the recently discovered elastomer from octopus a r t e r i e s (Shadwick 1980) i s shown i n table 2. 1. . (b) E l a s t i n cross-links Since e l a s t i n i s a protein that has a mechanical function, i t has to be cross-linked in order to prevent the polypeptide chains that make up the tissue from 'flowing' under stress. In the case of natural rubber t h i s i s accomplished by forming covalent bonds between carbon residues of adjacent polymer chains (Flory 1953).. With regard to e l a s t i n , the cross-links of t h i s elastomer were f i r s t discovered by Thomas e t . a l . (1963) who showed them to be pyridinium derivatives and named them desmosine and i s o - desmosine. These s t r u c t u r a l proposals were confirmed concurrently by nuclear magnetic resonance studies (Bedford and Katritzky 1 963) . These same authors (Partridge ______ 1965) l a t e r showed that these compounds cross-linked two polypeptide chains and 10 proposed a possible route for the formation of these l i n k s . I t i s currently thought that the desmosines and iso-desmosines are formed by the condensation of four l y s i n e s , three of which have been oxidized by the enzyme l y s l oxidase (Sandberg 1976). There i s also some evidence that residues other than the (iso)desmosines, such as lysinonorleucine, also serve as c r o s s - l i n k i n g agents i n the e l a s t i n network (Lent e t . a l . 1969) . Jc_ Soluble e l a s t i n As mentioned before, insoluble e l a s t i n results from the cross - l i n k i n g of s o l u t l e e l a s t i n precursors into a functional network. Due to t h i s relationship there has been some in t e r e s t i n the characterization of these precursor proteins. Although a number of methods for the s o l u b i l i z a t i o n of e l a s t i n are known,such as digestion with oxalic acid (Partridge and Adair 1955), elastase (Hall 1961), urea (Bowen 1953), KOH (Mcczar ______ 1979). The only method available at the moment for the i s c l a t i c n of an unbranched precursor i s the extraction cf tropoelastin from l a t h y r i t i c animals. This method i s based on the biochemistry cf c r o s s - l i n k formation which requires the oxidation of the lysine residues, involved in the formation of (iso)desmosines, by the copper requiring enzyme l y s l oxidase (Franzblau 1971). Inhibition of t h i s enzyme's a c t i v i t y , by r a i s i n g animals on copper defecient diets or by inducing lathyrism using agents such as B-amino- p r o p i o n i t r i l e , allows the extraction of soluble proteins from 11 the e l a s t i c tissue of the animal. The extraction procedure results i n a protein which has an amino acid composition that i s . s i m i l a r to mature e l a s t i n (with the exception of a high lysine content, due to the lack of cross-links) and i s termed tropoelastin i n analogy to the collagen- tropocollagen scheme (Sandberg 1976). It i s currently thought that tropoelastin, which has a molecular weight of 72,000, represents the building block of mature insoluble e l a s t i n . There i s some speculation that there exists a higher molecular weight species, which would be comparable to procollagen, and recent publications have stated the presence of such a protein, named proelastin, having a molecular weight of 130,000 to 140,000 (Foster e t . a l . 1976, 1977).. This speculation, however, has been put to rest by a recent paper that provides strong evidence that tropoelastin i s the primary precursor in e l a s t i n biosynthesis {Rosenbloom et^ali. 1980). The precursor-product relationship between tropoelastin and fibrous e l a s t i n has been demonstrated recently by the i n v i t r o cross-linking of the soluble proteins to give cross- linked e l a s t i n (Narayanan and Page 1976, Smith et. a l ^ .197 5).. P a r t i a l primary seguence data for porcine tropoelastin have also been published in the l i t e r a t u r e on e l a s t i n biochemistry (Sandberg e t ^ a L 1977). EU Composition of E l a s t i c Tissue 12 _[__ Water content Unpurified e l a s t i n samples from ligament nachae were blotted on paper towels to remove excess water and weighed at 240C..The same samples were then dried to constant weight i n an oven at 110<>C and reweighed. The results indicate that ligament e l a s t i n i s approximately 72% water by weight.. A similar value of 70% has been reported for a r t e r i a l e l a s t i n samples (Harkness ______.1957). (b) E l a s t i n _______ Unpurified ligament e l a s t i n samples were dried i n an oven to constant weight. The samples were then p u r i f i e d by repeated autoclaving (Partridge e t . a l . 1955), dried and reweighed. The value of e l a s t i n content so obtained was about 80% by dry weight. Values for the e l a s t i n content of thoracic a r t e r i e s occur in the range of 40% e l a s t i n (w/dry weight) (Charm e t . a l . 1974, Harkness ______ 1957).. Jc_ Neutral sugars Neutral sugar content of unpurified ligament e l a s t i n was evaluated using the phenol-sulphuric acid assay of Lo e t _ a l . (1970), using glucose (Sigma) as a standard. A value of 0.3% (w/dry weight of tissue) was obtained. _d_ Mucopolysaccharides I t i s almost impossible to determine the exact mucopolysaccharide content of e l a s t i c tissue due to the large Itr In IP- in> CHEMICAL COMPOSITION OF ELASTIC TISSUE. E Ligamentum nuchae Aorta Mucopolysaccharides: 2.3% 1.6% hexosamlnes (36%) (26%) uronic acids (41%) (34%) sulphate (15%) (12%) Collagen 20% 18% E las t in 78% 18% Neutral Sugars 0.3% ? l O II- 1 !/) H- In- IM- IS t 14 v a r i a b i l i t y from sample to sample. A general composition i s given i n table 2.2 for ligament (Meyer e t . a l . 1956) and a r t e r i a l tissue (Kirk 1959). In the case of ligament e l a s t i n the mucopolysaccharides are seen to make up about 2.3% of the whole tissue (w/dry wt. Tissue). Of t h i s approximately 36% i s present in the form of hexosamines, 41% as uronic acids with approximately 15% acid hydrolyzable sulphate. . Jej_ Collagen content After accounting for the various other components present in ligament tissue, the collagen content works out to be approximately 17.4% as compared to values of 18% reported for a r t e r i a l samples (Harkness et. a L . 1957) . The summary for the chemical composition of a r t e r i a l and ligament tissue i s given i n table 2.2 . E. Organization of E l a s t i c Tissue Since the main function of e l a s t i c tissue i s a mechanical one, i t i s not surprising that the organization of the e l a s t i n f i b r e s i n e l a s t i c tissue varies with the direction(s) of the stra i n that are imposed on the tissue i n vivo . In keeping with t h i s generally accepted hypothesis, the following i s a presentation of the microscopical organization of the two extremes of e l a s t i c tissue as demonstrated by ligament nuchae and a r t e r i a l e l a s t i n . Ja| Methodolc_g_y 15 Histology, Samples cf unpurified pig aorta were stripped of the a d v e n t i t i a l layer and other adhering tissue. Ligament e l a s t i n was also cleaned of adhering tissue, and both the a r t e r i a l samples and the ligament samples were treated with Bouins f i x a t i v e for 72 hours. They were then rinsed with d i s t i l l e d water, cut into small pieces and embedded i n paraffin wax using standard h i s t o l o g i c a l techniques. After embedding the tissue was cut into 5 to 7 um sections and stained. The procedure, which was a s l i g h t modification of that presented by Clark e t _ a l _ (1973), consisted of staining sections with orcein followed by a counter-stain of napthol green B. This protocol r e s u l t s i n red e l a s t i n f i b r e s with the collagen f i b r e s appearing f a i n t green. . Scanning electron microscopy: Instead of using f i x a t i v e s , the samples were frozen i n l i g u i d nitrogen and dried in a vaccum. They were then mounted onto stubs and coated with a fine layer of gold. The examination of the samples was conducted on a Cambridge Instrument Company, Stereoscan microscope (see methods, chapter 6 for more d e t a i l s ) . Jb_ Organization of ________ e l a s t i n As expected, ligament e l a s t i n in the l i g h t microscope shows a very d i s t i n c t alignment i n the longitudinal d i r e c t i o n , which i s the direction of the i n vivo s t r a i n (figure 2.1a)» The 1um collagen f i b r e s seem tc form a very fine network 16 Figure.2.1: Organization of ligament e l a s t i n . (a) l i g h t micrograph of sectioned, unpurified ligament nuchae . The e l a s t i n f i b r e s , E, are aligned in the dire c t i o n of the st r a i n (arrow) with the collagen (C) interspersed between the e l a s t i n . The bar represents 30um. . (b) s.e.m. of unpurified ligament showing the collagen. (c s.e.m. of unpurified ligament. The arrow indicates a branch point in the e l a s t i n network. The s o l i d bar in b, and c represents 10um. 17 Figure.2. 1. 18 around (between) the e l a s t i n f i b r e s which have a diameter .of about 6 to 8 um. There was no h i s t o l o g i c a l evidence for the presence of a collagen sheath, cr other such collagenous structure, associated with the i n d i v i d u a l e l a s t i n f i b r e s . Scanning electron microscopy e s s e n t i a l l y supported the findings of the l i g h t microscope study as well as giving a clearer picture of the fine d e t a i l s . It showed a very d i f f u s e network of collagen f i b r e s dispersed in the e l a s t i c network (figure 2.1b). There was also some evidence for the branching of the i n d i v i d u a l e l a s t i n f i b r e s (figure 2.1c). Jc_ Organization of a r t e r i a l e l a s t i n In contrast to the ligament samples, cross-sections of a r t e r i a l samples show a very d i s t i n c t lamellar organization of i t ' s constituents (figure 2.2a), with the lamella (which are about 2um i n thickness) running in the circumferential d i r e c t i o n . The e l a s t i n i n any given lamella seem to be organized u n i d i r e c t i o n a l l y , with the adjacent lamellae showing a successive change in this d i r e c t i o n (figure 2.2b). The presence of interlamellar f i b r e s i s also evident (figure 2.2c). The collagen seemed to occur between the e l a s t i n lamellae i n a dense f i b r i l l a r network. Although i t was not possible to determine the d i r e c t i o n of the collagen alignment (with respect to the longitudinal d i r e c t i o n of the artery) there have been reports that the collagen network actually follows a h e l i c a l path (Wolinsky and Glagov 1964). The composite s t r u c t u r a l organization of ligament and 1 9 Fig;ur;e__2, 2: Organization of a r t e r i a l e l a s t i n . (a) s.e.m. shewing the lamellar (L) organization of a r t e r i a l e l a s t i n . (b) Light micrograph of sectioned a r t e r i a l media showing the r e l a t i v e organization of adjacent lamellae. The tar represents 2Cum. (c) s.e.m. of artery showing the presence of the interlamellar f i b r e s (If) . The s o l i d bars in a, and c represents 10um. 20 2 1 a r t e r i a l tissue i s depicted i n figure 2.3. A more compelete analysis of the structure of e l a s t i c tissue can be found i n a r t i c l e s by Cotta-Pereira e t . a l . (1977), Carnes e t . a l . (1977) and Kadar (1977) . F«_. P u r i f i c a t i o n Techniques In order to study the properties of the e l a s t i n protein, i t i s necessary to i s o l a t e the protein from the rest of the tissue with which i t occurs in vivo . This can be done by the use of a number of p u r i f i c a t i o n technigues f o r which the methodology (Robert and Hornebeck 1976) and the chemical evaluation of the r e s u l t i n g e l a s t i n (Partridge 1962, Grant e t . a l . 1971) has been well documented. In t h i s thesis, which deals primarily with the physical properties of the e l a s t i n protein, the a l k a l i extraction procedure (Lansing et.al.,1952) and the repeated autoclaving method of (Partridge e t ^ a l ^ 19 55) were used exclusively. The following section i s therefore presented as a comparison of these two technigues on a chemical and s t r u c t u r a l basis. J§1 Amino acid compositions In evaluating the amino acid composition one has to f i r s t decide on a standard against which to compare the r e s u l t s of the p u r i f i c a t i o n product. This standard i s usually taken to be the precursor molecule, tropoelastin. Table 2.3 l i s t s the amino acid composition for tropoelastin, autoclaved e l a s t i n and a l k a l i extracted e l a s t i n as presented by Grant et. al.. 22 Figure.2.3: The _______ tissue __________. (A) The a r t e r i a l media showing the r e l a t i v e organization of the collagen (C) and the e l a s t i n (E) . (B) A schematic diagram of ligamenturn ______ showing the organization of the e l a s t i n , collagen, and the collagen sheath (CS) . 23 Figure.2 24 (1971). Both procedures are seen to give comparable e l a s t i n preparations with regard to the amino acid composition. jb_l Hexosamine and neutral sucjar content The data obtained by Grant e t . a l . . (1971) indicate that a l k a l i extracted e l a s t i n contains about half as much hexosamine as autoclaved samples (both are below 0.04% w/w). The neutral sugars were guantitated with the phenol-sulphuric acid assay of Lo e t . a l . . (1970) using glucose (Sigma) as a standard. The results show that autoclaved e l a s t i n had a neutral sugar content of 0.02% (w/w). There were no detectable sugars i n a l k a l i p u r i f i e d e l a s t i n . l£L Evaluation by_ scanning electron microscopy Samples of autoclaved and a l k a l i p u r i f i e d e l a s t i n were frozen i n l i g u i d nitrogen and dried i n a vaccum. The pieces of tissue were mounted onto stubs, coated with gold and observed in a Stereoscan microscope as described before. Both p u r i f i c a t i o n procedures gave clean preparations as observed in the scanning electron microscope, with no i n d i c a t i o n of collagen or 'matrix' substances (figure 2 .4 , a and b). However, there was some indicati o n of a l k a l i attack of the e l a s t i n f i b r e s prepared by 0.1N NaOH extraction (figure 2.4c). No such degradation was observed for autoclave p u r i f i e d e l a s t i n . G. Mechanical Properties of E l a s t i n Bundles 25- TaLl_g.2.3: _____o a c i d composition of e l a s t i c p r e p a r a t i o n s . AMINO ACID COMPOSITION OF ELASTIN PREPARATIONS* Tropoelastin Autoclaved . A l k c l i asx 3.3 6.4 5.4 thr 13.2 7.4 5.3 ., ser 9.2 8.7 6.6 glx 15.8 15.7 11.9 pro 101.1 118.4 96.4 hyp 6.6 8.7 12.9 giy 333.4 310.5 316.2 ala 237 238.6 243.1 cys — — — val 125.4 143.9 154.3 met — — -- i le 16.1 23.3 24.6 leu 47.5 59.5 63 tyr 14.1 10.7 13 phe 28.3 28.6 32.1 his — -- -- lys 45.1 3.1 3.1 4.3 7.7 2,7 *from Grant et.al. 1971. 26 fi^ureiJ,. Evaluation of £urification techniques. (a) s.e.m. of a l k a l i p u r i f i e d e l a s t i n . (b) s.e.m. of autoclave p u r i f i e d e l a s t i n . (c) Higher magnification of a l k a l i p u r i f i e d e l a s t i n showing the hydrolytic attack of the e l a s t i n f i b r e . The s o l i d bars represent 10um. 27 28 (a) Methods Samples of ligament e l a s t i n were dried and their ends were embedded into threaded s t e e l cups with epoxy glue. They were then hydrated i n d i s t i l l e d water over a period of seven days under s t e r i l e conditions before testing. The stress- s t r a i n properties of the e l a s t i n were determined with an Instron t e n s i l e testing machine, with the samples (of about 1 .5 cm length) being extended at a rate of 1mm/min. The unstrained cross-sectional area was measured as follows. The length of the sample between the anchoring points was measured with ca l i p e r s before the start of a test. This was taken to be the value of L°, used for the subsequent ca l c u l a t i o n of the extension r a t i o and unstrained cross- se c t i o n a l area. Immediately aft e r the mechanical test, the sample was cut at the anchor points, dried to constant weight and weighed. The volume of the protein was calculated from t h i s weight and a value of 1. 33g/cc for the density of the protein. The volume of the hydrated sample was clculated by using a volume fr a c t i o n of 0.65 f c r the protein at 24°C (Gosline 1978) , which was the temperature at which the tests were conducted. The ncminal cross-sectional area could then be calculated by dividing the value for the hydrated volume by LO. _(b) Results Figure 2.5 shows the results of the s t r e s s - s t r a i n 29 Figure.2.5; Mechanical __________ of e l a s t i n ________ Plots of nominal stress versus s t r a i n f o r : (a) autoclaved ligament e l a s t i n bundles. (x ) represents the f a i l u r e s t r a i n . (b) unpurified ligament e l a s t i n bundles. 30- figure.2.5. 50 100 150 Extens ion % 31 experiments cn unpurified and autoclave p u r i f i e d ligament e l a s t i n . The unpurified samples show a biphasic curve with an i n i t i a l t e n s i l e modulus of 6.87 X 10 s Nm-2 and a f i n a l modulus of 3.8 X 10 6 Nm-2..These unpurified bundles could be extended by about 150% cf the i r i n i t i a l length before f a i l u r e . In contrast to t h i s , autoclaved e l a s t i n samples f a i l e d at about 50% extension, and upto f a i l u r e exhibited a single value for the t e n s i l e modulus cf 8.6 X 10 s Nm-2 which i s higher than the modulus of the unpurified bundles i n the same region of extension. These values obtained for autoclaved e l a s t i n samples are in close agreement with the values attained by Mukerjee e t . a l . (1976).. Although t h i s result, of a higher modulus for the puri f i e d ligament as compared to the unpurified t i s s u e , appears ri d i c u l o u s at f i r s t glance, i t can be explained i f one assumes that only the e l a s t i n component of the unpurified samples i s contributing to the mechanical properties for elongations approaching 50% extension. Since the cross- sec t i o n a l area i s calculated for the whole tissue, of which only 80% i s e l a s t i n , the nominal stress values w i l l be an underestimate due to the overestimate of the 'contributing' cross-sectional by approximately 20%. This i s borne out by the absolute magnitude of the two modulii with the r a t i o of the unpurified modulus/purified modulus being about 0.75 .. H_.Mechanical Properties of ______ E l a s t i n Fibres The f i r s t studies on the mechanical properties of 32 Fi^are_. 2_. 6j_ Mechanical properties of single e l a s t i n fibres., j?lots of nominal force versus extension f o r : (a) unpurified fibres {Carton et^al.. 1962). (b) autoclaved f i b r e s . 50 100 150 Extension % 34 unpurified single e l a s t i n f i b r e s were reported by Carton e t . a l . . (1960, 1962). Their r e s u l t s are presented along with the mechanical properties obtained for p u r i f i e d single e l a s t i n f i b r e s i n t h i s study in figure 2.6 (the methodology i s discussed in chapter 7). The relationship obtained by Carton also exhibited a biphasic s t r e s s - s t r a i n curve s i m i l a r to that obtained for unpurified bundles of e l a s t i n . In comparison to t h i s , autoclaved e l a s t i n f i b r e s show a f a i r l y l i n e a r relationship upto approximately 100% extension, beyond which they also tend to deviate upwards, but by an amount that i s much smaller than that shown by the unpurified f i b r e s . In contrast to the autoclaved bundles, purified single f i b r e s could be extended to 120-150% extension before f a i l u r e . The Young's modulus for the i n i t i a l extension region, of both the unpurified and autoclaved single f i b r e s , had a value of about 1 X 106 Nm-2. I_. Discussion Ja_ Variation in e l a s t i n biochemistry Although" there i s a general concensus about the composition of e l a s t i n , there i s also some ind i c a t i o n that a few d i s t i n c t differences ex i s t in the amino acid p r o f i l e s and/or primary sequence of e l a s t i n from d i f f e r e n t sources. Recently there have been reports on the e l a s t i n from auricular c a r t i l a q e , which have shown t h i s protein to have an unusually large content of polar residues (twice as much as a r t e r i a l 3 5 elastin) with a 20% reduction i n the amount of valine residues (Field e t ^ a l i 1 9 78) . The sequential v a r i a b i l i t y was pointed out by Kieth et.. Al._ (1979), who analyzed the valine-proline seguence content of e l a s t i n from di f f e r e n t tissue. They showed that t h i s p a r t i c u l a r seguence occurs about 41 times/1000 residues i n ao r t i c e l a s t i n and only 9 times/1000 residues i n auricular e l a s t i n . On the basis of these r e s u l t s they favoured the existence of more than one gene for the e l a s t i n protein. This aspect of e l a s t i n biochemistry i s worrisome since i t raises doubts about the use of e l a s t i n from ligament nuchae to make generalizations about e l a s t i n from other sources e.g., a r t e r i a l elastin.These differences i n the primary seguence would be very c r u c i a l , i f as suggested by several authors (Urry et^al.. 1S77a) , the val-pro seguence i s a major determinant of e l a s t i n structure. Jb}_ The composite tissue E l a s t i c tissue, l i k e most other b i o l o g i c a l t i s s u e , occurs as a chemical and mechanical composite in vivo . Its organization i s seen to vary from tissue to tissue depending on i t ' s exact functional state. In general, i t seems evident that the e l a s t i n protein i s probably the dominant mechanical component at low strains with the collagen contributing the more s i g n i f i c a n t component at higher elongations. Comparison of single f i b r e r e s u l t s f o r purified and unpurified e l a s t i n , seems to show that another mechanical 36 component i s intimately associated with the single e l a s t i n f i b r e . There i s seme speculation that this other component consists of collagen which i s present as a sheath around the i n d i v i d u a l e l a s t i n f i bres (Finlay and Steven 1973, S e r a f i n i - Fracassini et_ A l . 1977). This aspect of e l a s t i n organization which i s based on mechanical properties, could not be supported by the r e s u l t s of the h i s t o l o g i c a l and scanning electron microscope studies conducted for t h i s report. Although the ground substance i s unlikely to be a major mechanical component i n . e l a s t i c tissue ( i t probably contributes a small amount as shown by Banga and Balo 1960), i t ' s chemical properties are probably important i n determining the environment within the e l a s t i c tissue. Alterations i n these environmental properties would ine v i t a b l y e f f e c t the mechanical properties of the e l a s t i c component. Recent studies cn the dynamic mechanical properties of e l a s t i n (Gosline and French 1979) indicate that the functional properties of the e l a s t i n protein are adversely altered by s l i g h t degrees of dehydration. In view of t h i s the c r u c i a l r o l e , of 'controlling the environment 1, which i s performed by the embedding matrix i n vivo cannot be ignored. _c_ Evaluation of _ u r i f i c a t i o n techniques In comparing the f e a s i b i l i t y of any given p u r i f i c a t i o n procedure one has tc f i r s t consider the experiment that i s to be conducted on the p u r i f i e d sample. It i s evident from t h i s study that a l k a l i p u r i f i c a t i o n yields a more pure e l a s t i n 3 7 (chemically closer to tropoelastin) than the autoclaving procedure. On the other hand, scanning electron microscopy showed that, s t r u c t u r a l l y , both preparations gave s i m i l a r r e s u l t s in grcss observation. However, closer examination of the preparations revealed that there was some evidence of hydrolytic attack of the e l a s t i n f i b r e i n the samples that had been p u r i f i e d by a l k a l i treatment. In contrast, there was no in d i c a t i o n of such unwanted side e f f e c t s in the autoclaved preparations. This brings me back to the f i r s t statement. If the experiment that one had i n mind was biochemical i n nature, then i t would be advisable to use the cleaner preparation afforded by the a l k a l i technique. Alternately, i f the experiment involves the measurement of physical properties, such as stress- s t r a i n r e l a t i o n s h i p s , i t i s more feasi b l e to use an autoclaved sample which, though less pure chemically, i s probably more appropriate for such purposes since there i s no evidence of e l a s t i n degradation. 38 C h a p t e r ^ I I I i . CONFOJiMATION OF ELASTIN^ THE CONTROVERSY.. Introduction Several models for e l a s t i n have been proposed over the years. These range from the extreme of the collagen-like t r i p l e helix structure, proposed by Ramachandran (1963), to the other extreme of a t o t a l l y amorphous random network si m i l a r to other k i n e t i c elastomers (Hoeve and Flory 1 9 5 8 ) . The remaining p o s s i b i l i t i e s l i e somewhere in between these two extremes, such as the Liguid-drop model of Weis-Fogh and Anderson ( 1970) , the O i l e d - c o i l model of Gray e t . a l . (1973) and the recently published cross-Beta s p i r a l of Urry (1976a) . These models for e l a s t i n structure are depicted i n figure 3 . 1 i This chapter w i l l be directed towards presenting the various proposals for e l a s t i n conformation, and w i l l t r y to evaluate the evidence on which they are based. . The Random Network Model The random network model for e l a s t i n was e s s e n t i a l l y an extension from the work of polymer chemists on the properties of hydrocarbon elastomers. Since these materials exhibited mechanical behaviour that could not be accounted for by the standard theories cf s o l i d e l a s t i c i t y present at the time, a new t h e o r e t i c a l framework, commonly referred to as the Kinetic Theory of Rubber E l a s t i c i t y , was developed (Guth e t . a l ^ 1946 ). The theory was based on Gaussian s t a t i s t i c s reguiring a random conformation for the material i n guestion. 39 Figure. 3 . 1: Proposals for the conformation of e l a s t i n (A) Collagen-like t r i p l e - h e l i x (Samachandran 1 9 6 3 ) . (B) Beta-spiral structure (Urry 1 9 7 8 b ) . (C) O i l e d - c o i l model (Gray e t ^ a l ^ 1 S 7 3 ) . (D) Liquid-drop model (Weis-Fogh and Anderson 1 9 7 9 ) . (E) Random network conformation (Hoeve and Flory 1 95 8) . 4 0 Figure. 3.1. B rigid t B-spiral I | dynamic j_-spiral c 41 This Kinetic theory was already in existence when people started to study the protein rubbers, and i t i s not surprising that the i n i t i a l attempts to characterize the properies of these protein elastomers involved the application of the k i n e t i c theory relationships. This was done f o r the bivalve hinge ligament Abductin (Alexander 1966) and the insect protein rubber R e s i l i n (Weis-Fogh 1961a). Both were convincingly shown tc be entropic elastomers as predicted by the k i n e t i c theory. Since t h i s theory i s based on a random network structure, i t was l o g i c a l to then extrapolate to the conformation of these elastomers and state that they were random at the molecular l e v e l . The idea of a random conformation f o r the e l a s t i n protein was also based on the analysis of i t ' s macroscopic properties according to the k i n e t i c theory of rubber e l a s t i c i t y . _a_ E l a s t i n thermoelasticity The i n i t i a l work on the thermoelastic properties of e l a s t i n , using the constant length experimental technique (appendix 1), indicated that a large energy component was associated with the e l a s t i c mechanism which was i n contradiction to the notion of an entropy elastomer (Meyer and F e r r i 1936, Wohlisch e t . a l . 1943). It was l a t e r shown that these investigators f a i l e d to account for the temperature dependent swelling of e l a s t i n , which has a large energy component associated with i t (Hoeve and Flory 1974). Hoeve and Fiery (1958) had gotten around t h i s 4 2 experimental d i f f i c u l t y , of temperature-deswelling, by studying the thermoelasticity of e l a s t i n in a mixed diluent system, of 30:70 ethylene-glycol:water, where the volume of e l a s t i n was independent of temperature. Their experiments showed that, under these conditions, e l a t i n behaved as a t y p i c a l k i n e t i c rubber with the energy component being close to zero. This conclusion was l a t e r supported by Volpin and C i f f e r i (1970) who conducted a si m i l a r experiment i n the temperature range of 50°-70°C, where the volume of e l a s t i n i s also independent of temperature. The mixed diluent systems u t i l i z e d by Hoeve and Flory was l a t e r c r i t i c i z e d by Oplatka _t_al__£ (1960) , who pointed out that although t h i s approach circumvented the problem of a temperature dependent volume change i t did not test for a composition change i . e . the changes i n the d i s t r i b u t i o n of the types of solvent molecules bound as a function of temperature. It was therefore necessary to make some changes in the basic t h e o r e t i c a l relationships to explain the thermoelastic behaviour of open systems such as e l a s t i n . This was accomplished by Oplatka e t . a l . , (1960), and Bashaw and Smith (1968) for pclymer systems of highly swollen rubbers. The s p e c i f i c application of these relationships to the e l a s t i n protein was provided by M i s t r a l i _______ (1971). Hence with regard to e l a s t i n thermoelasticity, at any rate, i t seems f a i r l y safe to conclude that the results point to e l a s t i n being a t y p i c a l k i n e t i c elastomer as described by the k i n e t i c theory cf rubber e l a s t i c i t y . Since t h i s theory i s 4 3 based on a random network conformation, i t seems reasonable to expect e l a s t i n to be random i n i t ' s conformation as well, Jb_ D i f f e r e n t i a l scanning calprimetry Kakivaya and Hoeve (1975) used the technique of d i f f e r e n t i a l scanning calorimetry to study the glass point of e l a s t i n . They were able to show that e l a s t i n undergoes a second order t r a n s i t i o n , in a temperature range that depends on i t ' s water content.. Such second order t r a n s i t i o n s are t y p i c a l cf the glass t r a n s i t i o n s that are observed for most amorphous polymers..They did not see any evidence for f i r s t order t r a n s i t i o n s that might be associated with the 'melting' of stable secondary structures. The values f o r the glass t r a n s i t i o n temperatures correlated well with the observed mechanical behaviour of e l a s t i n which i s characterized by a sudden drop i n the modulus as i t goes frcm being a r i g i d glass to an extensible polymer (Gotte ______ 1965). It was also found that diluents, such as ethylene glycol and water, depressed the glass t r a n s i t i o n temperature equally, on a volume basis, which argues against the objection of Oplatka (1960) regarding the use of a mixed diluent system for thermodynamic studies of e l a s t i n . They also argued that these r e s u l t s were consistent with a random network model, and only a random network model consisting of a single homogeneous phase. _c_ _____ And X-ray d i f f r a c t i o n studies 44 Early examination of negatively stained e l a s t i n f i b r e s , i n the electron microscope, showed e l a s t i n to be an amorphous protein with no detectable s t r u c t u r a l features (Cox and L i t t l e 1962, Eoss and Bornstein 1969, Karrer and Cox 1961)..This however was not an unanimous conclusion as various other investigators at that time also reported seeing f i b r i l l a r structures within the e l a s t i n f i b r e (Lansing e t . a l . 1952, Gross 1949). Similar structures have been seen i n recent studies, and these observations form the basis for a filamentous model of e l a s t i n , that w i l l be discussed l a t e r on in t h i s chapter. . The X-ray d i f f r a c t i o n studies of e l a s t i n conducted at that time were also i n support of a random network model for e l a s t i n (Astbury 1940, Cox and L i t t l e 1962).. Both papers reported diffuse d i f f r a c t i o n haloes at 4.6A0 and 7.8A° which did not change upon stretching. This type of pattern i s t y p i c a l of amorphous polymers and indicated a lack of c r y s t a l l i n e structure. Again the interpretations were not unanimous with some reports of strong d i f f r a c t i o n patterns for e l a s t i n (Kolpack 1 935, Bear 1942 and 1944)..All of, these r e s u l t s were la t e r shown to be a r t i f a c t s caused by collagen contamination. J d l N.M. B. Evidence The rationale for testing the random netwcwrk model using nuclear magnetic resonance l i e s i n the high s e n s i t i v i t y of thi s technigue i n measuring the the mobility of tihe components 45 involved. Torchia and Piez (1973) obtained a l 3C-nmr spectrum of ligament e l a s t i n and they were able to show that the c o r r e l a t i o n times, which are taken to be indicators of backbone movements (with the small times representing f a s t k i n e t i c motion), as obtained from the l i n e widths of the various resonances, had values i n the 10 nanosecond range i n d i c a t i n g a high mobility for about 80 percent of the backbone carbons ( l y e r l a and Torchia 1975). The remaining 20 percent were proposed to be involved in the c r o s s - l i n k i n g region which would be expected to have lower m o b i l i t i e s . These data also support the random network conformation. C_. Liquid Drop Model (a) Preliminary evidence The i n i t i a l idea of a two-phase model for e l a s t i n was seeded by Partridge (1967a, 1967b) who hypothesized a corpuscular structure for e l a s t i n on the basis of gel f i l t r a t i o n experiments conducted on columns packed with e l a s t i n f i b r e s . He was able to show that these e l a s t i n packed columns could seperate molecules on the basis of t h e i r molecular weights, and that these separation c h a r a c t e r i s t i c s were consistent with the matrix (elastin) being a material that contained pores of about 32A° in diameter, or a sytem of randomly distributed rods having a length of 16A°. Partridge also noticed that t h i s system would adsorb alcohols, and that t h i s adsorption of alcohols was d i r e c t l y related to the 46 hydrocarbon chain length. I t therefore seemed reasonable, at the time, to think of e l a s t i n as a two-phase system with discrete regions of hydrophobic clusters and interspersed solvent, especially since these structures were supported by electron microscope evidence (Partridge 1 968). A si m i l a r type of model was eluded to by Kornfeld- Poullain and Robert (1968) using evidence from a l k a l i degradation studies of e l a s t i n . Jhey guantitated the a l k a l i digestion of e l a s t i n as a function cf solvent hydrophobicity and showed that the addition of alcohols to the reaction mixture greatly f a c i l i t a t e d the degradation of e l a s t i n . They then proposed a series of steps i n the degradation process of e l a s t i n which involved the i n i t i a l dispersion of hydrophobic regions by the organic solvent followed by a l k a l i n e attack of the peptide moieties. Although i t i s not stated e x p l i c i t l y , there i s an i m p l i c i t assumption i n t h i s type of degradation process of a two-phase system. Jb_ Thermodynamic evidence Using the constant temperature thermoelastic experiment (appendix 1) Weis-Fogh and Anderson ( 1970) observed a heat during elongation that was many times larger than the work of extension. This was viewed as a res u l t that was inconsistent with the random network model for e l a s t i n since, for an entropy based e l a s t i c i t y , the heat released during extension should be egual to the mechanical work done to elongate the sample (see appendix 1). It was also observed that t h i s e f f e c t 47 of 'excess heat' cculd be reduced by adding long chain hydrocarbon molecules l i k e alcohols. On the basis of these results they proposed a l i g u i d drop model for e l a s t i n structure, which consisted of corpuscular units having a hydrophobic core with the h y d r o p h i l l i c groups on the surface. Extension of t h i s network would force parts of the hydrophobic core to the surface and the r e t r a c t i v e force was hypothesized to a r i s e from t h i s i n t e r f a c i a l enercjy. e f f e c t . Further support for t h i s model was obtained from n.m.r. Data ( E l l i s and Packer 1976) and by Gosline §tj,al. (1975) who used flourescence probe analysis to confirm t h i s reversible exposure of hydrophobic groups, from the corpuscular units, to the interspersed h y d r o p h i l l i c solvent during extension. p. Oiled C o i l Model The oi l e d c o i l model was proposed by Gray e t ^ a l ^ (1973), and i s based on the amino acid seguence data accumulated by these investigators for porcine tropoelastin (Foster et..al.. 1973, Sandberg et^al... 1971, 1 972) . They delineated their data into two catgories: (a) residues involved in the cross l i n k region and (b) residues involved in the extensible regions. To the f i r s t category were assigned the alanine and lysine r i c h areas, with the glycine, proline and valine residues being assigned to the f l e x i b l e regions. It i s thought that the alanine residues form alpha-helices which alig n the l y s i n e residues into a position that favours t h e i r cross l i n k i n g into desmosine and isodesmosine residues. 48 The o v e r a l l model envisaged a system having h e l i c a l cross l i n k regions with the extensible regions forming a broad o i l e d - c o i l . I t was further proposed that the glycine residues would occupy the exterior of these o i l e d - c o i l s i n a solvent exposed position, with the proline, valine, and other hydrophobic residues buried inside the c o i l s away from the solvent. As i s obvious, t h i s type of model i s conceptually si m i l a r to the l i g u i d drop model of Partridge (1967). The only difference l i e s i n the proposal for the shape of the hydrophobic c l u s t e r s . The model of Partridge seems to presume these clusters to be globules, whereas the o i l e d - c o i l proposes them to be f i b r i l l a r . Both of them would behave i d e n t i c a l l y , in the thermodynamic sense, and the calorimetric data obtained by Weis-Fogh and Anderson (1970) was also cited as evidence for the o i l e d - c o i l model. Ii F i b r i l l a r Models The tone of the f i b r i l l a r models for e l a s t i n was set by the intense a c t i v i t y in the collagen f i e l d that was prevelant at the time when investigators started to study e l a s t i n structure. These workers approached i t from a point of view which c l a s s i f i e d i t as being part of the collagen family, but with a very low melting temperature (Astbury 1940). It was taken to an extreme in a publication by Samachandran and Santhanam (1957) who proposed a collagen-like t r i p l e h e l i x for e l a s t i n . This idea of wanting to f i t e l a s t i n into a s t r u c t u r a l hierarchy, as borrowed from the collagen group, i s s t i l l 49 prevelant today and forms the major impetus for the increased i n t e r e s t i n the f i b r i l l a r models. (a) Electron microscope evidence The f i b r i l l a r appearance of e l a s t i n i n the electron microscope was f i r s t noted by Gross (1949) who stated that the e l a s t i n f i b r e was a composite of a twisted rope structures of 80A° diameter, which were cemented together via an amorphous matrix. Other studies also reported such f i b r i l l a r components in the e l a s t i n f i b r e (Lansing et^al^. 1952, Ehodin and Dalhamn 1955). A flood of papers on e l a s t i n ultrastructure have taken up this theme of a f i b r i l l a r model for e l a s t i n with some success. These investigators have u t i l i z e d drastic technigues such as sonication and heavy metal staining to v i s u a l i z e ordered arrays of f i b r i l s , 30A° i n diameter (Gotte et.al..1965, S e r a f i n i - F r a c a s s i n i and Tristam 1966). Recent publications of electron microscope studies have also confirmed the existence of sub-structure in the e l a s t i n f i b r e (Quintarelli e t ^ a l . 1973, Gotte e t . a l . 1974, S e r a f i n i - F r a c a s s i n i e t . a l . 1976,1978). The l a s t authors have also reported a r e f l e c t i o n of 4 0 - 5 0 A O p e r i o d i c i t y in the X-ray d i f f r a c t i o n patterns of ligament e l a s t i n that had been stretched by 60%.. This along with a recent study (Pasquali-Eonchetti e t ^ a l ^ 1979), u t i l i z i n g freeze fracture and etching technigues, which show the presence of an extension related alignment of the s t r u c t u r a l components, present strong evidence for the 5 0 f i b r i l l a r model. In general, the electron microscope evidence seems to support a f i b r i l l a r model which when taken to an extreme, can be vi s u a l i z e d as a twisted rope array of two 15A° filaments that, as a unit, are visualized i n the elctron microscope as 30A° f i b r i l s running along the long axis of the e l a s t i n f i b r e (Gotte ______ 1976). There i s also some evidence that these 30A° f i b r i l s are clustered together into a higher order of sub-fibre, 150 to 20Cnm in diameter, as observed i n . the scanning electron microscope (Hart ______ 1978). J__ Nuclear magnetic resonance ________ I t has been pointed out in seme recent publications that the primary seguence cf tropoelastin, which i s thought to be the precursor of the fibrous protein, contains recurring sequences of certain amino acids (Sandberg _t_al.,1971, 1972). Based on these primary sequence data Urry and h i s co-wprkers have synthesized a number of repeat peptides corresponding to the seguences i n tropoelastin, and have attempted to decipher the conformation of the e l a s t i n protein by using these synthetic peptides as models. Their e f f o r t s have concentrated on three such model peptides: 1. A tetrapeptide (Val-Pro-Gly-Gly) n 2. A pentapeptide (Val-Pro-Gly-Val-Gly) n 3. A hexapeptide (Ala-Pro-Gly-Val-Gly-Val) n For the sake of sim p l i c i t y a short discussion of the tetrapeptide studies follows. 5 1 £ia^re_. 3̂ 2.2 B e t a - t u r n s . (A) The t e t r a p e p t i d e ( v a l - p r c - g l y - g l y ) s h o w i n g t h e r e s u l t s o f nmr s t u d i e s : (•) s o l v e n t s h i e l d e d m o i e t i e s , (o) s o l v e n t e x p o s e d m o i e t i e s (from U r r y and Long 1976a) . (B) B e t a - t u r n p r o p o s a l based on t h e s e r e s u l t s . Dashed l i n e i n d i c a t e s t h e n y d r o g e n bond (from U r r y and Long 1977b). 5 2 - Figure. 3.2. 53 Figure 3.2 (a S b), shows the tetrapeptide and the summary of the proton and carbon nuclear magnetic resonance data obtained from solvent t i t r a t i o n technigues (DMSO- Triflouroethanol), which involves the monitoring of the resonances as a function of solvent composition (Urry and Long 1976a). I t was found that some moities showed a marked solvent dependence of their chemical s h i f t s , while ethers were r e l a t i v e l y uneffected. These uneffected residues were proposed to pa r t i c i p a t e in hydrogen bonds. In the case of the tetrapeptide, proton resonace data showed the GlyU NH to be a solvent shielded moiety which was involved i n a hydrogen bond with the carbonyl oxygen of the Val1 residue. In comparison to t h i s , the Gly3 and the Val1 NH were found to be solvent exposed, and were not thought to partic i p a t e i n hydrogen bonds. Carbon resonance data also supported these r e s u l t s , showing that the Val1 CO was i n fac t solvent shielded with the Gly4 CO being solvent exposed (Urry and Long 1976a) . .  On the basis of these findings they have proposed a Beta-turn stucture (figure 3.2b) for thi s tetrapeptide (Urry and Long 1977b). Studies of the high polymers of t h i s tetrapeptide repeat indicated that, above 50oc, i n addition to the Gly4 NH, the Gly8, 12, and 16 NH*s also behaved in a solvent shielded manner. .Furthermore, the Gly4 CO behaved as though i t too was involved in a hydrcgen bond. To account for these additional findings a Beta-spiral structure (figure 3.3) was proposed to exist above 50oc (Urry ej^al... 197 7a) . . 5 4 Farther support for these Beta-turn structures was obtained through conformational energy calculations (Khaled et.al_,1976) and nuclear overhauser enhancement studies, which showed that the i r r a d i a t i o n of the £ro2 CH protons resulted i n an enhancement of the Val1 (CH ) proton resonances. This indicated the close association between the side chains of these two residues after ring closure (formation of a Beta- turn) (Urry et. a l . 1977a) . Similar studies cn the poly-pentapeptide (Urry ______ 1976b) and the poly-hexapeptide (Urry e t . a l . 1974, 1978a,b) indicated that these polymers were also involved in the Beta- turn as a preferred conformation. A number of additional hydrogen bonds are present in the hexapeptide which make i t a more stable (rigid) structure than the ether two repeats (Urry 1978a) . Assimilating t h i s nuclear magnetic resonance data with the electron microscope evidence, Urry has proceded to define a h i e r a r c h i a l model for e l a s t i n which i s depicted i n fig u r e 3.1. This structure consists of dynamic Beta-spirals in the extensible regions with the r i g i d hexapeptide Beta-spirals occuring in the cross-link regions, f a c i l i t a t i n g the condensation of the lysine residues into the (iso) desmosines. _. Discussion Ja_ _________ models Under t h i s c l a s i f i c a t i o n I have grouped the liquid-drop 55 model (Partridge 1967, Weis-Fogh and Anderson 1970) and the o i l e d - c o i l model (Gray e t . a l . 1973) since both of these propose d i s t i n c t regions of hydrophobic clus t e r s surrounded by solvent. The major support for these two-phase models i s based on the thermodynamic evidence of Weis-Fogh and Anderson (1970) and i t i s cn t h i s very evidence that i t has been refuted. In a communication by Grut and McCrum (1S74) i t was pointed out that the data obtained by Weis-Fogh and Anderson was predictable from the kin e t i c theory of of rubber e l a s t i c i t y (which supports a random conformation). They argued that the excess heat observed during elongation was a resu l t of the adsorption of water onto the non-polar groups and that the energy term represented the heat of d i l u t i o n from t h i s process. This also explained the decrease i n the energy component with the addition of organic solvents to the diluent (which would have a lower heat of d i l u t i o n ) . . The point was elaborated further b y Dorrington ______ (1975) who arived at the same conclusions. This absorption of the water by the e l a s t i n network during extension (Hoeve and Flory 1974) could also explain the re s u l t s of the flcurescence probe analysis (Gcsline e t . a l . 1975) which could, in retrospect, be predicted by the k i n e t i c theory relationships (Mark 1976). A two-phase model would also be inconsistent with the glass t r a n s i t i o n temperature studies of Kakivaya and Hoeve (1 975) . These authors explained that i f this glass t r a n s i t i o n occured inside these •hydrophobic globules', then i t would be 56 hard to account for the dependence of the t r a n s i t i o n temperature on the amount of water that was present outside these globules.. I t has also been pointed out i n recent studies that the temperature-swelling behaviour of e l a s t i n i s consistent with the Flory-Behner model for network swelling which, again, i s based on an amorphous, single phase network for the polymer (elastin) i n guestion (Gosline 1977, 1978) . Carbon nuclear magnetic resonance studies (Torchia and Piez 1973), have indicated a very rapid back-bone motion for the e l a s t i n protein.This i s also hard to reconcile with these two phase models for e l a s t i n struture, since the protein would be expected to have a reduced mobility inside such compact structures. IhL Evidence for secondary structures Investigators using d i f f e r e n t spectroscopic technigues have confirmed that approximately twenty per-cent of the residues in e l a s t i n are involved i n secondary structures (Torchia and Piez 1973, Lyerla and Torchia 1975, Starcher e t . a L . 1 973, Tamburro et.a. JU . 1977, Marami efj.aU . 1970) . . The controversy that i s present at the moment involves the location of these secondary structures: do they occur i n the c r o s s - l i n k regions of e l a s t i n or are they present i n the extensible parts of the polymer chains. Urry and his co-workers have argued i n numerous publications that these ordered regions are an i n t e g r a l part 5 7 of the e l a s t i n chain in the extensible regions, and as discussed previously, these investigators believe that most of the valine, proline, and glycine residues are involved i n Beta-turn structures. On the other hand, a recent study by Fleming ______ (1980), who also used nuclear magnetic resonance technigues, have shown that almost a l l of the valine residues in e l a s t i n are characterized by rapid movements. In judging between these two views one has to ask a number of questions: (a) what percentage of the amino acid residues occur as the repeat peptides, which form the basis of Urry's work? (b) can r e s u l t s from other methods be used tc d i f f e r e n t i a t e between he two p o s s i b i l i t i e s ? In answer to the f i r s t guestion, only about 20% of the residues occur as repeat peptides in the e l a s t i n primary sequence (Foster et . a l . 1973). Urry has argued that although t h i s i s i n fact a reasonable estimate, the structures proposed by him would occur as a major conformation assuming that the repeat peptides could t o l e r a t e substitutions and s t i l l r e t a i n t h e i r secondary structure. This assumption, however, i s not borne out experimentally since even a r e l a t i v e l y conservative substitution (val--pro) results in the disruption of the Beta- turns (Urry ______ 1977a). In reference tc the second question, i t i s possible to assimilate thermodynamic data with the nuclear magnetic resonance results tc obtain a clearer story. Kakivaya and Hoeve (1S75) using the technigue of d i f f e r e n t i a l scanning calorimetry, f a i l e d to observe any f i r s t order t r a n s i t i o n s . 58 arguing against the presence of Beta-spiral structures, which would be expected to give a sharp peak i n the region of the t r a n s i t i o n (from the s p i r a l s to the random c o i l as induced by high temperatures). Their results would tend to favour the view that e l a s t i n i s essentially an amorphous protein. It must also be kept in mind that most of the proposals for the Beta- structures are based on studies involving small peptide fragments, which may not be s a t i s f a c t o r y models for insoluble e l a s t i n . Jc]_ Hydrogen bonded structures It i s evident that the Beta-turn and the Beta-spiral are structures that depend on hydrogen bonding for t h e i r s t a b i l i t y , and as pointed out before, the thermodynamic data of Kakivaya and Hoeve (1975) are inconsistent with these interpretations of e l a s t i n structure. There are a number of p o s s i b i l i t i e s that could account for the f a i l u r e of these investigators to observe a f i r s t order t r a n s i t i o n : (a) the energy change associated with the t r a n s i t i o n i s very small or (b) these structures are extremely stable over the temperature range of the experiment (0O-200OC) . This again creates a dilema. I f the energy for the t r a n s i t i o n from the Beta-spiral structure to the random c e i l conformation i s very small, then there i s no reason to assume that the peptide-peptide hydrogen bonds (needed for Beta- turns) should be favoured over the peptide-solvent i n t e r a c t i o n s . The system i s then very close to being an 5 9 amorphous, single-phase network with rapid back-bone movements. This view i s also consistent with the Beta-spiral structures, i f they are present, being very dynamic ones. On the other hand, i f these structures are very stable, then protein-protein interactions can be considered to be favoured over protein-solvent interactions. That th i s could occur i s net surprising since they are known to be present i n other fibrous proteins such as collagen, s i l k , and keratin, which have hydrogen bended organizatons l i k e the alpha-helix and beta-sheet structures. This i s where the dilema arises. Any hydrogen bonded system that would so overly favour peptide-peptide interactions has to, as an ine v i t a b l e conseguence, exhibit r e l a t i v e l y s t i f f mechanical properties as do the above mentioned group of proteins. E l a s t i n also exhibits such properties, when i t i s dry. The dry state (or low hydration conditions) can be considered to be analogous to a system where, due to the lack of the p l a s t i c i s i n g water, the peptide-peptide interactions dominate to a point where the normally rubbery material behaves as a glass. The function cf water i n systems such as these i s to compete with the bonding i n t e r a c t i o n s , e s s e n t i a l l y •dissolving' the peptide back-bone and allowing i t to be a functional e l a s t i c tissue. In view of the high e x t e n s i b i l i t y and low modulus of e l a s t i n , i t must be concluded that the hydrogen bonded structures cannot be very stable ones, and i t seems reasonable to expect e l a s t i n to be an amorphous protein..The p o s s i b i l i t y 60 for the s t a b i l i z a t i o n of the Beta-type structures in diseased states i s a more plausible idea, which at the moment remains an open guestion. (d) F i b r i l l a r models As was pointed cut before, the f i b r i l l a r model as based on the electron microscope studies, i s thought to consist of 3 to 5 nm filaments organized along the long axis of the e l a s t i n f i b r e . . Since t h i s type of organization involves the presence of discrete regions cf protein and water, a l l the arguments presented against the o i l e d - c o i l model and the l i q u i d drop model also apply here. The only possible way to get around t h i s objection i s to propose that the filaments are themselves an i s o t r o p i c system L e ^ that the protein making up the filaments i s in a random conformation. This type of structure would be consistent with a l l the evidence pesented in support of the k i n e t i c theory explanations for e l a s t i n e l a s t i c i t y . . B u t i s t h i s a reasonable statement? At the moment there exists no evidence to support t h i s assumption. On an i n t u i t i v e basis, i t i s hard to vi s u a l i z e the 3 to 5nm f i b r i l s as being capable of accomodating random c o i l s of proteins, e s p e c i a l l y considering the size of i n d i v i d u a l amino acid residues. G. Conclusions Examination of the various s t r u c t u r a l models and the dif f e r e n t methods cf study seems to indicate that e l a s t i n structure can be assimilated most s a t i s f a c t o r i l y i n terms of a 6 1 conformation that i s very close to the k i n e t i c a l l y agitated, random network structure that i s demanded by the k i n e t i c theory of rubber e l a s t i c i t y . The following chapters try to further . delineate (confuse, disguise) the c o n f l i c t s presented here, and to test the v a l i d i t y of the random network as a viable description of e l a s t i n conformation. 62 Chapter^IV.. CO N FOB MAT ION OF ELASTINl COACEBV ATE STB UCTUB E^ A i IHt^oduction The analaysis of the conformational state of the e l a s t i n protein i s generally confined to the insoluble, fibrous form of t h i s protein,.Another approach i s to study the conformation of the soluble proteins, and to extrapolate from these to the f i n a l product. Although t h i s i s an i n d i r e c t approach to the problem i t could provide some interesting r e s u l t s . The Phenomenon of Coa eery ation An operational d e f i n i t i o n cf coacervation was given by Bungenberg de Jong (1949) as: "I f one starts from a s o l , that i s a solution of c o l l o i d in an appropriate solvent, then according to the nature cf the c o l l o i d , various changes (temperature, pH, addition of a substance) can bring about a reduction of the s o l u b i l i t y as a r e s u l t of which a larger part of the c o l l o i d seperates out i n a new phase. The o r i g i n a l one-phase system-the s o l - thus divides into two phases, one of which i s r i c h i n c o l l o i d , the ether poor............ Macroscopic or microscopic investigation allows one to distinguish between c r y s t a l l i z a t i o n when obviously c r y s t a l l i n e individuals are formed and coacervation, when amorphous l i g u i d drops are formed, which l a t e r coalesce more or less readily into one clear homogeneous c o l l o i d - r i c h l i g u i d layer, c a l l e d the coacervate layer". Soluble e l a s t i n s display this phenomenon of coacervation..Both tropoelastin and alpha-elastin are fre e l y soluble i n water at room temperature, but upon r a i s i n g the temperature to about 37oc, they both show coacervation properties..If l e f t standing for about 20-24 hours, t h i s coacervate becomes insoluble, 6 3 presumably due to the entanglement cf the protein chains (Wood 1958) . Electron microscope studies of the coacervates of soluble peptides and synthetic peptides of e l a s t i n , show a very f i b r i l l a r appearance when visualized with negative stains (Volpin ______ 1976, a, b, c ) . This raises another controversy, F i r s t , are these f i b r i l s a true representation of the coacervate organization, or, are they a result of procedural a r t i f a c t s ? Second, i f these f i b r i l s do e x i s t , would they be capable of accomodating random c o i l s of proteins within such a r e s t r i c t e d domain? In attempting tc answer these types of questions, i t seems that one should choose experimental methods that w i l l allow an examination of the sha_e and the ________ of the soluble e l a s t i n coacervates... In view of t h i s purpose, the present chapter deals with vi s c o s i t y and nuclear magnetic resonance studies which can, i n p r i n c i p l e , t e s t for shape and mobility, respectively. The discussion w i l l be limited to the 66,000 m.w. peptide fragment commonly referred to as alpha- e l a s t i n , but the arguments presented here are expected to apply to the precursor protein, tropoelastin, as well. C_ Preparation Of Alpha-Elastin Instead of using the usual method of producing soluble e l a s t i n s by oxalic acid digest (Partridge e t . a l . 1955), the alpha-elastin used in t h i s study was prepared by enzyme hydrolysis of insoluble e l a s t i n as outlined by H a l l (1976) and 64 Hall and Czerkawski (1961). The enzyme method of preparation i s advantageous since the product i s a monomeric peptide as compared to the pclydisperse mixture that i s otherwise attained through acid digests. Ja_ ______ hydrolysis Borate buffer: of ph8.5 and i o n i c strength of 0.384 was prepared by mixing 50ml of a mixture of 0.2M H3 B°3 a n <^ 0.2M KCl with 10ml of 0.2M NaOH, and d i l u t i n g to 250ml..The borate/chloride solution was prepared by dissolving 12.369g H-̂ BO- and 14.911 KCl in 1L of d i s t i l l e d water.. Preparation of e l a s t i n : Ligamentum ______ obtained from mature beef c a t t l e was stripped of free adhering tissue and p u r i f i e d by repeated autoclaving (Partridge _t_a1_ 1955). The resultant tissue was f i n e l y minced, washed with several l i t r e s of bo i l i n g d i s t i l l e d water and dried. About 5g of t h i s autoclaved tissue was suspended in 380ml of the borate buffer, 0.5,g of SDS was added and the mixture was s t i r r e d at 37°C for 1hr. It was then c h i l l e d to 4<>C and 20ml of borate buffer, which contained 5mg of elastase (Sigma chemical company), was added. The reaction vessel was put into a shaking, water-bath at 370C and the reaction was allowed to proceed u n t i l a l l the e l a s t i n had been dissolved (usually about 5hrs). At the end of t h i s period the solution was brought tc a b o i l to stop the reaction. The entire sample was freeze-dried and stored at - 70°c u n t i l further use. 6 5 Jb_ Removal of SDS The SDS was removed from the peptide by a modification of the ion-pair extraction method proposed by Henderson ______ (1979). About 100mg of the alpha-elastin powder was dialysed exhaustively against d i s t i l l e d water and freeze-dried.. This peptide was redissolved in a solution of triethylamine:acetic acid:water (5ml:5ml:5ml) and s t i r r e d at room temperature for 1hr. The mixture was then cooled to 4"C and 85ml of anhydrous acetone was gradually added to the solut i o n . This results i n the p r e c i p i t a t i o n of the protein while the SDS s a l t i s extracted i n the acetone. The prec i p i t a t e was redissolved in a •washing' solution, which consisted of 5ml of water, and 95ml of the acetone was added to precipitate cut the protein. This •washing' was repeated 4 to 5 times and the f i n a l product was blown dry in an oven to remove a l l traces of acetone. N.m.r. Spectroscopy showed t h i s preparation to be t o t a l l y free of any SDS. J_L Characterization of the alpha-elastin The resultant peptide was a monomeric preparation of 66,000 M.W., as shown by SDS-polyacrylimide gel electrophoresis (Weber and Osborn 1969, Chrambach and Robard 1971). It also displayed the property of coacervation as monitored by the absorbance of the peptide solution at 380nm as a function of temperature (figure 4.1). The amino acid composition was shown to be si m i l a r to insoluble e l a s t i n by comparing the n.m.r. spectrum of the alpha-elastin 6 6 hydrolysate with a spectrum of ligament e l a s t i n hydrolysate. The hydrclysates were prepared by acid hydrolysis in vacuo i n 6M HC1 at 1150C for 24hrs. D_i Viscosity and Shape i§l The relevant equation If one adds a number of s o l i d p a r t i c l e s , which are much larger than the solvent molecules, to a solvent of v i s c o s i t y , n, one observes an increase i n the macroscopic vi s c o s i t y of the solution due to the d i s t o r t i o n of flow patterns, which i s induced by the solute p a r t i c l e s . The relationship for t h i s e f f e c t , assuming that the p a r t i c l e s are far enough apart to prevent overlap of the distorted flow l i n e s , was given by Einstein i n 1906 as (Tanford 1961) : n'=n (1+v>0) 4.1 where n and n' are the v i s c o s i t i e s of the solvent and the solution respectively, with Vs and representing. the 'geometry' and volume f r a c t i o n of the solute. The e f f e c t of the solute on the viscosity of the solvent i s usually expressed as the s p e c i f i c v i s c o s i t y , n": n"= (n'-n) /n 4.2 The experimental manipulation for non-ideal molecules, to account for the concentration e f f e c t s , involves the empirical evaluation of the reduced v i s c o s i t y , n"/c, as a function of the concentration, c. The graphical plot cf n"/c versus c, as c-*0, gives a value termed the i n t r i n s i c v i s c o s i t y [n] : 6 7 ___________ Coaceryat ion p r o f i l e of alpha-elastin. Plot of normalized absorbance at 360nra versus temperature °C, for a solution of alpha-elastin at a concentration cf 6.8nag/ml, ph 7 . This sample was subsequently used for the nmr experiments.  69 [n]=n"/c, as c-»0 4.3 Substituting eguation 4.3 into eguation 4.1 gives: [n]=¥^/c 4.4 since the volume fr a c t i o n of the sclute can be represented by: jzS=Nv c/molecular weight of the solute 4.5 h where N i s Avogadro's number, c i s the concentration i n g/ml, and v i s the hydrodynamic volume, defined according to h Tanford as (196 1) : v, = (m. w./N) ( v+SvO) 4.6 n where v i s the p a r t i a l s p e c i f i c volume of the solute molecule, vO i s the p a r t i a l s p e c i f i c volume of the solvent molecule, and & i s the hydration expressed as g solvent/g solute. Combining eguations 4.6, 4.5, and 4.4, gives: [ n]=v(v+kvO) 4.7 In the case of water, v« i s egual tc unity, and eguation 4.7 reduces to: £n] = v(v+ S ) 4.8 IhL Evaluation of the shape parameter The symbol, v-, in eguation 4.7 represents the shape of the solute molecule and has been evaluated for e l l i p s o i d s of varying a x i a l r a t i o by Simha (1940). For the s p e c i f i c case of prolate e l l i p s o i d s of a x i a l r a t i o , J, i t takes the form (Tanford 1961): v=J2/15 (ln2J-3/2) 4.9 Since v> can be calculated i f the values of [ n ] , v, , and v° 70 are known. Ihis method can be used to empirically evaluate the geometry of the solute molecules. The dependence of \r, on the shape of the solute molecule, i s shown i n figure 4.2. Jc_ Application to soluble e l a s t i n s I f the process of coacervation does not result i n a change of shape, to a more anisotropic f i b r i l l a r form, then the value of v-, as given by the r a t i o (v+ ̂ >)/[n], should remain constant over the entire temperature range. The exact value of v- w i l l probably l i e i n the range of 12.18 for tropoelastin, as calculated from eguation 4.9 and the sedimentation data of Schein e t _ a l _ (1977). On the other hand, i f these peptides do i n fact form filamentous systems, then the value of v- should increase dramatically at the c r i t i c a l temperature at which the t r a n s i t i o n takes place. The absolute magnitude of T, a f t e r the t r a n s i t i o n , can be predicted for the tropoelastin molecule assuming that the f i b r i l l a r font i s made up of Beta-spiral structures as suggested by Urry and Long (1977b). According to Urry (1976a), the Beta-spirals are expected to have a diameter cf 15nm with a translation length of 0.44nm/6aa residues. In the case of tropoelastin which has approximately 850 residues (Sandberg 1976) , the expected dimensions are 62nm and 1.5nm for the length . and diameter respectively. Assuming a prolate e l l i p s o i d as being grossly representative of t h i s shape, the a x i a l r a t i o of 41.3 amounts to a Simha factor, Vs, of 39.3 (eguation 4.9). A sim i l a r 71 _iaJJ£__i i_2 i 3___jn^__ce of the Simha factor on a x i a l _a_i°*. Evaluation of the Simha fac t o r , v> as a functxon of a x i a l r a t i o f c r prolate (a) and ofclate (b) e l l i p s o i d s , according to equation 4 . 9 . Simha factor, \r Jvj - * N > U T o o - — j — i > X cr cn 73 p r e d i c t i o n f o r a l p h a - e l a s t i n cannot be made due to i t ' s branched nature, but i t i s a l s o expected t o show a d r a s t i c i n c r e a s e i n r i f i t too forms f i b r i l l a r s t r u c t u r e s as re p o r t e d i n the e l e c t r o n microscope s t u d i e s (Cox e t . a l . .1973). H i V i s c o s i t y S t u d i e s Of A l p h a - E i a s t i n _[a]_ V i s c o s i t y measurements A l l the v i s c o s i t y experiments described here were conducted with an Ostwald type c a p i l l a r y viscometer, with t r a n s i t times f o r water i n excess of 300sec. These long t r a n s i t times should o b v i a t e the need f o r kinematic c o r r e c t i o n s . In d e a l i n g with an Ostwald type viscometer the v i s c o s i t y of the s o l u t i o n , n', i s given by: n'=Bpt 4. 10 where B i s an apparatus constant, p i s the d e n s i t y and t i s the t r a n s i t time i n seconds. The apparatus c o n s t a n t , B, was evalu a t e d at each temperature by c a l i b r a t i o n with d i s t i l l e d water, using d e n s i t y and v i s c o s i t y values from standard p h y s i c a l t a b l e s . . A standard s o l u t i o n of p r o t e i n was made up by d i s s o l v i n g about 260mg of p u r i f i e d a l p h a - e l a s t i n i n about 250ml of d i s t i l l e d water. T h i s s o l u t i o n was c e n t r i f u g e d at 12,000X f o r 15min., t o remove p a r t i c u l a t e matter and l a r g e aggregates, and the c l e a r supernatant was used f o r the subseguent v i s c o s i t y experiments. The exact c o n c e n t r a t i o n was measured by t a k i n g a 1ml a l i g u c t s of the p r o t e i n s o l u t i o n , d r y i n g them i n an oven, 74 ____________ __________ of e l a s t i n water content 211 temperature. Plot of r e l a t i v e volume (taken to be 1 at 30OC) versus temperature °C for ligament e l a s t i n (from Gosline 1978).  76 and measuring the residue weight. . 10ml of the alpha-elastin solution was pippetted into the visccmeter and the t r a n s i t times were measured with an e l e c t r o n i c stop-watch (±0.1 sec). The v i s c o s i t y measurements were repeated on s e r i a l l y d i luted solutions of alpha-elastin and the data at each temperature were plotted i n the form of n"/c versus c (see eguation 4. 3) . This allowed the evaluation of the i n t r i n s i c v i s c o s i t y , [ n ] , for alpha-elastin at each temperature. Jb_ Calculation of p a r t i a l s p e c i f i c volume and hydration The p a r t i a l s p e c i f i c volume of the alpha-elastin, which was calculated according to the method of Cohn and E d s a l l (1943) from the amino-acid composition for alpha-elastin given by Starcher et. a l . (1973), had a value of 0.739. The hydration of the alpha-elastin, at each temperature, was calculated from the temperature dependence of hydration for insoluble e l a s t i n given by Gosline (1978) (figure 4.3). This seems valid since C e c c o r u l l i ______ (1977) have shown that the two types of e l a s t i n exhibit similar temperature dependence f o r their water contents. Jc_ Besults and discussion The results of the viscosity studies on alpha-ealstin are summarized in table 4.1 and figure 4.4. I t i s evident that the i n t r i n s i c v i s c o s i t y , Xn3, of alpha-elastin remains r e l a t i v e l y unchanged with temperature (figure 4.4,a). I f anything, there i s a s l i g h t decrease in [n] with increasing temperature, which VISCOSITY OF ALPHA-ELASTIN. TUC n sp^ c v e r s u s c P l ° t s I n ] slope correlation coeffecient, r 12 0.67 0.99 3.7 22 1.30 0.84 4.1 36 1.20 0.97 3.1 41 1.40 0.84 3.6 78 F igurei4..4j. Visccsity of alpha-elastin^ (A) Plot of i n t r i n s i c v i s c o s i t y , £n], versus temperature °C. The clashed l i n e represents the expected relationship calculated from equation 4 . 9 , assuming no change of shape, and the hydration values from figure 4. 3. (B) Plot of the Simha factor, v, versus temperature oc calculated according to equation 4.8 using the needed values from table 4 .1 and figure 4.3. o O ro o u> o o tn o O O Simha factor, tn to <*> r axial r a t i o . o ' b rO o o o o o tn o (n] (cc/g) ro <*> *^ tn " i i i i i / /• / / / 9! 8 0 i s expected from the decrease in the water content of the peptide (figure 4.3) and eguation 4.8 (figure 4.4, a dashed l i n e ) . Evaluation of the Simha fact o r , v, according to eguation 4.8 indicates that the alpha-elastin molecule i s more or l e s s spherical, and that there i s no change with temperature (figure 4.4,b). Incorporating the values of the molecular weight, p a r t i a l s p e c i f i c volume, and hydration i t i s possible to calculate the dimensions of the alpha-elastin molecule, assuming a spherical shape, to be i n the order 20nm in diameter. Interestingly, however, a glance at table 4.1 shows that the slopes of the n"/c versus c plots tend to increase with increasing temperature. This can be interpreted i n the following way. I f the p a r t i c l e s in solution do not in t e r a c t with each other, then the slope of the n"/c versus c plots should be zero (Tanford 1961, Huggins 1942). The fact that the slopes for the alpha-elastin solutions are posit i v e supports the presence of aggregation processes (Tanford 196 1) which, i n general, seem to be favoured with increasing temperatures (table 4. 1) . F_ Nuclear Magnetic Resonance (a) Theory The possession of both spin and charge confers on a p a r t i c l e , such as a proton, a magnetic moment. Thus, a spinning proton can be regarded as a bar-magnet along the axis 8 1 _ i _ u r j _ _ _ _ _ _ Precession of a _roton in a magnetic f i e l d . A prcton of magnetic moment, u, i s placed i n an external magnetic f i e l d , H Q, causing i t to precess around the z axis at an an angle 0 . H, i s the orthogonal magnetic f i e l d that i s used to determine the frequency cf precession. 8 2 F i g u r e . U . 5 8 3 of spin, and the strength cf t h i s magnetic dipole i s expressed as the nuclear magnetic moment, u. I f placed in an external magnetic f i e l d , H0 , the in t e r a c t i o n of t h i s f i e l d with the magnetic moment of the proton w i l l influence i t to orientate i t s e l f i n the di r e c t i o n of the f i e l d , tut the eff e c t of the spin creates a torque which causes the i t to precess around the z axis at a frequency, v° , and angle 0 (Stothers 1973, figure 4.5). The anqular velocity of t h i s precession, coa , i s given by : = % H o 4. 11 where & i s the gyromagnetic r a t i o defined as: * =2ur/h 4.12 where u^ i s the projection of the vector u i n the direction of the f i e l d H Q, and h i s Planck's constant. The angle 0 i s determined by the spin number of the nucleus, I, which for protons has the value of 1/2. The angle of the precession for this spin number i s r e s t r i c t e d to two orientations, at 54.9° and 1 8 0 O - 5 4 . 9 O respectively (Metcalfe 1970). The poten t i a l energy, E, of the interaction of tihe magnetic moment u^ i s given by: E =— U^He.....o 4. 1c and the separation between the two energy levels f o r the proton i s : AE=2u^H0 ,.4.14 I f a small rotating f i e l d H( i s generated orthogonal to H 0 (figure 4.5), u would experience the combined effects of both H( and H c. The nucleus would alsorb energy from Hj i f the 84 angular frequency of H( i s egual to v°, changing 8. The el e c t r o m a g n e t i c r a d i a t i o n which w i l l e f f e c t such t r a n s i t i o n s i s given by: hv°=AE=2u_H_ 4. 15 Since only two values of 8 are p o s s i b l e the a b s o r p t i o n , or emmision, of energy guanta causes the nucleus to f l i p from one o r i e n t a t i o n to another when H, i s i n "resonance 1 with v° (Metcalfe 1970). N.m.r. Spectroscopy i s based on the d e t e c t i o n of the absorbed energy when the n u c l e a r s p i n s of the system come i n t o resonance (chemical s h i f t ) with H| . J i l l The chemical s h i f t I f a l l the protons were t o undergo magnetic resonance at i d e n t i c a l f r g u e n c i e s no c o n f o r m a t i o n a l i n f o r m a t i o n c o u l d be de r i v e d from t h i s technique.. F o r t u n a t e l y , however, the resonance frequency of each proton i s s e n s i t i v e to i t ' s e l e c t r i c a l and magnetic environment (Walton and B l a c k w e l l 1973). The n u c l e i can be s h i e l d e d from the a p p l i e d magnetic f i e l d by the e x t r a - n u c l e a r e l e c t r o n s , hence, the e f f e c t i v e magnetic f i e l d experienced by the nucleus, H e , i s not the same as the a p p l i e d f i e l d and can be expressed as (Metcalfe 1970) : H e =H 0(1-rj-) 4.16 where CT i s the s h i e l d i n g f a c t o r which i s a r e f l e c t i o n of the e l e c t r o n d i s t r i b u t i o n around the observed proton. I t i s t h e r e f o r e expected t h a t changes i n e l e c t r o n d i s t r i b u t i o n around the n u c l e i , i . e . co n f o r m a t i o n a l or 85 bonding changes, would effect the system because of shielding e f f e c t s , and one would expect to observe a seperate s i g n a l , with a c h a r a c t e r i s t i c chemical s h i f t for each group of equivalent protons i n the molecule. The chemical s h i f t s , S , are measured in a dimensionless number expressed as parts per m i l l i o n (ppm) , and i s defined as: £> = (HS-H^ /Ĥ , ) X 106. 4.17 where H5 and are the resonance f i e l d s for the sample and reference compound respectively. JcJL Belaxation processes As stated before, protons are re-orientated in an applied magnetic f i e l d by the absorption of electromagnetic radiat i o n of the resonance freguency from . The induced t r a n s i t i o n s , however, have an egual probability in either d i r e c t i o n so that i f there were egual populations cf nuclei i n the two energy l e v e l s , there would be no net absorbtion of energy. In f a c t , however, there i s a small excess of nuclei i n the lower energy state which i s determined by the energy difference between the two l e v e l s (equation 4.13) and the r e l a t i v e populations (n'/n) are given by: n'/n=exp (- A E/BT) 4.18 where n' and n are the number cf protons in the upper and lower l e v e l s respectively. The excess of protons i s only in the order of 7ppm in a f i e l d of 10kG (Metcalfe 1970), but t h i s i s s u ffecient to give a net absorption of energy since the number of upward t r a n s i t i o n s are s l i g h t l y greater than in the 86 other d i r e c t i o n . These t r a n s i t i o n s that determine the l i f e t i m e of the excited nuclei are termed relaxation processes. S p i n - l a t t i c e relaxation (longitudinal relaxation), T j , results i n the dissipation of the absorbed energy as thermal energy to the other nuclei and electrons i n the sample, c o l l e c t i v e l y referred to as the l a t t i c e . This process acts d i r e c t l y to maintain an excess of nuclei i n the lower energy state. T 2, the other process of relaxation, l i m i t s the l i f e t i m e of an excited nucleus by a mutual exchange of orientation with a neighbouring nucleus of the same kind. This i s termed spin- spin relaxation (transverse r e l a x a t i o n ) . This process results in the r e - d i s t r i b u t i o n of absorbed energy and does not contribute to the maintainance of the excess nuclei in the lower state. Both of these relaxation processes determine the spectral line_widths. Beth relaxation processes are influenced by molecular motion . For example, the rapid thermal motions i n l i q u i d s leads to large s p i n - l a t t i c e relaxation times (Metcalfe 1970). The e f f e c t i v e f i e l d experienced by the nuclei i s also averaged out by the rapid i s o t r o p i c movements, which r e s u l t s i n narrow line-widths and large values for T_. The reason for t h i s i s that the a b i l i t y of two nuclei to exchange their spins depends d i r e c t l y on how long their motions remain i n phase with each other. This time during which r e l a t i v e orientation persists i s ca l l e d the cor r e l a t i o n time, > -nd i s the parameter that characterizes the molecular motions. Thus nuclei that exhibit 87 f a s t , i s o t r o p i c motions w i l l be characterized small co r r e l a t i o n times and narrow line-widths. The analysis of the n.m.r. spectra can therefore be used to deduce protein mobility. G_ ______ Studies Of Alpha,Elastin In comparison to the viscosity experiment whose main result i s an evaluation of a molecule's shape, a nuclear magnetic resonance experiment allows the evaluation of the molecules mobility (Dwek 1973)Furthermore, since any n.m.r. spectrum i s a ccmposite of the sum of i t ' s component chemical s h i f t s and line-widths, i t should be possible to predict n.m.r. spectra i f the composition and the mobility (conformation) of the constituent molecules i s known. Alternately, i f the ccmpcsition i s known one can assume a conformation, and predict a spectrum based on such an assumption, which i s then tested by obtaining experimental values. _a_ Materials and method The p u r i f i e d alpha-elastin was dissolved in D_0 at a concentration of approximately 6mg/ml. Proton nmr spectra were obtained cn a Bruker WD-400 FT-NME spectrometer at the University of B r i t i s h Columbia, Department of Chemistry with the kind co-operation of Dr. . A. G. .. Marshall and Dr. .P. D. . Bur ns. Buns were made at 10°C i n t e r v a l s over a temperature range of 15°C to 75°C, which spans the region of alpha-elastin 88 gigjjg! 5s.i*.i6l The L o r e n t z i a n l i n e shape.. The L o r e n t z i a n l i n e , as determined by equation 4.19, where 1/t i s the h a l f - w i d t h at h a l f - h e i g h t . w" i s the resonant p r e c e s s i o n frequency of the nucleus.. 89 F i g u r e . U . 6 : 1/t w. 90 T a b l e . . _ _ 2 : _ S p e c t r a l p a r a m e t e r s u s e d f o r r a n d o m - c o i l PARAMETERS FOR THE i _ _ _ B . ' g - » 8 ! f e B f t SPECTRA AT 400MH1. FROM OSS. CHEMICAL PPM SHIFT Hz LINE-HI DTH Hz NUMBER OF PROTONS a.a C l a t t l n j IN: Albumin^ ala CH3 1.34 534 25 3 783 138 arg «-CM2 1.75 700 35 2 10 46 J-CH2 1.67 667 32 2 10 46 3.11 1240 21 2 10 46 asn O-CHj 2.69 1074 28 2 " 20 asp a-CH 2 2.73 1092 28 2 9 76 gin 8-CH2 1.96 780 30 2 43 32 <-CH2 2.43 971 43 2 43 32 glu »-CH2 1.95 780 30 2 - 118 i -CH 2 2.35 942 35 2 118 gly*-CH 2 3.92 1569 18 2 610 30 his C2-H 8.54 3414 18 1 - 17 C4-H 7.09 2835 18 1 ** 17 H-CH2 3.05 1222 23 2 " 34 ile a-CH2 1.59 634 39 2 34 28 l-CH 2 1.59 634 39 2 34 28 (CH3)2 0.81 322 30 6 103 84 leu fl-CH2 1.59 634 39 2 107 122 l-CH 1.59 634 39 1 54 61 (CH3)2 0.84 322 30 6 322 366 lys 3-CH2 1.70 680 40 2 11 118 ir-CH2 1. 39 554 30 2 11 118 S-CH2 1.62 647 30 2 11 118 S-CH2 2.92 1167 25 2 11 118 net «-CH2 2.17 870 24 2 - 8 <-CH2 2.60 1040 31 2 -- 8 S-CHj 2.02 807 36 3 -- 12 pre t(C«2)2 7.20 2880 19 5 147 130 o-CM2 2.93 1172 10 2 59 52 pro NCH2 3.56 1423 59 2 234 56 o-CH2 1.92 769 36 2 234 56 l-CH 2 2.00 796 36 2 234 56 ser 3-CH2 3.81 1520 36 2 22 56 tf.r CH3 1.14 454 21 3 46 102 P-CH 4.13 1652 10 1 15 34 tyr i»(CH2)2 7.03 2811 14 .5 10 10 7.00 2798 15 1.5 30 29 6.70 2678 15 1.5 30 29 6.66 2654 14 .5 10 10 0-CH2 2.90 1160 17 2 41 38 val Q-CH2 2.20 878 40 2 232 72 (CM3)2 0.87 347 32 6 696 216 âmlno add composition from Starcher et.al. âmino acid composition from Dayhoff 1976. 1973. 91 coacervation (see figure 4.1).Typical spectral parameters were: 16k f . i . d data set, 1.4 sec. acg u i s i t i o n time with a 4.6 sec. delay between successive acquisitions to avoid resonance saturation. A spectral width of 6000 Hz was used with quadrature detection and phase a l t e r a t i o n sequence and exponential apcdizaticn equivalent to 2 Hz l i n e broadening, to enhance the signal to noise r a t i o . A l l assignments were made from the residual H2 0 peak taken to be at 4.68ppm from DSS at 250C. Jb_ Prediction of randc m-coil spectra In approaching t h i s problem I have opted for an empirical route which u t i l i z e s various parameters that have been obtained f c r proteins which are known to be i n the random c o i l conformation. The shape of the curves was f i t t e d to the eguation for a Lorentzian l i n e , which takes the form (Marshall 1979, and figure 4.6): A (w) =t/ (1* (w°-w) z _ 2 4.19 Where A(w) represents the absorption at the freguency w, w° i s the resonance freguency (i.e. the chemical s h i f t of a residue) and t describes the line-width such that: 1/t=1/2 (width at half-height) 4.20 The values used for the predictions involve the amino acid composition, the chemical s h i f t data, and line-width parameters frcm Bradbury and Rattle (1972) (see table 4.2). The predicted spectra were calculated according to eguation 4.19 on a D i g i t a l Equipment pdp11 computer, using a Fortran 92 program. J S l Jesuits and discussion Figure 4.7 shews the 400HHZ proton spectra for alpha- e l a s t i n in Dz0 at 25°C. The assignments (see legend table 4.3) were made on the basis of the predicted spectra (figure 4.8a) and other published spectra (Egmond e t . a l . 1979, Cozzone et. a l . 1 980) . Examination of figures 4.7 and 4.8 shows that the main absorption peaks i n the n.m.r. spectrum correspond to the valine, alanine, and glycine residues..This aspect of the r e s u l t i s valuable because the d i s t r i b u t i o n of these amino acids i s very d i s t i n c t , i n that the alanine residues occur almost exclusively i n the cross-link regions, whereas the glycine and valines are r e s t r i c t e d to the extensible parts of the e l a s t i n chains (Gray et_^aJU_. 1973, Anwar 1977). These residues can therefore serve as probes for the conformation i n the d i f f e r e n t regions of the e l a s t i n network. The general s i m i l a r i t y between the predicted spectra (assuming a random- c o i l conformation) and the experimentally observed r e s u l t s supports the presence of a random cenformation for alpha- e l a s t i n . However, there are also some very d i s t i n c t differences i n the two spectra which must be explained s a t i s f a c t o r i l y before the above statement can be accepted with confidence. The differences in the two spectra seem to be r e s t r i c t e d to three types of residues: the alanine residues (1.3ppm), the glycine residues (3.9ppm) and the aromatics (broad envelope at 3ppm and the peaks between 6.7 and 7.2ppm). 93 Figure.4.7 : 400 MHz nmr spectrum of alpha-elastin. Pmr spectrum of alpha-elstin i n D20 at a concentration cf 6.8 mg/ml, ph 6.8, at 25°C (see table 4.3 for peak assignments) .. The large peak at 4.68ppm i s from the residual H,0.  >3 laJUj§.i J*.*i;i P^i.k assignments f o r a l p h ^ - g i a s t i n a t 4 0 0 MHz^ 1. lie. , leu, val : ( C H 3 ) 2 2 . thr : C H 3 l > s : V - C H 2 3 . ala : C H 3 4., ile , leu : <3-CH2, tf-CHj 5. s r n lys : S - C H _ , Jf -CH_ : ? - C H 2 , g -CH^ 6 . pro : P - C H 2 , * - C H 2 glu : P - C H 2 7. val : P - C H 2 8 . glu : tf-f.H2 9 . asp : P - C H 2 1 0 . phe : (i - C H 2 lys : « - C H 2 tyr : |3 - C H 2 11 arg : S - C H 2 1 2 . pro : N - C H 2 1 3 . ser : P " C H 2 I** • gl y : - < - C H 2 1 5 . envelope of alpha-carbon 1 6 . tyr ring : H 3 , 5 1 7 . tyr ring : H 2 , 6 l f i . phe : c ? < C H 2 ) 2 1 9 . i 30-desmosine PEAK ASSIGNMENTS FOR ALPHA-ELASTIN. 20. iso-desmosine a n d d e s m o s i n e 96 Interestingly, a l l the deviations seem to be of one type, i . e . the observed line-widths are broader than the predicted ones. As mentioned before, the alanine residues i n the e l a s t i n network are l o c a l i z e d in the cross-link regions. The r e s u l t s of seguence studies indicate that these alanines are grouped in seguential runs of 4-5 residues, and there i s some evidence that these alanines form a <x-helix (Foster et. a l . 1976). The presence of h e l i c a l structures would be expected to r e s u l t i n r e s t r i c t i o n of the molecular motions i n the cross-link regions, as r e f l e c t e d i n the broadening of the spectral l i n e s . Since the the aromatics are also concentrated around the c r o s s - l i n k regions a similar explanation, as the one forwarded for the alanine residues, can be invoked for the l i n e broadening of these residues as well. Eut what about the glycines? There are two alternatives that can explain the broadening of t h i s peak. It i s possible that the glycines are invoved in secondary structures which could re s u l t in l i n e broadening due to r e s t r i c t e d motions. This i s probably not the case since the glycine residues occur with the valines i n the e l a s t i n seguence (Sandberg e t . a l . 1977) and t h i s l a t e r group does not shewn any presence of secondary structures, as visualized in the narrow line-widths (figure 4.7). Alternately, the observed glycine peak could be a composite of a broad d i s t r i b u t i o n of narrow resonances a r i s i n g from a number of d i f f e r e n t glycine populations. This i s a reasonable statement since the glycine protons are d i r e c t l y bonded to the alpha-carbon of the residue and would therefore be markedly 97 Figure.4.8: Predicted nmr spectra for alpha-elastin. (A) Predicted randcm-coil spectra for alpha-elastin using the spectral parameters from table 4.2, and the amino acid composition from Starcher e t . a l . 1 973. (B) The same spectrum with the phe, t y r , and ala residues broadened to 50 Hz to simulate their p a r t i c i p a t i o n i n secondary structures. The gly peak has been broadened to 30 Hz. 9 8 99 affected by the type of residue that occurred arcund i t i n the amino acid sequence. Hence di f f e r e n t primary sequences, with regard to the glycine residue, would be expected to re s u l t i n dif f e r e n t chemical s h i f t s for the glycine protons, and,since there i s no reason to believe that any cne type of amino acid always occurs with a glycine i n the primary seguence, t h i s explanation for the li n e broadening of the glycine peak seems acceptable. When the explanations for the differences between the randcm-coil spectrum (figure 4.8a) and the observed spectra (figure 4.7) are incorporated into the predictive process, one observes a closer agreement betweeen theory and experiment (figure 4.8b and legend). Figure 4.9 shows the spectrum obtained for a solution of hydrolyzed alpha-ealstin where the peaks have been broadened to 30hz. As expected the alpha-protons (3 to 4.5ppm) are affected the most by the hydrolysis of the peptide bonds, The upfield region of t h i s spectrum, f c r the free amino acids, i s seen to be i d e n t i c a l with the spectra obtained for in t a c t alpha-elastin (figure 4. 7)., This again supports the presence of a randcm-coil conformation for this e l a s t i n peptide. The p o s s i b i l i t y exists, however, that the f a s t motions of the protons deduced from the narrow line-widths, i s a r e s u l t of a rapid tumbling of the whole alpha-elastin molecule i n solution rather than being a r e f l e c t i o n of a k i n e t i c a l l y free protein chains. To ensure against t h i s a r t i f a c t a proton spectra was obtained for bovine serum albumin (Sigma Chemical Company). Albumin has a simi l a r molecular weight and i n t r i n s i c 100 Figure. 4.9:. Nmr spectrum of alpha-glastin i i l _ _ c 400 MHz spectrum of alpha-elastin hydrolyzed in vacuo i n 6M HCl at 115<>C for 24hrs. The spectrum has been broadened to 30 Hz. 101 Figure. U 102 v i s c o s i t y (Tanford 1961) as alpha-elastin (see v i s c o s i t y section of t h i s chapter) and, hence, should display s i m i l a r f r i c t i o n a l properties and tumbling properties i n solution. In contrast to e l a s t i n , however, albumin has been shown to contain stable secondary structures which are mostly alpha- h e l i c a l in nature (Timasheff e t . a l . . 1967) and t h i s should be r e f l e c t e d in the n.m.r. spectrum. Figure 4.10,a, shows the 400MHz spectrum obtained for albumin i n D̂ O at 25°C. The mass of broad resonance envelopes, as compared to the predicted spectrum for the random-coil conformation (figure 4.10,b), i s consistent with the results of CD. studies of t h i s protein (Timasheff e t ^ a l ^ 1967), more importantly, t h i s demonstrates that the tumbling motion of t h i s molecule does not mask the presence of secondary structures. Broadening a l l the resonances used i n the prediction of the spectra (amino acid composition from Dayhoff 1976) to 100hz r e s u l t s i n a better approximation of the observed r e s u l t s (figure 4.10,c). On the basis of the r e s u l t s presented here i t seems reasonable to state that the conclusicn of a random conformation for the alpha-elastin molecule based on the n.m.r. analysis i s not a r e s u l t of experimental a r t i f a c t but, rather, i t i s a true r e f l e c t i o n of t h i s proteins conformational state. Furthermore, since t h e i r was no observable temperature dependence of the spectra, i t seems that the coacervate of alpha-elastin i s also characterized by a random conformation. 103 Figure. 4.10: Nmr spectra of albumin.. (A) 400 MHz spectrum of bovine serum albumin at 250C. . (B) Predicted random-coil spectra for albumin using spectral parameters from table 4.2 and amino acid composition from Dayhoff 1976. . (C) Same as spectrum B, but with a l l the resonances broadened to 100 Hz to simulate the presence of stable secondary strucutres. 104 F i g u r e . 4 . 1 0 _ A n 105 !!•. Conclusions Because of the d i f f i c u l t i e s involved i n working with insoluble materials such as fibrous e l a s t i n , a number of researchers have chosen to look at the s t r u c t u r a l properties of the soluble e l a s t i n s : alpha-elastin (Partridge e t . a l . 1955), and the precursor protein, tropoelastin (Partridge and Whiting 1 979) . Since e l a s t i n has a very high hydrophobic amino acid content, and since there i s an increase i n hydrophobic interactions with temperature (Tanford 1973), the soluble e l a s t i n s exhibit the phenomenon of coacervation at high temperatures ( 3 7 0 C ) . This coacervation, which i s characterized by a phase separation of the solution into a protein dense phase and an eguilibrium solution, i s thought to be a c r u c i a l step for concentrating and cro s s - l i n k i n g the precursor molecules into the fibrous tissue (urry 1978c). Several workers have shown that both tropoelastin and alpha-elastin exhibit a f i b r i l l a r structure i n the coacervate state. (Cox et^al^.1973, 1974, Cleary and C l i f f 1978), with s i m i l a r p e r i o d i c i t i e s as the ones observed for insoluble e l a s t i n preparations (Gotte e t . a l . . 1976). Urry and his co- workers have also supported t h i s view by demonstrating the presence of these f i b r i l s in the ccacervates of the synthetic repeat peptides (Rapaka and Urry 1978, Volpin e t . a l . 1976a, b, c) . Although the concentrating e f f e c t , of coacervation, i s very obvious, the 'aligning' aspect implies an ordered 106 ( f i b r i l l a r ) structure for these soluble e l a s t i n s i n the coacervate states..Taken a step further, the presence of the filaments argues against an entropic e l a s t i c i t y for the e l a s t i n protein, and this forms the basis of the controversy currently surrounding the structure of e l a s t i n coacervates. This controversy i s further fueled by the c o n f l i c t i n g evidence obtained by the various workers who are addressing t h i s question. The n.m.r. data i s contradictory i n that studies reported by Urry and long (1977b), for the synthetic peptides, indicate an increase i n order with temperature, whereas, Lyerla ______ (1975) report the converse for fibrous e l a s t i n . The analysis of the viscosity studies presented i n t h i s chapter indicates that the process of alpha-elastin coacervation i s dominated by aggregation processes with nc evidence for the formation of p a r a l l e l i s o t r o p i c filaments which are seen i n the transmission electron microscope. This in t e r p r e t a t i o n of the results i s i n excellent agreement with the the 1 3C-n.rn.r results of Lyerla e t . a l . . (1 975), quasi- e l a s t i c l i g h t scatterring experiments (Jamieson e t . a l . 1972, 1976) and sedimentation data (Schein ______ 1977) , which also support a random conformation for the soluble e l a s t i n coacervates. That there are some secondary structures i n the e l a s t i n i s c l e a r l y demonstrated by a number of studies (Starcher ______ 19 73, Tamburro ______ 1977, Foster e t . a l . 1976). The unresolved guestion deals with whether these areas of 107 secondary structures are l o c a l i z e d in the cross-link area or whether they are c h a r a c t e r i s t i c of the extensible regions. Since the analysis of the e l a s t i n as a kinetic rubber assumes that there are no stable secondary structures, i t i s important that t h i s c o n f l i c t be resolved. The results of the proton n.m.r. r e s u l t s presented i n th i s chapter suggest that the secondary structures i n the e l a s t i n network are r e s t r i c t e d to the c r o s s - l i n k regions, and that the majority of the residues are characterized by random, i s o t r o p i c motions. 108 Chapter^Vo. C 0 NFO EM ATICN OF EL ASTIN ± BIBEFBINGENCE PEOPEETIES^ Introduction As pointed out e a r l i e r , several recent electron microscope studies of negatively stained fibrous e l a s t i n and coacervates of soluble e l a s t i n , reveal a highly ordered, anisotropic structure consisting of 3 tc 5nm filaments that are presumed to run p a r a l l e l to the long axis of the e l a s t i n f i b r e ( Q u i n t a r e l l i e t . a l . 1973, S e r a f i n i - F r a c a s s i n i e t . a l . 1976 and 1 978, Gotte ejUal.. 1974, Cox et.a lg_. 1 974, Cleary and C l i f f 1978). The mechanical properties of e l a s t i n , however, can be accurately explained by the Kinetic Theory of Bubber E l a s t i c i t y (Hoeve and Flory 1958, Dorrington e t , a l . . 1975 and 1977, Gosline 1978, Volpin and C i f f e r r i 1970, Aaron and Gosline 1980), and t h i s theory i s based on an i s o t r o p i c , random network structure that i s very d i f f e r e n t from the anisotropic, filamentous systems seen in the electron microscope. Given these two possible types of structures for e l a s t i n , one should be able to distinguish between them by observing the birefringence of single e l a s t i n f i b r e s i n the p o l a r i z i n g microscope. This approach was chosen because the technique does not require any physical disturbance of the protein, thus reducing the chance of any procedural a r t i f a c t s . . F u r t h e r , the technique i s very v e r s a t i l e , allowing the evaluation of structure at two l e v e l s of organization: (a) at the molecular l e v e l as r e f l e c t e d by the i n t r i n s i c birefringence, and (b) at 109 the sub-microscopic l e v e l ( i _ e _ , at the l e v e l of the 3 to 5nm filaments) as indicated by the form birefringence. If e l a s t i n i s i s o t r o p i c i n i t s organization, then t h i s should be reflected as a lack cf birefringence when observed between crcsed polarizers. On the other hand, i f e l a s t i n i s i n fact filamentous, as implied by the elctron microscope evidence, then t h i s should be manifested as an observable form birefringence. Similar studies on collagen ( P f e i f f e r 1943) and Chitin (Diehl and Iterson 1935) attest to the r e l i a b i l i t y of t h i s type of analysis. This chapter deals with the birefringence properties of single e l a s t i n f i b r e s and i t ' s analysis in terms of the implications for e l a s t i n conformation. M i Phengmengloqical e_p_.anat.ion of double __________ The basis for the phenomenon cf birefringence, and i t ' s use as an indicator of molecular ccnformation, eventually rests on the interaction between the e l e c t r i c properties of p a r t i c l e s and the wave nature of l i g h t . Hence i f one can explain and account,for the observed interaction between the two mediums, then, by comparing the nature of the incident l i g h t to the r e s u l t i n g radiation, one can make some deductions as to the s p a t i a l arrangement of the interacting p a r t i c l e s . It seems f i t t i n g then, that something should be said about the processes of molecular interactions that give rise to the phenomenon of double r e f r a c t i o n . 110 (a) Retardation of polarized l i g h t The retardation of polarized l i g h t i s based on the concept that chemical bonds w i l l interact with that component of l i n e a r l y polarized l i g h t whose e l e c t r i c vector i s p a r a l l e l to the bond d i r e c t i o n , 'retarding' i t ' s transmission v e l o c i t y . This axis i s referred to as the slow axis cf transmission. Upon emerging frcm the birefringent object the retarded vector w i l l add up with the ncn-retarded vector (which propagated along the f a s t axis of transmission) to give e l l i p t i c a l l y polarized l i g h t , a component of which i s passed by the analyzer (Wilkes 1971). Consider a beam of l i n e a r l y polarized l i g h t with i t ' s e l e c t r i c vector V as shown in figure 5.1&, and i t ' s d i r e c t i o n of propagation along the y axis ( i _ e _ out of the plane of the paper). This e l e c t r i c vector can be represented as a resultant of the vector l y i n g in the xy plane (figure 5.IB), and the vector lying normal to i t in the zy plane (figure 5.IC). I f t h i s plane polarized l i g h t was to pass through a non-birefringent object, the r e s u l t i n g l i g h t would be i d e n t i c a l to that depicted in figure 5. 1A. Since there i s no component p a r a l l e l to the analyzer axis (which i s at 90° r e l a t i v e to the p o l a r i z e r ) , none of i t w i l l be transmitted by the analyzer. Hence the specimen w i l l appear dark when viewed through the analyzer. However, for a p o s i t i v e l y birefringent, c y l i n d r i c a l object, orientated along the z axis, the plane polarized l i g h t in the zy plane w i l l be p a r a l l e l to the slow axis of 111 ____________ Propagation of _________ l i g h t through i_:otro^ic material^ (A) Looking down the axis of propagation, showing the r e l a t i v e positions of the polarizer (pol and the analyzer (an) . (B) The e l e c t r i c vector i n the xy plane. (C) The e l e c t r i c vector i n the zy plane. . 112 Figure. 5,J. 113 Figure.5.2: Propagation of polarized l i g h t through anisotropic material. (A) The e l e c t r i c vector in the xy plane, normal to the slow axis. (B) The e l c t r i c vector i n the zy plane i s p a r a l l e l to the slow axis and i s retarded by an amount 0 r e l a t i v e to the vector i n the xy plane. (C) The resultant l i g h t i s e l l i p t i c a l l y polarized with a ccmponent (V) that i s transmitted by the analyzer (an) . 1 1 " 115 transmission (figure 5.2B), re s u l t i n g i n the retardation of i t ' s transmission velocity. On the other hand, the vector in the xy plane w i l l be normal to t h i s slow axis of transmission and w i l l therefore, remain unaltered (figure 5.2A). Upon emerging frcm the birefringent object the retarded vector, which propagated along the slow axis, w i l l be out of phase by an amount ^ with the unaltered one. As stated before, these two vectors w i l l add up to give e l l i p t i c a l l y polarized l i g h t which has a component V p a r a l l e l to the transmission axis of the analyzer (figure 5.2C). For any given retardation, the maximum transmission at the analyzer ( at 90° to the polarizer) w i l l occur at a specimen orientation of 45° with respect to the polarizer (Bennett 1950). J p l Quantitating the retardation The retardation of the e l e c t r i c vector propagating along the slow axis cf transmission can be guantitated by ins e r t i n g a c a l i b r a t e d compensator between the birefringent object and the analyser. This compensator, as the name implies, functions by reversing the e f f e c t s of the birefringent specimen. It changes the e l l i p t i c a l l y polarized emergent l i g h t back to l i n e a r l y polarized l i g h t , with i t ' s e l e c t r i c vector p a r a l l e l to that of the pol a r i z e r . Since the analyzer w i l l not pass any component that i s at 90° to i t ' s transmission axis, the experimental manipulation involves rotating the compensator to extinction (cf the bright specimen), at which point the 'reverse' retardation of the compensator, i s egual to the 116 i n i t i a l retardation cf the specimen. The absolute value of the retardation can then be tabulated by u t i l i z i n g the appropriate equations for any given compensator and wavelength of l i g h t . This value of retardation i s divided by the o p t i c a l path length, d, usually equated to the thickness of the specimen, to y i e l d a value f o r the birefringence, which i s unitless. Figure 5.3 i s a summary figure showing the r e l a t i v e positions of the various components used in the study of the birefringence properties of materials. A more detailed account of the theory and methodology involved i n polarized l i g h t microscopy can be found in a r t i c l e s by Bennett (1950), Frey- Wyssling ( 1953) and Wilkes (1971). C_ __________ The Types of Birefringence J _ l I n t r i n s i c birefringence The concept of i n t r i n s i c or c r y s t a l l i n e birefringence, which is thought to be the r e f l e c t i o n of the organization at the molecular l e v e l , i s best introduced by considering an i s o t r o p i c material. In such a material the chemical bonds are di s t r i b u t e d egually over a l l angles, r e l a t i v e to the incident l i n e a r l y polarized l i g h t . As a r e s u l t of t h i s homogeneous d i s t r i b u t i o n of bond angles there i s no selective retardation of the component e l e c t r i c vectors, and the emergent plane polarized l i g h t w i l l have i t ' s e l e c t r i c vector p a r a l l e l to that of the incident beam. Hence the i s o t r o p i c material w i l l appear dark when viewed between crossed-polars. 117 Ficj.ure._5_.j:. The b i r e f r i n gence experiment. Pol: p o l a r i z e r . S: specimen. D: path length. C: compensator. An: analyzer. Ob: observer. The arrow shows the direction cf propagation. 118 _ _ _ _ _ _ _ _ _ j o Z 119 This system however, cannot d i f f e r e n t i a t e between an isotropy that i s a result of the random thermal motion of anisotropic segments (such as the k i n e t i c elastomers) and the isotropy that arises from a homogeneous d i s t r i b u t i o n of r i g i d segments (such as glass). Fortunately, t h i s aspect of the molecular characterization can be elucidated on the basis of the mechanical properties of the materials under study. Kinetic elastomers are characterized by a Young's modulus i n the order of 106 Newtcns/m2, whereas glassy substances usually have modulus values that are atout three tc four orders of magnitude higher than the rubbery materials. Now consider a material whose molecular structure shows a predominant d i r e c t i o n a l i t y of i t ' s units. Such a preferred orientation of bond angles, with reference to the incident l i g h t , w i l l r e s u l t i n the transformation of the l i n e a r l y polarized l i g h t to e l l i p t i c a l l y polarized l i g h t , and the object w i l l appear bright when examined between crossed polars. The amount of retardation as a function of the path length, i s an indicator of the extent of molecular organization. The sign of the birefringence, which refers to the d i r e c t i o n of the slow axis of transmission, has been conventionally defined as being p o s i t i v e when the slow axis i s p a r a l l e l to a 'given dimentional feature' such as the long axis of a c y l i n d r i c a l object. Conversely, i f the object has i t ' s fast axis p a r a l l e l to the 'given dimentional feature', i t i s said to be negatively birefringent (figure 5 . 4 ) . 1 2 0 F igure,_J5_. 4^ The s i g n of the b i r e f r i n g e n c e . (A) N e g a t i v e l y b i r e f r i n g e n t specimen: f a s t a x i s p a r a l l e l to the ' d i m e n s i o n a l f e a t u r e 1 . (B) P o s i t i v e l y b i r e f r i n g e n t m a t e r i a l : slow a x i s p a r a l l e l to the ' d i m e n s i o n a l f e a t u r e ' .  122 Z i g u r e ^ S i S . : Form birefringence. (A) A composite of f i b r e s with r e f r a c t i v e index n̂- , i n a matrix of r e f r a c t i v e index n m. (B) Evaluation of the form birefringence according to eguation 5.2: (a) for f i b r e s with positive birefringence (b) for i s o t r c p i c f i b r e s (c) for negatively birefringent f i b r e s . The inset shows the r e l a t i v e dependence of the form birefringence on the volume f r a c t i o n cf the f i b r e s , \ . 123 F i g u r e . 5 . 5 . R 124 It) Form birefringence Unlike i n t r i n s i c birefringence which arises from an anisotropy at the molecular l e v e l , form birefringence arises from an anisotropy of a scale that i s larger than the dimensions of molecules but smaller than the wavelength of l i g h t . The l i m i t to their 'smallness* i s set by the requirement that the structural units should be big enough to possess true phase boundaries (Frey-Wyssling 1953). Consider the structural composite shown i n figure 5.5A.. This represents a system of p a r a l l e l , i s o t r o p i c f i b r e s of r e f r a c t i v e index n^ , surrounded by an i s o t r o p i c medium of r e f r a c t i v e index n^. A structure such as t h i s w i l l exhibit form birefringence a r i s i n g from the preferred orientation of assymetric bodies in a medium of d i f f e r i n g r e f r a c t i v e index (Bennett 1950). The f i r s t t h e o r e t i c a l r e l a t i o n s h i p for such composites was presented by Weiner in 19 12, and i t has formed the basis of most of the form birefringence studies to date. For a system of rods, the the o r e t i c a l expression has the form (Bear ______ 19 37) : n u 2-n_2= (n^ 2-n m 2 ) 2 f ( 1 _ f ) / A f 2 ( 1 _ f ) « . n _ 2 ( 1 + f _ 5 . r where. n u and n_ represent the r e f r a c t i v e index of the system p a r a l l e l to and normal to the dominant axis of the specimen respectively, with f representing the volume f r a c t i o n of the f i b r e s . Another relationship for a si m i l a r type of system, based on a modification of the Weiner equation, has been 125 proposed by Bear et.al.. (1937). If n_c and n,̂  are nearly egual, the above relationship can be s i m p l i f i e d to an experimentally more useful one of the form (Stokes 1 963) : n,v-nJ_= ( n r n m ) 2 f ( 1 - f ) / i i 5.2 A graphical representation of th i s eguation i s shown i n figure 5.5B, curve b. Since the two types of birefringence (form and i n t r i n s i c ) are thought to be additive, the t o t a l birefringence of a system can be characterized as shown i n figure 5.5B, curve a, for rods of positive birefringence, and curve c, for rods having negative i n t r i n s i c birefringence. In a l l cases, the form birefringence goes to zero when n+=nn_, and the birefringence cf the system eguals the i n t r i n s i c birefringence of the f i b r e s . It also follows from eguation 5. 2 that for any given r e f r a c t i v e index difference the maximum form birefringence occurs at a volume f r a c t i o n of f i b r e s , f , egual to 0.5 (figure 5.5 i n s e t ) . _ [ £ L Strain birefringence When an i s o t r o p i c non-birefringent object i s subjected to a s t r a i n along one of i t ' s axis, i t w i l l exhibit a birefringence which i s commonly l a b e l l e d accidental or s t r a i n birefringence.. The imposed s t r a i n has the effect of inducing a preferred d i c r e c t i o n a l i t y on the d i s t r i b u t i o n of the bond angles, i n the o r i g i n a l l y i s o t r o p i c material, which i s subsequently r e f l e c t e d as an o p t i c a l anisctropy.. The molecular changes responsible for the observed s t r a i n 126 birefringence depends on the type of material in guestion. The o p t i c a l anisoptropy that results when c r y s t a l l i n e s o l i d s are strained, arises from the deformation of electron o r b i t s i n the material, which i n turn i s a conseguence of the altered inter-atomic distances (Frey-Wyssling 1953). When considering the s t r a i n birefringence of rubbery polymers however, a di f f e r e n t t h e o r e t i c a l basis has to be invoked. Rather than a r i s i n g from the displacement of electrons, the s t r a i n birefringence of these materials i s thought to re s u l t from the alignment of the 'random l i n k s ' that make up the k i n e t i c a l l y free chains of the polymer network. The optical properties of the l i n k s themselves .remain uneffected. The t h e o r e t i c a l eguations for the st r a i n birefrinigence of rubbery polymers w i l l be more appropriately discussed i n chapter 7. For the moment i t s u f f i c e s to simply state the qu a l i t a t i v e differences between the two types of s t r a i n birefringence. D_ Materials and Methods Single, 6 to 8 un, e l a s t i n f i b r e s from untreated bovine 2i3§ i™en_u_ nuchae and from ligament that had been p u r i f i e d by repeated autcclaving (Partridge et_ ___ 1955) were i s o l a t e d with f i n e forceps on a dissecting micrcsccpe. The f i b r e s were viewed between crossed polars on a Wild M-21 pol a r i z i n g microscope, and the retardations were determined at 546nm using a Zeiss 1/30 wavelength rotary compensator. Illumination was provided by a 100 watt quartz lamp which was used i n 127 conjunction with an interference f i l t e r to give the monochromatic green l i g h t . Birefringence was calculated by dividing the retardation at the center of the f i b r e by the fi b r e diameter. The form birefringence curves were . obtained from measurements on single e l a s t i n f i b r e s and rat t a i l tendon collagen f i b r e s that had been swollen in l i g u i d s of known r e f r a c t i v e index. These are l i s t e d i n table 5.1.. The temperature controlled stage was b u i l t in the lab to f i t the Wild microscope. It consisted of an aluminium piece that had been chanelled out to allow an i n t e r n a l closed c i r c u l a t i c n of water. The outlets from the stage were connected to a thermostated c i r c u l a t i n g water bath. The temperature at the sample was monitored with GB32 thermistor bead that had been calibrated with a mercury thermometer. Unless otherwise stated, a l l experiments were conducted at 240C. I i Birefringence Properties of Single E l a s t i n Fibres iJLL Form birefringence Single e l a s t i n f i b r e s were either swollen i n l i g u i d s of known r e f r a c t i v e index (water, ethylene g l y c o l , glycerol) or ai r dried and immersed in organic solvents of d i f f e r e n t r e f r a c t i v e index. In a l l cases the e l a s t i n f i b r e s (purified and unpurified) , gave a constant low birefringence value of about 2 X 10-* (table 5.1, figure 5.6 curve a). There was no 128 Iable.5.1; Form bit e f r i n qsncQ of single e l a s t i n f i b r e s . 0} •«- i <t- — 01 — «3- rr u >> 129 Figure.5.6: Form _____________ of e l a s t i n f i b r e s . (a) For single e l a s t i n f i b r e s in (x) water n=1.33 (+) ethylene gly c o l n=1.43 and glycerol n=1.48„ {•) for organic solvent of d i f f e r e n t r e f r a c t i v e index.. (b) Theoretical birefringence calculated for i s o t r o p i c f i b r e s according to eguation 5.2. (c) Results obtained for single e l a s t i n f i b r e s by Se r a f i n i - F r a c a s s i n i e t . a l . 1976. The inset shows the temperature dependence for the birefringence of single e l a s t i n f i b r e s .  13 1 indicat i o n of form birefringence, which i s usually visualized as a U-shaped curve f c r the graph of birefringence versus r e f r a c t i v e index of imbibing l i g u i d . This U-shaped curve i s c h a r a c t e r i s t i c of fibrous proteins with filamentous sub- structure, such as collagen, which shows a very d i s t i n c t form birefringence curve (figure 5.7 curve a ), and reported r e s u l t s for s i l k , c h i t i n , and keratin (Frey-Wyssling 1948, 1953) . I f e l a s t i n was in fact filamentous, as suggested by the electron microscope studies (S e r a f i n i - F r a c a s s i n i e t . a l . 1976), then one should expect a res u l t that closely follows the t h e o r e t i c a l prediction afforded by eguation 5.2, as shown i n figure 5.6 curve b. This t h e o r e t i c a l curve was calculated by assuming the filaments (if present) to be i s o t r o p i c , and using a value of 1.55 for the r e f r a c t i v e index cf the e l a s t i n protein. The volume f r a c t i o n , f, was taken to have a value of 0.65 (Gosline 1978). It should be mentioned that e l a s t i n swollen in water, ethylene g l y c o l , and glycerol retains i t ' s e l a s t i c properties, hence the experimental re s u l t s obtained under these condition are considered to be a more accurate r e f l e c t i o n of e l a s t i n ' s functional conformation, as compared to the data points which were obtained for dry e l a s t i n f i b r e s i n the di f f e r e n t organic l i q u i d s . Nevertheless, i t i s encouraging, and prcbably relevant, that both sets of data showed i d e n t i c a l r e s u l t s . L a s t l y , i t should be pointed out that the re s u l t s obtained in these experiments are inconsistent with the 132 reports of positive form birefringence curves ( S e r a f i n i - F r a c a s s i n i et. a l . . 1976), figure 5.6 curve c, and a l a t e r report of a negative form birefringence curve (Bairati and Gotte 1977) for single e l a s t i n f i b r e s . A possible explanation f o r these c o n f l i c t i n g r e s u l t s w i l l be presented i n the discussion section of t h i s chapter. Jb_ I n t r i n s i c birefringence Since there i s no indication of form birefringence, i t i s reasonable tc assume that the constant value of 2 X 10-* represents the i n t r i n s i c component of the birefringence. This value i s seen to be temperature independent f o r e l a s t i n f i b r e s in water (figure 5.6 i n s e t ) , and i s extremely small as compared to other known c r y s t a l l i n e proteins (Schmitt 1950). It i s therefore tempting tc conclude that e l a s t i n i s an amorphous protein. One might argue however, that t h i s low value for the i n t r i n s i c birefringence i s actually an a r t i f a c t . Since the t o t a l birefringence cf a system i s a sum of the i n t r i n s i c and form birefringence, i t i s possible that these two types of birefringence counteract each other when they are of egual magnitude and have opposite signs, to yield a zero (or nearly so) t o t a l birefringence. This i s demonstrated in figure 5.7, where the birefringence of dried collagen f i b r e s and tannic acid f i x e d collagen f i b r e s ( P f e i f f e r 1943) i s plotted as function of the r e f r a c t i v e index of the immersion medium. Collagen f i b r e s , which are known to be anisotropic at the 133 Figure .5.7: Form birefringence of collagen.. (a) unpurified collagen f i b r e s . (b) e l a s t i n f i b r e s . (c) Tannic acid fixed collagen fibres ( P f e i f f e r 1 943) . 134 135 molecular and the sub-microscopic lev e l s , show both positive form and positive i n t r i n s i c birefringence {figure 5.7 curve a). Tannic acid reverses the sign of the i n t r i n s i c birefringence, and as a res u l t of t h i s , tannic acid fixed collagen appear i s o t r o p i c (show zero t o t a l birefringence) at n m egual to 1.40 and 1.67 (figure 5.7 curve c) . That the lack of birefringence i n single e l a s t i n f i b r e s does not occur as a r e s u l t of t h i s phenomenon i s supported by the following. In order for the t h e o r e t i c a l form birefringence curve to intersect the observed data point for e l a s t i n i n water, one has to either (a) assign an unusally large negative (or positive) i n t r i n s i c birefringence to e l a s t i n , or (b) assume a r e f r a c t i v e index for the protein that i s much smaller than the value of 1.55 used i n t h i s study. Both of these p o s s i b i l i t i e s are unreasonable. A high i n t r i n s i c birefringence would indicate a very c r y s t a l l i n e structure and t h i s i s not supported by the X-ray d i f f r a c t i o n reports (Astbury 1940). The second choice, that of a low r e f r a c t i v e index, would be an exception to the known r e f r a c t i v e indeces for a wide variety of proteins, with values that l i e between 1.50 and 1.55. Most importantly however, the p o s s i b i l i t y of an a r t i f a c t i s precluded by the fact that, as presented in the l a s t section, e l a s t i n f i b r e s show a very low, constant birefringence over a bread range of r e f r a c t i v e index l i g u i d s (figure 5.6). This i s taken to mean that there i s no form birefringence component associated with the observed value of 2 X 10-*, which i s taken to be the value of the apparent 136 i n t r i n s i c birefringence. The timely i n t e r j e c t i o n of the word •apparent' i s discussed i n the following section. _[cl Explanation for the apparent birefringence Although the magnitude of the residual birefringence i s extremely small, i t might be i n t e r e s t i n g to speculate about the source of t h i s ' i n t r i n s i c ' birefringence. In doing so one i s faced with three p o s s i b i l i t i e s : 1. The birefringence i s an inherent property of the f i b r e . 2. That the birefringence i s a r e s u l t of an anisotropic residue around the f i b r e . 3. That i t i s due to the r e f r a c t i v e index difference at the interface of the swollen protein and the surrounding water. Inherent birefringence: Since the retardation i s proportional to the path length through the f i b r e , then, i f the birefringence was inherent to the f i b r e one would expect i t to be greatest at the center of the f i b r e where the path length i s at i t ' s maximum value.. For a c i r c u l a r cross-section one can predict the r e l a t i v e retardations across the f i b r e of radius, r, i n terms of the path length,p, according to: p= 2 (cos6r) 5.3 This r e l a t i o n s h i p i s depicted in figure 5.8.. l i r e f r i n g e n t coating: In the case of the unpurified f i b r e s i t i s possible that there exists a sheath of birefringent material (such as the 137 ___________ Expected rela t i o n s h i p for i n t r i n s i c ^i^ef£i£ 3 s n c e _ Graphical representation of the r e l a t i v e i n tensity versus distance across f i b r e calculated according to eguation 5.3. M i s the middle of the f i b r e . 138 Figure.5.8. 2 139 glycoprotein m i c r o - f i b r i l s ) around the central amorphous core of the f i b r e . S i m i l a r l y , i n the case of the autoclaved e l a s t i n one might expect that a residue could be l e f t covering the f i b r e s , which i f c r y s t a l l i n e , would give r i s e to an observable birefringence. Again assuming a c i r c u l a r cross-section for the e l a s t i n f i b r e , i t should be possible to predict the r e l a t i v e retardations through the analysis of the path length across the f i b r e . Consider a beam of polarized l i g h t propagating through a cylinder of radius r ' , containing within i t a cylinder of radius r " , with both having common centers (figure 5 . 9 A ) . .The t o t a l path length, p, transversed by the ray at a distance, x, away from the center i s given by: p= 2(r« 2 - x 2 ) V 2 5 . 4 The component, p 1, of t h i s t o t a l path length which l i e s inside the inner cylinder of radius, r M , at a distance, x, away from the center, i s given by: p i - 2 ( r " 2 - x 2 ) V 2 5 . 5 Since only the coating matrix i s birefringent, the e f f e c t i v e path length, p", which i s the path length contributing to the retardation, at a distance, x, away from the center, f o r x<r", i s given by: p"=p-p« 5 . 6 For values of x>r", p' goes to zero, and the above eguation reduces to: p"=p 5.7 These equations were evaluated for various r a t i o s of r ' : r " . 140 F iqure.5.9: Expected _____________ fo r an a _ _ _ _ t _ _ _ i _ coating. (A) Isotropic material of radius r" surrounded by a b i r e f r i n g e n t coating of radius r * . M represents the middle of the f i b r e . (B) Graphical representation of the r e l a t i v e i n t e n s i t y versus distance across f i b r e calculated according to eguations 5.6 and 5.7, for d i f f e r e n t r a t i o s of r n / r ' : (a) 0.75 (bj 0.90 (c) 0.95.  142 and the results are shown in figure 5. 9E. This analysis of the system predicts that the birefringence should be greatest at the edges, f a l l i n g to a f i n i t e value towards the middle. I n t e r f a c i a l effects.: In t h i s case, the source of the birefringence i s thought to be the interface at the border of the protein f i b r e and the surrounding water. The hydrated protein would be expected to have a r e f r a c t i v e index somewhere between that of pure water and pure protein, probably around 1.5. The exact value for the r e f r a c t i v e index of the hydrated protein i s inconseguential as long as i t different from the value of the surrounding l i q u i d (which i t obviously i s ) . Although i t was not possible to derive a t h e o r e t i c a l r e l a t i o n s h i p for a system such as t h i s , i t was possible to make some predictions based on empirical observations. Towards t h i s purpose, observations were made on the birefringence patterns at oil-water interfaces. In a l l cases i t was possible to observe a very evident birefringence zone at the interface. Unfortunately however, i t was not possible to extract any useful information from these observations since the interfaces were e s s e n t i a l l y two dimentional. Totally by chance, in the case of a few of these experiments, a number of a i r bubbles were trapped in the o i l . Since these a i r bubbles represent a three dimentional system, and show a d i s t i n c t birefringence, an e f f o r t was made to study the pattern of t h i s structure. The r e s u l t s showed that the 143 birefringence was greatest at the edges , f a l l i n g off towards the center cf the bubble. In analogy to t h i s , one might expect that the e l a s t i n f i b r e surrounded by water should behave i n a similar manner. Furthermore, within the resolution of the experimental methods, i t seems that the birefringence should resemble the t h e o r e t i c a l prediction for a system which consists of an amorphous core surrounded by an anisotropic sheath, as described in figure 5.9.. Figure 5.10A shows the birefringence pattern observed for an e l a s t i n f i b r e i n water. It i s clear that the retardation, as indicated by the in t e n s i t y , i s greatest at the edges of the f i b r e . This conclusively rules out the p o s s i b i l i t y that the •apparent' birefringence i s an inherent property of the e l a s t i n protein. In dealing with last two choices, i t would be possible to distinguish between them, i f one could manipulate the experiment by changing the r e f r a c t i v e index of the surrounding l i g u i d without a l t e r i n g the hydrated protein. If the birefringence i s caused by the presence of an anisotropic sheath, then i t should remain unchanged. Cn the other hand, i f the birefringence i s a result of i n t e r f a c i a l e f f e c t s , then i t should be considerably reduced as the system approaches uniformity (as the r e f r a c t i v e index of the surrounding l i g u i d approaches that of the protein). For p r a c t i c a l purposes, i t i s possible to achieve this by hydrating the e l a s t i n f i b r e i n the vapour phase over a d i l u t e 144 £i_u££i5__0_ Birefringence pattern of ______ e l a s t i n _______ (A) The birefringence pattern of single e l a s t i n f i b r e s n=1.55, in water n=1.33, between crossed polars. The birefringence i s seen to be highest at the edges. The bar represents 10uni. (B) Densitometer tracings for negatives of single e l a s t i n f i b r e s i n : (1) water, n=1„33. (2) immersion o i l n=1.52. (3) control tracing of blank f i e l d . 145 DISTANCE ACROSS FIBER 146 s a l t solution, and then covering the f i b r e with immersion o i l which w i l l not interact with the hydrated protein, but has a value of r e f r a c t i v e index (1.52) that i s close to that of the swollen f i b r e . E l a s t i n fibres treated in t h i s manner were mounted between crossed pclars and photographed. The negatives were then scanned on a Joyce-Loebel scanning densitometer. Figure 5.10B, curve 1, i s a densitometer tracing for an e l a s t i n f i b r e in water, confirming the previous observation of the higher i n t e n s i t i e s at the edges of the f i b r e . Curve 2, shows a tracing of the same f i b r e after reduction of the r e f r a c t i v e index difference at the interface, as described above. It i s observed, that the retardations are reduced d r a s t i c a l l y , and i s indistinguishable from the control tracing of a blank f i e l d (curve 3). Hence i t seems safe to state that the 'apparent' residual birefringence i s a result of the r e f r a c t i v e index difference at the fibre-water i n t e r f a c e , and that the actual value for the birefringence of e l a s t i n i s very close to zero, as expected for a random protein. F. Discussion (a) Previous studies There are a number of studies present i n the l i t e r a t u r e that deal with the birefringence properties of e l a s t i n and the i n t e r p r e t a t i o n of these properties i n terms of the molecular structure of t h i s protein (Schmidt 1939, Dempsey 1952, Eomhanyi 1958, Gotte 1965, B a i r a t i and Gotte 1977, S e r a f i n i - 147 Fraca s s i n i ______ 1976, Fischer 1979). .Most of these studies are i n agreement with reference to the low birefringence of e l a s t i n (with the exception of Serafini-Fracassin e t . a l . 1976), reporting values around 2 X 10-* as obtained i n t h i s study.. A l l of these other studies have assumed th i s low birefringence to be an inherent property of the e l a s t i n f i b r e , and on the basis cf this assumption have proceeded to • v i s u a l i z e 1 the structure u t i l i z i n g the phenol reaction (Eomhanyi 1958) and the permanganate-bisulfite-toluidine blue reaction (Fischer 1979). . Using the results from these manipulations they favoured the anisotropic, ordered s t r u c t u r a l models for e l a s t i n . It should be pointed out that the above studies were conducted on unpurified e l a s t i n which could r e s u l t i n a r t i f a c t s from the chemical interactions of components other than the e l a s t i n protein, which also occur i n e l a s t i c tissue. In order to prevent the occurence of these a r t i f a c t s t h i s study has concentrated on the analysis of p u r i f i e d e l a s t i n f i b r e s in water, a condition that most close l y resembles the i n vivo rubbery state. As mentioned before the results obtained i n t h i s study, with regard to the form birefringence, are in c o n f l i c t with those reported by S e r a f i n i - F r a c a s s i n i e t . a l . (1976) and B a i r a t i and Gctte (1S77). This study f a i l e d to reveal any form birefringence for e l a s t i n , whereas S e r a f i n i - F r a c a s s i n i and his co-workers obtained a very d i s t i n c t i v e curve as well as a high i n t r i n s i c birefringence of 1 X 10 - z, which they interpreted as being i n favour of the f i b r i l l a r models. In t r y i n g to explain 148 t h i s c o n f l i c t a clue i s afforded by glancing at the form birefringence curve obtained by these authors (figure 5.6 curve c) and comparing i t with the results obtained in t h i s study for the form birefringence of collagen (figure 5.7 curve a). The resemblance i s guite marked and one might speculate that these investigators were actually dealing with a collagen f i b r e . This objection i s not too far fetched considering the fact that these authcrs used collagenase to purify t h e i r protein, and that t h i s technigue has been shown to r e s u l t i n high l e v e l s of collagen contamination in the p u r i f i e d tissue (Kadar 1977). Furthermore, both B a i r a t i and Gotte (1977) and Se r a f i n i n - F r a c a s s i n i (1976), used experimental preparations that contained more than one e l a s t i n f i b r e . On the basis of t h i s i t could be argued that these authors were reporting a r t i f a c t s , caused by the i n t e r f a c i a l e f f e c t s , which would be very large for ccnglcmorations of f i b r e s . Jb_ The f i b r i l l a r models In trying to interpret the birefringence results i n the context of the f i b r i l l a r models for e l a s t i n one i s faced with two p o s s i b i l i t i e s : 1. E l a s t i n i s made up of an array of p a r a l l e l filaments aligned in the d i r e c t i o n of the long axis, and that these filaments are themselves i s o t r o p i c , possibly accomodating random c o i l s of protein. 2. That e l a s t i n i s filamentous in i t ' s organization, 149 as mentioned above, and in addition to thi s these filaments contain secondary structures such as the Beta-turns. In the f i r s t case, that of i s o t r o p i c filaments, one would expect e l a s t i n to exhibit a marked form birefringence which would reduce to zero when the r e f r a c t i v e index of the immersion medium equalled that of the protein filaments. In the second case, cne would also expect a marked form birefringence a r i s i n g from the well defined s p a t i a l arrangement of the filaments along the long axis of the f i b r e . But i n addition, to thi s form birefringence, one should see an i n t r i n s i c birefringence associated with the c r y s t a l l i n e structure of the filaments themselves. In the s p e c i f i c case of the occurence of the Beta-turns in the filaments (Drry 1978c) , one would predict a negative i n t r i n s i c birefringence since the peptide back-bone involved in this type of structure would be running normal to the long axis of the filaments. As i s guite evident from the previous section, single e l a s t i n f i b r e s do not show any form birefringence nor do they seem to have any i n t r i n s i c birefringence. These results argue against the presence cf any filamentous organization, or the occurence of stable secondary structures in the e l a s t i n protein. Furthermore, the fact that e l a s t i n does not display any temperature dependence of i t ' s birefringence also argues against any d r a s t i c , temperature, induced change of i t ' s conformation to a more ordered state as suggested by some workers {Urry 1976a). F i n a l l y , since the synthetic f i b r e s of 150 e l a s t i n (Orry and Long 1977b) are known to be highly birefringent (Long 1979) one might be j u s t i f i e d i n questioning the use of these materials as models for e l a s t i n structure. G_.Con elusions In view cf the evidence presented in t h i s chapter, i t i s probably j u s t i f i a b l e to conclude that e l a s t i n possesses a random network structure which i s t y p i c a l cf other known k i n e t i c elastomers. However, the technique of polarized l i g h t microscopy can only deal with the 'average conformation', and i s not able to distinguish between the homogeneous d i s t r i b u t i o n of s t a t i c units and the random k i n e t i c movements of mobile anisotropic units. Hence i t cannot argue against the presence of secondary structures as long as these are thought of as being dynamic (in rapid movements). One can probably disregard the presence of s t a t i c , c r y s t a l l i n e structure on the basis of the p l i a n t mechanical properties of e l a s t i n , which are inconsistent with glassy structures, and the nmr analysis presented in chapter 4. The filamentous organization visualized in the electron microscope may well be the result of drying e l a s t i n i n the presence of the heavy-metal s a l t s used for negative staining. It i s also plausible that the samples of e l a s t i n prepared for electron microscope studies were inadvertently extended to high elongations by the receding water during the drying process. This could result in the alignment of the polypeptide chains along the axis of the f i b r e , which as a result of the 151 • s t a t i c ' view afforded by the electron microscope technique, would be a mis-interpretation of the normally agitated, k i n e t i c a l l y free structure, as being a highly organized filamentous system that i s irrelevant tc the in vivo rubbery condition. This point i s supported by electron microscope studies, which, using the freeze-etching technigues to avoid the gradual drying down of the t i s s u e , could only demonstrate filamentous structure i n samples that had been extended 150 to 200% of the i r i n i t i a l length (Pasquali-Eonchetti et.al..1979). I t i s also my opinion that the 200nm sub-fibres which have been observed i n the scanning electron microscope (Hart e t . a l . 1978) are large enough to accomodate an i s o t r o p i c random network structure, and are not in c o n f l i c t with the idea of e l a s t i n being a t y p i c a l k i n e t i c elastomer. This aspect of e l a s t i n organization i s examined in the next chapter. 152 Chapter^VI^ CONFORMATION OF ELASTINj. SCANNING ELECTRON MICROSCOPY^ A. Introduction In the previous chapter I have examined the sub- microscopic and the mclecular structure of the e l a s t i n protein. . Having done so i t seems appropriate at t h i s time t c evaluate the organization of e l a s t i n at a l e v e l of order that approaches the range of scanning electron miscroscope techniques, which should allow an examination of the 200nm sub-fibres i f they are in fact present. This i s a fe a s i b l e proposition since the procedures u t i l i z e d i n t h i s study make use of quick freezing technigues in the presence of a solvent (water) that cl o s e l y resembles the in vivc environment. These procedural aspects should protect against organizational a r t i f a c t s . The disadvantage l i e s i n the fact that the technigue of scanning electron microscopy only allows a v i s u a l i z a t i o n of the surface texture, which can easily lead to mis-interpretation of the images. . I i Methods E l a s t i n , from unpurified ligament, a l k a l i extracted ligament, and autoclaved ligament, was cut with a sharp razor into cubes (largest dimension less than 1mm) and l e f t i n d i s t i l l e d water at 4°c overnight. The preparation for scanning electron microscopy was done by dropping the hydrated pieces of tissue into a butanol bath 153 that was cooled in l i g u i d nitrogen. Some of the samples were fractured while frozen. A l l samples were dehydrated i n a freeze dryer (operating at -50°C) and mounted onto stubs with conducting s i l v e r paint. These stubs were subsequently coated, in vacuo , with a thin layer of gold approximately 100A° i n thickness. The specimens were viewed in a Cambridge Instrument Company, Stereoscan microscope, at a filament accelerating voltage of 50kv. The images were recorded on Polaroid 655 f i l m , and the negatives were preserved by treatment with sodium sulphite for one hour. C_ Results Preparations of unpurified ligament e l a s t i n showed some indi c a t i o n of a ' f i b r i l l a r ' arrangement when viewed i n the long i t u d i n a l d i r e c t i o n (figure 6.1)..These structures, which had diameters of 80 tc 100nm, could represent the the collagen sheath that i s thought to occur around the i n d i v i d u a l e l a s t i n f i b r e (Finlay and Steven 1973). The fracture surfaces of these f i b r e s , however, f a i l e d to support such an organization, in d i c a t i n g that i t i s probably a feature that i s confined to the periphery of the e l a s t i n f i b r e s . Autoclaving the el a s t i n f i b r e s resulted i n the removal of the 'surface coating' revealing what appears to be a clear i n d i c a t i o n of sub-structure (figure 6.2), that i s visualized at the surface as a granular appearance with ridges thrown in for good measure. But again, cross-sectional views of the same 154 Figure. 6._1i S.E.M. Of unpurified ligament e l a s t i n f i b r e _ (E) E l a s t i n . (CS) Collagen sheath. The bar represents 10um, 155 156 preparations f a i l e d tc show any sub-structure. A l k a l i extracted specimens showed a very d i s t i n c t surface structure, with 40nm fi b r e s that were spaced approximately 200nm apart, similar tc these observed for a r t e r i a l e l a s t i n by Carnes et^al.. (1977) (figure 6.3a). Higher magnification of the fracture surfaces showed a very smooth appearance, without any i n d i c a t i o n of in t e r n a l structure (figure 6.3b). M>. Discussion Almost a l l of the studies reported i n the l i t e r a t u r e , dealing with the scanning electron uicrcscopy of e l a s t i n , have supported the 200nm sub-fibre arrangement for the e l a s t i n protein. In objection to thi s point of view, i t should be stated that most of these investigators have looked at the appearance of the f i b r e s in the longitudinal direction. As i s shown by t h i s study, t h i s approach to the problem can be mis- leading since i t cannot distinguish between surface features and inherent organization..The few papers that have dealt with the cross-sectional view of the e l a s t i n f i b r e s (Minns and Stevens 1974) are i n agreement with the findings of t h i s study that there i s nc indicati o n of sub-structure. It i s int e r e s t i n g to note that these sub-fibres have never been observed i n the transmission electron microscope. The following are a few mere aspects of the r e s u l t s which also argue against the presence of these 200nm sub-fibres. Since the fracture of a substance r e s u l t s from the propagation of a crack through the material, and since the 157 ___________ Surface texture of autoclaved e l a s t i n _______ There i s seme ind i c a t i o n of substructure as re f l e c t e d in the surface texture of these f i b r e s . The bar represents 2um. 158 ___________ 159 F iqure.6.3: Fracture surfaces of e l a s t i n f i b r e s . A l k a l i p u r i f i e d fibxes showing the 200nm spaces on the e l a s t i n f i b r e s . ..The fracture surfaces, howver, are smooth and do not indicate the presence of 200nm sub-fibres. The tar represents 2um. 160 Figure.6. 16 1 path of crack propagation follows a route that reguires the least expenditure of energy, one wculd expect the fracture properties of a homogeneous substance, such as glass, to be noticeably d i f f e r e n t from a composite material, such as f i b r e - glass which i s made up of glass f i b r e s embedded i n an epoxy matrix. Applying t h i s analogy to the fracture properties of e l a s t i n , one would expect a •homogeneous' e l a s t i n f i b r e (at 90°C) to fracture in a smooth manner s i m i l a r to glassy materials. I f , however, these sub-fibres do e x i s t the material could be represented as a composite consisting of the e l a s t i n protein (2C0nm) embedded in a cementing matrix. The fracture of t h i s material (at -90°C) should give results s i m i l a r to the fracture of fibre-glass materials, with some of the cracks propagating between these 200nm sub-fibres resulting i n a •splayed' appearance cf the fracture surface. As mentioned before, the fracture surfaces of the e l a s t i n f i b r e s were always smooth, with no evidence f o r the presence of 'splayed' f i b r i l s . It i s possible though, that the embedding matrix material has mechanical properties that are i d e n t i c a l to the prctein f i b r e s as well as being s t r u c t u r a l l y continuous with these sub-fibres. In which case, the material would fracture in a homogeneous manner. This leaves me with one l a s t point: how to explain the presence of these 200nm surface texture. The reports that have observed these sub-fibres often state that they are only seen aft e r the p u r i f i c a t i o n of the 162 protein {Hart ______ 1978). On the basis of t h i s comment i t i s inte r e s t i n g to speculate-that the 200nm structures are an a r t i f a c t caused by the removal cf the glycoprotein f i b r i l s during p u r i f i c a t i o n , and actually represent the spaces that would r e s u l t from their removal. This explanation i s plausible because the glycoproteins do i n fact occur i n 100 to 200nm bundles around the e l a s t i n f i b r e , as seen i n the transmission electron microscope (larenbach ______ 1966). Having presented the arguments against the presence of the sub-fibres, I would l i k e to put the controversy into i t ' s proper perspective by stating that i t i s guite i r r e l e v a n t . If they are in f a c t r e a l attributes of the e l a s t i n f i b r e , they would s t i l l not be i n contradiction to the idea of e l a s t i n being a t y p i c a l kinetic elastomer, since their s i z e i s large enough to accomodate random c o i l s of protein. Their presence however, would a l t e r the interpretations of the f a i l u r e properties of e l a s t i c tissue since they would be an introduction of another a r c h i t e c t u r a l factor i n the network composite. 163 Chapter.VII. ELASTIN AS A KINETIC ELASTOMER. A_o_ Introduction In the previous chapters I have attempted to rigourously examine the conformational state of the e l a s t i n protein. The reason for that exercise (in f u t i l i t y ? ) was to validate the use of the kinetic theory of rubber e l a s t i c i t y as an adeguate basis for the elastomeric properties of t h i s protein. This chapter, then, deals expressly with the evaluation of e l a s t i n as a t y p i c a l k i n e t i c elastomer, assuming that a l l the conditions of the k i n e t i c theory are met. But why do these tests on single e l a s t i n fibres? Aside from i r r e l e v a n t e x i s t e n t i a l and philosophical arguments, there are a number of p r a c t i c a l reasons for suffering through the single f i b r e studies. The analysis off the elastomeric properties of e l a s t i n upto now, has been based on experiments using bundles of e l a s t i n f i b r e s . Although t h i s i s without guestion the more convenient form of experiment, i t ' s disadvantage l i e s i n the fact that using a sample with sub- structure w i l l make i t d i f f i c u l t to distinguish the properties of the 'architecture' from the properties of the material. This study was undertaken with the purpose of getting around the a r c h i t e c t u r a l interference by studying the physical properties of s i n g l e , 6 to 8um, e l a s t i n f i b r e s . This allowed an interpretation cf the properties at the molecular l e v e l , which was attempted by using the various k i n e t i c theory r e l a t i o n s h i p s . Hence the major product of t h i s study was the 1 6 4 evaluation of the various k i n e t i c theory parameters, as they apply to the e l a s t i n protein. Furthermore, since the f i b r e s in the e l a s t i n bundles are not continuous, p u r i f i e d bundles of e l a s t i n f a i l at low s t r a i n (about 50% extension). As w i l l be shown i n t h i s chapter, e l a s t i n i s s t i l l i n the •Gaussian 1 region of i t ' s macroscopic properties, at 50% extension, and therefore, bundle studies do not allow one to examine the additional information that i s r e a l i z e d by the analysis of the non-Gaussian properties of the polymer network. It i s these properties that need to be evaluated, i f one i s to extrapolate from the e l a s t i n system to other proteins i n the random conformation. L a s t l y , the photc-elastic experiments demand that the experimental setup u t i l i z e single f i b r e s , since bundles of f i b r e s have a large form birefringence, due to the i n t e r f a c i a l e f f e c t s , that masks the properties of the single f i b r e s , making the evaluation of the bundle properties impossible. __troa_ ___________ The Kinetic ______ of ______ E l a s t i c i t y J i l l Gaussian chain s t a t i s t i c s a_d entropy In trying to understand the s t a t i s t i c a l mechanics of random c o i l e d molecules i t i s convenient to represent one end of the chain, a, to be fixed at the o r i g i n , and to characterize the 'random walk' to the other end, b, via the quantity termed the end-to-end distance r, (figure 7 . 1 ) . The probability density function, of finding b i n the v i c i n i t y of 165 point P w i t h i n the volume element [dx,dy,dz] (which can a l s o be r e p r e s e n t e d as the p r o b a b i l i t y d e n s i t y f u n c t i o n of r values) forms the b a s i s of the k i n e t i c theory r e l a t i o n s h i p s . The d e r i v a t i o n of these p r o b a b i l i t i e s depends on the e v a l u a t i o n of the number of conformations t h a t allow the p l a c i n g of the chain end, b, w i t h i n a given volume element. I f one assumes that a l l conformations are e g u a l l y probable, then the d e n s i t y f u n c t i o n i s d e s c r i b e d by ( T r e c l a r 1975): p[x,y,z ]= ( b 3 / T T 3 / 2 ) exp (-b 2r 2) 7. 1 where p[x,y,z] r e p r e s e n t s the p r o b a b i l i t y of f i n d i n g the c h a i n end b a d i s t a n c e , r (r 2=x 2+y 2 + z 2) , away from end, a. The term b i s d e f i n e d by: b 2= ( 3 / 2 ) s l 2 7.2 where s i s the number of random l i n k s i n the chain and 1 i s the l e n g t h of each random l i n k . The important r e s u l t of eguation 7*1, i s t h a t the f u n c t i o n has a maximum at r=0, which i s s i m i l a r t c saying t h a t most of the conformations are c o n s i s t e n t with the end, b, being at the o r i g i n (no pun i n t e n d e d ) . Since the entropy of the chain i s p r o p o r t i o n a l to the l o g a r i t h m of the number of conformations a v a i l a b l e to the system ( T r e l o a r 1975) the entropy i s a l s o seen to be at a maximum when r=0. For a chain whose point s are seperated by a d i s t a n c e , r , the entropy cf the c h a i n , S, i s given by (Treolar 1975): S=c-kb 2r 2 7. 3 where c i s an a r b i t r a r y c o n s t a n t , and k i s the Boltzmann c o n s t a n t . As i s evident from eguation 7.3, the i m p o s i t i o n of 166 F i g u r e _ 7 . 1 : The random-coiled chain The random c h a i n with end a at the o r i g i n and end b at a p o i n t P(x,y,z) w i t h i n a volume element _dx,dy , d z 2 . 167 F i g u r e . 7 . 1 . 168 s t r a i n on t h i s system w i l l increase r, resulting i n a decrease of the ccnfigurational entropy of the system. This decrease, i n entropy, forms the basis for the r e t r a c t i v e force of k i n e t i c elastomers. The int e r n a l energy term remains constant and does not contribute to the e l a s t i c properties. Jb_ The e l a s t i c _______ Any molecular interpretation of a rubber polymer, based on the k i n e t i c theory of rubber e l a s t i c i t y , assumes a network made up of a three dimensional array of idealized random chains. The chains are cross-linked at various points (this i s necessary i f the network i s to maintain i t ' s structural and mechanical i n t e g r i t y when strained) and one can therefore characterize the chain between cross-links as having an average molecular weight Mc, and consisting of s number of random l i n k s each of length 1. In the case of r e a l molecules, however, ideal random l i n k s cannot cccur since bond movements are r e s t r i c t e d by valence angles, potential b a r r i e r s , and s t e r i c hindrances. One therefore has to invoke the concept of a 'functional' random l i n k . This l i n k w i l l consist of a number of bonds, which as a unit, appear to s a t i s f y the s t a t i s t i c a l reguirements cf the 'i d e a l ' random l i n k . As stated before, imposing a s t r a i n on t h i s network w i l l decrease the configurational entropy, giving r i s e to the r e t r a c t i v e force. The basic assumptions of the k i n e t i c theory, which are discussed in d e t a i l by Treolar (1975) and Flory (1 953), are as fellows: 1. The network contains N chains per unit volume. 169 These chains, which were defined above, are held in a network by a few stable c r o s s - l i n k s . 2. The mean square end-to-end distance f o r the whole assembly of chains in the unstrained state i s the same as for a corresponding set of free chains. 3. Deformation does not lead to a change in volume. 4. The components of length of each chain change i n the same r a t i c as the corresponding dimensions of the bulk rubber. 5. The entropy of the network i s the sum of the entropies of the i n d i v i d u a l chains. Jc_ Mechanical properties of k i n e t i c rubbers Assuming an i d e a l Gaussian system, the k i n e t i c theory makes i t possible t c characterize the tensional e l a s t i c force, r e s u l t i n g from the s t r a i n induced free energy change (egual to the entropy change for an i d e a l rubber), by the following eguations (Treolar 1975): f=NkT ( > - > ~ 2 ) 7.4 and, T=NkT( > 2 - >-*) 7.5 where f i s the nominal stress (force/unit unstrained cross sec t i o n a l area), i s the true stress (force/unit strained cross sectional area), k i s the Boltzmann constant (1.38 X 10~ 1 6 erg K- 1), T i s the absolute temperature, N i s the number of random chains per unit volume of the material, and > i s the extension r a t i o expressed i n units of the unstrained length. In the case of swollen rubbers, a state that i s relevant to the prctein elastomers, a volume f r a c t i o n term i s incorporated to give: f=NkTv_-/3 ( > - > - 2) 7.6 170 and, 0- = NkTv 2 V 3 { X 2 - 7.7 where v z i s the unswollen volume/swollen volume. f and cr refer to the force per unit swollen unstrained cross-sectional area, and force per unit swollen strained cross-sectional area respectively. A l l four of these eguations define a term that characterizes the s t i f f n e s s of the material being tested, c a l l e d the e l a s t i c modulus, G, according to: G = N k T 7.8 Since G i s proportional to the number of chains, N (based on assumption 5), and since N i s i t s e l f related to the cross- l i n k i n g density, which for the case of one cross-link attaching four chains i s approximately egual t c : N=2 (#c) 7. S where #c i s the number of cross-links per unit volume. It seems i n t u i t i v e l y possible to obtain a measure of the molecular weight between cross - l i n k s (which for a given composition i s inversely proportional to the number of cross- l i n k s , M (1/2)#c), from the value cf the e l a s t i c modulus G, This i s realized in the following r e l a t i o n s h i p : G=Nkt= f ET/Mc 7.10 where p i s the polymer density, B i s the universal gas constant (0.082 X 10-3m3atm m o l - i K - 1 ) , and Mc i s the molecular weight between c r o s s - l i n k s . Water swollen e l a s t i n , being a hydrophobic protein i n equilibrium with a h y d r o p h i l l i c solvent, presents some 17 1 problems when one t r i e s to interpret i t ' s properties using the above relationships since i t ' s eguilibrium degree of swelling, contrary to the assumption of the k i n e t i c theory, changes as a function cf both temperature and elongation (Gosline 1978, Hoeve and Flory 1976). Oplatka e t . a l . (1960) and Bashaw and Smith (1968) extended the .kinetic theory to account for these compositional changes that take place in open systems, with M i s t r a l i e t^al.... (1971) providing the preliminary support for i t ' s application to e l a s t i n . These relationships, although they are mentioned here, were not tested rigourously since the physical measurements were not accurate enough to allow a c r i t i c a l appraisal of such refined d i s t i n c t i o n s . (d) Photo e l a s t i c i t y The k i netic theory also affords a relationship for the strain-birefringence behaviour of rubbery polymers swollen in an o p t i c a l l y neutral solvent (Treolar 1975): n„-n L= (fi2 + 2)22T H_«<,-o<J v^/ 3 ( \ 2 - >-i)/ii45 7.11 where and are the p o l a r i z a b i l i t i e s of the random l i n k p a r a l l e l to i t ' s length and normal to i t , respectively, n i s the average r e f r a c t i v e index of the protein, and N i s defined as before. The assumption i s made by t h i s theory that the o p t i c a l anisotropy of the polymer when i t i s strained arises from an alignment of the random l i n k s , whose inherent p o l a r i z a b i l i t y remains unchanged, in the direction of the s t r a i n . The birefringence i s not thought to represent an anisotropy 172 a r i s i n g from the displacement of electron o r b i t a l s (due to /the a l t e r a t i o n of the interatomic distances), as i s the case with c r y s t a l l i n e or glassy polymers. The relationship for the stress-birefringence can be derived from the above eguation by substituting i n equation 7.7, which describes the relationship between the true stress and the extension, into equation 7.11 to give: nn ~n_.= CT(n2+2) 22 TT /n45kT = CTC 7.12 Since (ot\ - °_) and n are presumed to remain constant, i t i s possible to incorporate a l l the parameters into an o v e r a l l constant, C , which i s termed the s t r e s s - o p t i c a l coeffecient. This coeffecient depends only on the mean r e f r a c t i v e index of the polymer and the p c l a r i z a b i l i t y of the random l i n k , and i t should be inversely proportional to the temperature. The value of (<*|-°_)» which represents the anisotropy of the random l i n k , i s t h e o r e t i c a l l y independent of s t r a i n , swelling, stress, and temperature. Hence i t ' s constancy over a number of parameters could be used as an indicator of the peptide back- bone s t a b i l i t y . Je_ Non-Gaussian effects and the evaluation of s Since the non-Gaussian effects cbserved for the mechanical and photoelastic properties of the e l a s t i c network i s the r e s u l t of the f i n i t e length of the chain between cross- l i n k s , and hence a f i n i t e number of random l i n k s , s. It seems i n t u i t i v e l y obvious that one should be able to work •backwards' and evaluate the value 's' by analysis of these 173 non-Gaussian properties. The Gaussian relationships mentioned upto now, assume that the end-to-end distance, r, i s small compared to the f u l l y extended length of the chains (r/sl«1). This assumption r e s t r i c t s t h e i r application to small s t r a i n s , and necessitates the use of the more accurate non-Gaussian relationships at higher l e v e l s of extension. For the case of the mechanical data, the relationship takes the form of a series expansion of which the f i r s t f i v e terms are (Treolar 1975) : f=Nkt ( X - X ~ 2 ) [ 1+ (3/25s) (3 > 2 + 4 / X ) + (297/6125s2) (5 X * + 8 % + 8/ X 2 ) + (123 12/2 20 5000s3) (3 5 X 6 + 6 0 X 3 + 7 2+6 4/ x 3 ) + (1261 1 7/693 (673750)s* (630 X 8 + 1 1 2 0 X 5 + 1 440 > 2+(1536/x ) + 128C/ X *) + ] 7.13 For the s t r e s s - o p t i c a l data, the non-Gaussian r e l a t i o n s h i p takes the form (Treolar 1975): ou-n^= (r\?+2) *4-rr (P, -Pz )/3fi 7. 14 where (pj -P Z) i s defined according to: (P, -P 2)=N(*.-* 2)[ (1/5) X 2 - ( 1 / X ) + (V150s) (6 X * + 2 X - 8 / x 2 ) + (1/35Cs2) (10 X 6 + 6 X 3 " 1 6 / x 3 ) 7.15 Comparison of the experimentally obtained curves (for the mechanical and phctoelastic properties) with these relationships should allow an evaluation of s. l i L Induction of e l a s t i n data to the unswollen form Since the protein elastomers such as E e s i l i n , Abductin, and E l a s t i n , exhibit their elastomeric behaviour only when 174 they are swollen by a diluent (such as water), i t might seem a l i t t l e i r r e l e v a n t to attempt the reduction of the data obtained for the swollen form to an 'unswollen' state. This i s necessary, however, i f cne i s make a val i d comparison between the t h e o r e t i c a l relationships f c r the non-Gaussian systems mentioned i n the l a s t section (which are derived f o r the unswollen network) and the e l a s t i n data. The data for e l a s t i n was reduced according to the relationships provided by Flory (1953): f o=f s/v//3. ... . . 7. 16 X °= > V v 2 V 3 7.17 q-°=g-Vv_2/3 7. 18 ( ^ - n j o=(n | |-n 1)/v|/3 7.19 where the superscripts 0 and s refer tc the 'unswollen' and swollen forms of the designated terms, which are defined as before. C. Materials and Method Jaj_ P u r i f i c a t i o n of e l a s t i n Bovine ligamentum nuchae of mature beef c a t t l e was obtained from a l o c a l slaughter house and p u r i f i e d by repated autoclaving i n d i s t i l l e d water according to the method of Partridge et__al_, (1S55). B r i e f l y , t h i s procedure involves •cooking' the e l a s t i n i n an autoclave operating at f i f t e e n pounds per sguare inch for a period of an hour. This removes the associated proteinaceous material, such as the collagen 175 and the matrix substances, leaving behind the insoluble e l a s t i n protein. The procedure i s repeated 6 to 8 times, with fresh changes of d i s t i l l e d water, to ensure a clean preparation. Tissue thus prepared can be stored f o r an i n d e f i n i t e period of time with occasional s t e r i l z a t i o n by autoclaving. The evaluation of t h i s p u r i f i c a t i o n procedure has been presented i n chapter 2. _b_ The experimental stacje The major piece of apparatus used i n the experiment, along with the r e l a t i v e positions of the various components, i s shown in figure 7.2..The stage was assembled from glass s l i d e s glued together with epoxy adhesive. The f l e x i b l e glass rod, which served as a microforce transducer, was made by pulling a 5mm diameter s o l i d . g l a s s rod ever a bunsen burner flame u n t i l the desired diameter of 80 to lOOum was. obtained. Dow Corning high vaccum grease was applied between the movable glass s l i d e and the stage to smooth out the manipulations during the experiment and to keep the extensions fixed at the desired pcint. The temperature control system was the same as the one described in chapter 5. Jc_ Preparation of expericental specimen A s t r i p of dried p u r i f i e d e l a s t i n (approximately 2mm X 5mm in dimension) was mounted d i r e c t l y onto the experimental stage by anchoring one end to the f l e x i b l e glass rod and the other end to the movable glass s l i d e using rubber 176 F i g u r e . 7 . 2 : T h e e x p e r i m e n t a l ______ D i a g r a m o f t h e e x p e r i m e n t a l s t a g e u s e d f o r f o r c e , e x t e n s i o n , a n d r e t a r d a t i o n m e a s u r e m e n t s . T h e e l a s t i n f i b r e , E , w a s e x t e n d e d b y m a n i p u l a t i n g t h e m o v a b l e s l i d e , S , a n d t h e e x t e n s i o n w a s m o n i t o r e d b y m e a s u r i n g t h e d i s t a n c e b e t w e e n t w o l a n d m a r k s o n t h e e l a s t i n f i b r e . T h e f o r c e e x p e r i e n c e d b y t h e e l a s t i n f i b r e w a s c a l c u l a t e d f r o m t h e m e a s u r e m e n t o f t h e d e f l e c t i o n , d , o f t h e g l a s s r o d , G , r e l a t i v e t o a f i x e d m a r k e r , M . 177 178 cement. D i s t i l l e d water was then added to the preparation, and the e l a s t i n fibres teased away, under a dissecting microscope with fine forceps, u n t i l a single connecting f i b r e was l e f t i n t a c t between the glass rod and the movable s l i d e (figure 7.2).. The preparation was then mounted between crossed polars, on a Wild M21 polarizing microscope, and the experiment was conducted by the manipulation of the mounted specimen at 300X magnification. The f i b r e was strained in a random steps, by s h i f t i n g the movable glass s l i d e , and the values f o r the extension, force, and retardation, were obtained as described below. (d) Measurement cf s t r a i n Two distinguishing marks were noted i n the unstrained state and the distance between them was calibrated using a F i l a r micrometer.. The separating distance was evaluated at each extension step, and the elongation could then be represented as an absolute value, cr as an extension r a t i o of the i n i t i a l length. _e_ Measurement of force The manipulation of the e l a s t i n f i b r e , which was done by moving the glass s l i d e , exerted an extending force on the e l a s t i n f i b r e which cculd be guantitated by measuring the def l e c t i o n of the f l e x i b l e glass rod with reference to the fixed marker (figure 7.2), using a F i l a r micrometer.. This 179 value could then be converted to an absolute force, F, by u t i l i z i n g the equation for the bending of a beam: F=d (3EI/13) 7.20 Where d i s the deflection of the glass f i b r e , 1 i s the length of the glass f i b r e (usually around 1.3 to 1.5 cm), and I i s the second moment of area, which for a rod of c i r c u l a r cross- section of radius, r, takes the form: I=TTr*/4 7.21 The value of E, which i s the Young's modulus cf glass, was obtained by measuring the deflection of a macroscopic glass rod, when experiencing a bending force from several c a l i b r a t e d weights. Substituting these values for the deflection and force into eguation 7.20 along with the dimension data, and solving for E gave a value of 6.2 X 10*ONm-2 f o r the Young's modulus of glass. The experimentally obtained force could then be represented as either the nominal stress (force/unit unstrained cross-sectional area) or the true stress (force/unit strained cross-sectional area). To insure against the presence of erroneous values r e s u l t i n g from the use of th i s method, one of the micro-force transducer glass rods was cal i b r a t e d by hanging known weights on i t ' s end and measuring the d e f l e c t i o n s . These deflections were always within 2% of the deflections predicted by use of eguations 7.20 and 7.21.. Jf1 Calculation cf cross-sectional area The unstrained diameters were measured with the F i l a r 180 micrometer and the cross-sectional areas were calculated by assuming a c i r c u l a r cross-section. The cress-sectional areas for the strained f i b r e s were calculated from the measured length change by assuming that the volume of the f i b r e s remained constant with extension. JLal Measurement of birefringence The specimen on the polarizing microscope was orientated between crossed polars (polarizer at 0°, analyzer at 90°) with i t ' s long axis at +45° to the polarizer (positive angles being counter-clockwise). Illumination was by a 100watt quartz lamp, which along with an interference f i l t e r provided a green monochromatic l i g h t (546nm) source. The retardations were measured, after each force- extension measurement, at the center of the f i b r e using a 1/30 wavelength Zeiss rotary compensator. This value of retardation was divided by the o p t i c a l path length (taken to be the diameter at each extension step) to give the value for birefringence. Jh_ Errors Since i t i s hard to evaluate the errors involved i n dealing with a system such as the one u t i l i z e d i n t h i s study, a s t a t i s t i c a l approach was thought to be more f e a s i b l e , and accordingly, the relevant s t a t i s t i c a l parameters are presented with the r e s u l t s . In general, the excellent r e p r o d u c i b i l i t y of the data and the minimum scatter, i s taken to be a good 181 indicator of the systems r e l i a b i l i t y . D_ Physical Properties of S J J__le E l a s t i n Fibres Ja_ General c h a r a c t e r i s t i c s The mechanical behaviour of single e l a s t i n f i b r e s was observed to be completely e l a s t i c with no observable hysteresis or change with r e p e t i t i v e cycling of the s t r a i n . T y p i c a l l y , the f i b r e s could be elongated to 100%-200% of t h e i r i n i t i a l length (X = 2 tc 3) before breaking. The cause of the f a i l u r e could not be determined with certainty. I t can only be stated that there was no observable p l a s t i c flew before f a i l u r e . The f a i l u r e i t s e l f occured either as a result of a breakage of the strained portion under observation, or a breaking away of the f i b r e from the residual bundles of e l a s t i n which were used for anchoring the preparation. The t e n s i l e strength i s probably i n the range of 10&Nm-2. IhL il§chanica_ properties and the derivation of Mc Eguation 7.6 indicates that i f the data obtained for the mechanical properties of single e l a s t i n fibres i s plotted as a graph of nominal stress versus ( X - X - 2 ) , i t should y i e l d a straight l i n e of zero intercept and a slope egual to Gv^/3. Figure 7.3,a, shows the mechanical data f o r eight single e l a s t i n f i b r e s in d i s t i l l e d water at 24°C. I t i s evident that the rel a t i o n s h i p obeys the prediction of a straight l i n e upto }\ =2 (an extension of 100%) .. Beyond t h i s extension, however, 1 8 2 the experimental points deviate from the straight l i n e , seen as an upturn i n the graph. This deviation i s the resu l t of the non-Gaussian properties of the network, which w i l l be analyzed in d e t a i l l a t e r . Fcr the moment, the analysis w i l l be r e s t r i c t e d to the linear portion of the s t r e s s - s t r a i n r e l a t i o n s h i p . The e l a s t i c modulus, G, obtained from the treatment of the data according to eguation 7.8, has a value of 4.1 X 105Nm-2. This was calculated from the slope of the line a r portion of the plot (figure 7.3,a), using a value of v^O.65 (Gosline 1978). .Similarly, the value of Mc, which i s the molecular weight cf the chain between cr o s s - l i n k s , was evaluated from eguation 7.10, the value of the e l a s t i c modulus, and ^ = 1.33. .This gave a value of 7,100 f o r the molecular weight of the chain between cr o s s - l i n k s . This use of eguation 7.10 for the calc u l a t i o n of Mc assumes id e a l tetra-functional c r o s s - l i n k s . This i s obviously an o v e r s i m p l i f i c a t i o n since, i n r e a l i t y , imperfections such as loose ends etc. w i l l be present in the network. It i s possible tc correct for these imperfections, assuming randomly di s t r i b u t e d c r o s s - l i n k i n g , by u t i l i z i n g a modification of eguation 7.10, according tc (Treloar 1975): G= f RT (1-2MC/M) /Mc 7.22 Where M i s the molecular weight cf the polymer before cross- l i n k i n g . This relationship cannot, in the s t r i c t e s t sense, be applied to e l a s t i n since the cross-linking s i t e s on the precursor protein, tropoelastin, are not randomly distributed 183 r_±2ii£!__Z___ Physical __o__rtj.es of single e l a s t i n _______. ,(A) Graph of the force extension data for single e l a s t i n fibres plotted according to equation 7.6. The l i n e a r regression, through the f i r s t 30 points, has a cor r e l a t i o n coeffecient r=0.91.. (B) Graph of the Birefringence-stress data for single e l a s t i n f i b r e s plotted according to eguation 7.12. The l i n e a r regression, through the f i r s t 30 points, has a correlation coeffecient r=0.88. 1814 185 but, rather, cccur in d i s t i n c t locations on the tropoelastin molecule (Sandberg et^al.. 1972). Nevertheless, the use of eguation 7.22 should give an estimate of the lower l i m i t of Mc. U t i l i z i n g the value obtained above, of 7100 for Mc, and eguation 7.22, along with a value of 72,000 for the molecular weight of the precursor tropoelastin (Sandberg 1976), gives a corrected molecular weight of 6000 for the chain between cr o s s l i n k s . Hence the data i s consistent with a Mc value i n the range of 7100 to 6000 g/mcle. This r e s u l t i s i n good agreement with the expected values calculated from the known biochemical composition and the cross-linking p r o f i l e s of insoluble e l a s t i n (Gray e t . a l . 1973) . lc) Photoelasticity According to eguation 7. 12, a plot of birefringence versus stress, should give a straight l i n e of slope C . Figure 7.3,b, represents the data for eight single e l a s t i c f i b r e s . As with the st r e s s - s t r a i n r e l a t i o n s h i p , the t h e o r e t i c a l r e l a t i o n s h i p i s followed to a certain point, aft e r which i t deviates towards the stress axis, with the birefringence approaching i t ' s ultimate value. This departure from the theory can again be explained in terms of the non-Gaussian properties of the e l a s t i n network: the f i n i t e length of the chain between cross-links i s responsible f c r t h i s non-Gaussian s h i f t , with the assymptotic l i m i t representing the state of 's' l i n k s in f u l l alignment. The. existence of th i s common causal element, i s supported by the observation that both the 186 Table.7 . 1: Kinetic theory parameters for e-lasti CO o o >— ra cn o o o o n CM E E O n o o r-H i — i X X o CO —t cvj 4- O t- ID c —- U —- 0 s-E 4- U o CU c 01 + J QJ to - C 0) S- O ) 2 CU •— -4-> ft3 U •— +-» •M C C L CU O - CU 4-s- CU *J o I to cu» M x: f— o o 187 s t r e s s - s t r a i n graph (figure 7.3,a) and the stress- birefringence graph (figure 7.3rb) enter the non-Gaussian region at the same l e v e l cf extension ( X = 2 ) . Concentrating on the li n e a r portion of t h i s graph (figure 7. 3,b), the slope of t h i s relationship yields a value for C' of 1.0 X lO-^mZN-1. U t i l i z i n g t h i s value of C and a value of n=1.55, i t i s possible to solve for the p o l a r i z a b i l i t y of the random link i ^ r ^ z ) ° Doing t h i s one obtains a value of 2.84 x 10- 3Om3 for the p o l a r i z a b i l i t y of the random li n k at 24°C. The value calculated for the st r e s s - o p t i c a l coeffecient, C 1, i s comparable to the resu l t s obtained by Weis-Fogh (196 1b) for the invertebrate elastomer, r e s i l i n (1.0 X 10- 9m 2N _ l for e l a s t i n as compared to 1.1 X 10- 9m 2N _ l for r e s i l i n ) . This i s not surprising since they are both protein elastomers and would, therefore, be expected to have similar values for the average r e f r a c t i v e index and the p o l a r i z a b i l i t y of the random l i n k . The k i n e t i c theory parameters derived f or e l a s t i n are summarized in figure 7. 1. . I t should be mentioned that the data for e l a s t i n birefringence extrapolate to a positive birefringence at zero stress. This i s consistent with the results obtained for unstrained single e l a s t i n f i b r e s , which appear to have a small residual birefringence of about 2.4 X 10~*. This r e s i d u a l birefringence has been shown to arise from the r e f r a c t i v e index difference at the water/fibre interface. The "true" birefringence of the unstressed f i b r e i s indistinguishable from zero (Aaron and Gosline 1980).. 138 Jdj_ Temperature dependence of the o p t i c a l anisotropy Eguation 7.12 alsc states that the s t r e s s - o p t i c a l coeffecient, C , should be inversely proportional to the absolute temperature, T, assuming that the mean r e f r a c t i v e index and the p o l a r i z a b i l i t y of the random link remain unchanged. Figure 7.4,a, shows the experimentally obtained r e s u l t s for the temperature dependence of C*. It seems that the predictions of the k i n e t i c theory (figure 7.4,a, dashed line) are followed q u a l i t a t i v e l y but the decrease with temperature i s larger than that which would be predicted by eguaticn 7.12. There are two possible sources for t h i s deviation. F i r s t , since e l a s t i n decreases i t ' s volume with increasing temperature (Gosline 1978), the mean r e f r a c t i v e index i s expected to increase with temperature. However, the extent of th i s increase i s small, and in any case increasing the re f r a c t i v e index should increase the value of the stress- o p t i c a l coeffecient. Second, i t i s possible that the p o l a r i z a b i l i t y of the random link i°<^-°<2) changes with temperature, which could account for the large decrease i n C . Figure 7.4,b (open c i r c l e s ) , shows the value for the p o l a r i z a b i l i t y of the random l i n k assuming that n remains unchanged with temperature. I t i s evident that the p o l a r i z a b i l i t y of the random l i n k decreases by approximately 30% over the temperature range studied. The implication of th i s change i s discussed below. 189 F i g u r e . 7 _ 4 : Temperature dependence of photoelasticity. (A) Graph of the s t r e s s - o p t i c a l coeffecient, C , versus 1/T. ( f i l l e d c i r c l e s ) : experimental points. The linear regression has a co r r e l a t i o n coeffecient of r = 0 . 9 2 . (dashed l i n e ) : expected relationship for the temperature dependence of C* calculated from eguation 7 . 1 2 . (E) (open c i r c l e s ) : dependence of the random l i n k anisotropy on temperature. ( f i l l e d c i r c l e s ) : the same data plotted as an Arrhenius relationship according to eguation 7 . 2 3 . . The activation energy calculated from the slope of the linear regression ( r = C 9 4 ) has a value of 1 . 6 kcal/mole. . 190 F i g u r e . 7 . U . 2 - 3.0 12 3.4 3.5 10>K 6 — 1 — 1 1 i 3.0 12 3.4 3.6 3.8 i a 3 /°K 191 As pointed out before, when dealing with r e a l polymer chains, the presence of energy barriers to bond rotation implies that a number of chemical bends are required to exhibit the properties of a random l i n k . This also implies that as these enerqy barriers are overcome with an increase i n temperature, the number of bonds needed to give a random l i n k should decrease. Hence the length of t h i s 'functional* l i n k and consequently the polymer chain dimensions are expected to be temperature dependent.. Studies on the temperature dependence of the o p t i c a l properties cf polymers other than e l a s t i n , have shown that the o p t i c a l anisotropy of the random l i n k usually decreases with increasing temperature (Saunders 1957), and t h i s decrease i s thought to r e f l e c t a reduction i n the conformational r e s t r i c t i o n s cn the molecules i n the network, as discussed above.. It has been shown that the variation of the l i n k anisotropy with temperature i s consistent with an Arrhenius type relationship of the form (Morgan and Treloar 1972) : (°<r*_.)= Aexp(E^/RT) 7.23 Where E^ i s an activation energy . Hence i t i s possible tc obtain the value of E froa a plot of l n H , - ^ versus 1/T. The value of the a c t i v a t i o n energy should r e f l e c t the magnitude of the energy barriers of the r e s t r i c t i o n s on the polymer chains. A plot of the e l a s t i n data for the l i n k anisotropy as a function of temperature i s shown i n figure 7.4,b ( f i l l e d c i r c l e s ) . From the slope of the graph one obtains a value of 192 1.6 kcal/mole for the activation energy. This value i s comparable to the value obtained from nuclear magnetic resonance data for e l a s t i n peptides (Urry e t . a l . 1978d). The magnitude of t h i s activation energy i s too small to represent the melting or the formation of stable secondary structures, since the ac t i v a t i o n energies for these systems would be expected to have values in the range of 15 to 20 kcal/mole (Fraser and McCrae 1973). It i s more plausible that t h i s value, of 1.6 kcal/mole, i s representative of the t o r s i o n a l energy barr i e r s about the cf*—CO and C^—N bonds of the polypeptide backbone. This explanation i s consistent with the t h e o r e t i c a l analysis of random polypeptides presented by Brant and Flory (1965 a and b). _(e]_ Non-Gaussian properties of the e l a s t i n network The non-Gaussian properties of the e l a s t i n network were analyzed graphically by comparing the experimental results to the curves generated according to eguations 7.13 to 7.15 for various values of s, which i s the number of random l i n k s between cross - l i n k s . Figure 7.5,a and 7.5,b, presents t h i s evaluation for the non-Gaussian mechanical and photoelastic properties of polymer networks. In each case the s o l i d l i n e s represent the experimental data obtained for single e l a s t i n f i b r e s , reduced to the unswollen form according to eguations 7.16 to 7.19. These lines were obtained by a least sguares f i t of the e l a s t i n data to regressions upto a tenth degree polynomial. The mechanical and photoelastic data were found t c 193 Figure.7.5 : Non-Gaussian properties of the e l a s t i n network. (A) Analysis of the non-Gaussian mechanical properties. The th e o r e t i c a l curves (open c i r c l e s ) were generated by evaluating eguation 7.13 for various values of s. The f i l l e d c i r c l e s represent the e l a s t i n data f i t to a seventh degree polynomial: y= -0.15 X 109 +0.62 X 10*x -0.11 X 10io X2 +0.11 X 10io x3 -0.65 X 10«x* +0.22 X 10^x5 0.41 X 108x6 +0.32 X 107x*. The s o l i d v e r t i c a l bar represents t standard deviation. (B) Analysis of the non-Gaussian photoelastic properties. The the o r e t i c a l curves (open c i r c l e s ) were generated by evaluating eguaticns 7. .14 and 7.15 for various values of s, using N=1.0 X 10 2 8chains/m 3 and H«-*_) =2.84 X 10-3o m3. _he f i l l e d c i r c l e s represent the e l a s t i n data f i t to a fourth degree polynomial: y= 0.33 X 10~3 +0.82 X 10-«x +0.36 X 10-15x2 -0.2 X 1 0 - 2 i x 3 +0.26 X 10-z«x*. The s o l i d v e r t i c a l bar represents + standard deviation. 194 • F i g u r e . 7 , 5 . 6 r 5 10 A f /NkT 195 have the best f i t to seventh and fourth degree polynomials respectively (see legend for figure 7.5). The graphical evaluation in both cases yi e l d s a value of approximately 10, for the number of random l i n k s , s, between cross-links. As indicated above, the value cf the e l a s t i c modulus, G, i s consistent with a molecular weight between cross-links of 6,000 to 7,100 g/mole. Since the average residue weight for e l a s t i n i s about 84.5 g/mole (Sandberg 1976), t h i s amounts to 71 to 84 amino acid residues between cr o s s - l i n k s . Furthermore, since the analysis of the non-Gaussian behaviour indicated that 10 random l i n k s were present between c r o s s - l i n k s , dividing the number of amino acid residues between cross-links by 10 gives a value of 7.1 to 8.4 amino acids per random l i n k . That i s , i t takes about 7 to 8 amine acid residues to exhibit the properties of the s t a t i s t i c a l , freely rotating links which form the basis of the kinetic theory relationships. The v a l i d i t y of these numbers obtained from the properties of the e l a s t i n network w i l l be evaluated in the next chapter. J i Conclusions This chapter has presented convincing evidence that the properties of the e l a s t i n network are best described in terms of the k i n e t i c theory r e l a t i o s h i p s derived for entropy elastomers. Furthermore, since these theories of rubber e l a s t i c i t y are based on an assumption of a random network conformation, i t i s reasonable to state that the protein chains which make up the e l a s t i n f i b r e s are themselves devoid 196 of any stable secondary structures. This conclusion i s in agreement with the results of birefringence (Aaron and Gosline 1980) and nuclear magnetic resonance studies (Fleming e t ^ a l ^ 1980, Aaron et^al.. 1980) of the e l a s t i n network. The evaluation of the activation energy for the network • r e s t r i c t i o n s ' indicates that, under normal conditions, the —CO and the C*—N t o r s i o n a l angles are the major determinants of protein random-coil dimensions, as proposed previously (Schimmel and Flory 1968, M i l l e r and Goebel 1968). c 197 _____________ A __________ TEST FCR ELASTIN ____________ A_ Introduction Having obtained some 'concrete* numbers from the analysis of the physical properties of e l a s t i n , according to the ki n e t i c theory of rubber e l a s t i c i t y , i t i s tempting to extrapolate from the ch a r a c t e r i s t i c s of the e l a s t i n protein to other proteins in the random c o i l conformation. In order to do so one must f i r s t define the 'char a c t e r i s t i c variable' which i s to be predicted using the experimentally obtained values (and manipulations of these values) , and i d e a l l y one must then stumble across some l i t e r a t u r e that has 'observed values' for the same variable, preferably attained through a d i f f e r e n t technique of experimental analysis. The r e s u l t s of t h i s comparison should allow one to evaluate the assumption of a random conformation for e l a s t i n . B_ Characterization of Random Proteins _a_ The measurable dimension I f a person i s qiven an object and asked to describe i t ' s physical shape, he w i l l usually respond with the appropriate adjective such as round, square, oblcng, weird! etc. If asked to be s p e c i f i c , this person might then proceed to describe their dimensions i n the usual terms l i k e length, width, radius, a x i a l r a t i o , but he probably w i l l not make any progress with that 'weird' object. Random c o i l e d polymers 198 would be c l a s s i f i e d by t h i s frustrated person as belonging to the 'weird' category.. In practice however, there i s a dimension that can be u t i l i z e d to describe a random c o i l e d polymer. . This dimension, as you know, i s referred to as the root-mean-sguare (r.m.s.) distance, <r 2> 1/ 2.. But what does thi s r.m.s. value represent in terms of a measurable dimension? Consider an i d e a l gas molecule located at the o r i g i n at time t=0. Suppose that the movement of the molecule can occur in any d i r e c t i o n (randomly) i n discrete steps of length, 1. If the molecule moves a steps per second, then after b seconds i t w i l l have moved s number of steps. Where s=ab. He-evaluation of the molecules position a f t e r s steps w i l l show that i t has been displaced away from the o r i g i n by a distance characterized by the linear vector between the o r i g i n and t h i s new p o s i t i o n . This vector represents the end-to-end distance, <r> (figure 8.1a).. In transforming this analogy to r e a l polymers one has tc simply replace the spaces between two sucessive positions cf the gas molecules by an * id e a l ' l i n k , made up of a number of chemical bonds, of length, 1. This allows i t ' s characterization by the measurable dimension <r>, which now represents the distance between the two ends of the molecule (figure 8.1b). The clue, as to the conceptual framework that forms the analysis of 'random walk* systems, can be found i n the d e f i n i t i o n of the adjective 'random'.. The Miriam-Webster dictionary describes the word random in two ways. One i s to 199 £igi?rei8_.Jj_ The random walk. (A) A 10 step random walk for a gas molecule s t a r t i n g at the o r i g i n and moving in d i s t i n c t steps of length 1 . <r> i s the end-to-end distance. (B) Replacing the distance between two successive positions by an 'i d e a l ' link of length 1, allows the extension of the Gaussian derivations f o r random walks to r e a l polymers. . 2 0 0 - < 20 1 think of random as being haphazard. The other i s to think of i t i n terms cf chance. It i s this second d e f i n i t i o n of random that describes i t i n terms of a 1 probability of observing an occurrence' , which allows a p r a c t i c a l method for describing the random walk. Evaluation of the problem through Gaussian s t a t i s t i c s yields a f a i r l y simple res u l t which characterizes the end-to-end distance, <r 2> 1/ 2, in terms of the number of l i n k s , s, and the length of the l i n k , 1 , as (Flory 1 9 5 3 ) : <r 2>=sl 2 8 . 1 Jb__ Accounting for the non-ideality The above derivations for random c o i l s assumes an i d e a l system. This obviously does not occur i n re a l polymers, which because cf th e i r f i n i t e volume, w i l l display an 'excluded volume e f f e c t ' . This e s s e n t i a l l y states that no two r e a l molecules can occupy the same volume element. The r e s u l t of t h i s e f f e c t i s tc expand the domain of the random c o i l by a factor oL , which accounts for the ncn-ideality of the system ( i . e . polymer-polymer in t e r a c t i o n s , and polymer-solvent interactions) : <r2> V 2 = < K < r 2 ^ V 2 8 . 2 where r 0 and r are the unperturbed and actual dimensions of the molecule respectively. In the case of a theta solvent, where the non-ideality of the polymer-solvent interactions balances out the non-ideality of the polymer-polymer i n t e r a c t i c n s , <=< i s egual tc unity, and the above eguation 202 reduces to eguation 8.1. C_ ______ ______ From Viscosity (a) Viscosity of random c o i l s There are present in the l i t e r a t u r e a vast number of papers that deal with the visccsuty of random hydrocarbon polymers (Flory 1949, Fox ______ 1951, Kurata ______ 1960, Kurata and Stockmayer 1963, Stockmayer and Fixman 1963). These studies have developed t h e o r e t i c a l relationships and have obtained experimental parameters for the prediction of random- c o i l dimensions. Only recently have there been any attempts made to extend these vi s c o s i t y relationships to the study of random protein polymers (Brant and Flory 1965, a and b, Tanford ______ 1966,1967, Reisner and Eowe 1969), and the resu l t s indicate the a p p l i c a b i l i t y of t h i s type of approach to proteins - Tanford e t . a l . (1966) have published studies on the vis c o s i t y of proteins i n 6M GuHCl, where these polymers behave as random c o i l s . Applying the appropriate realtionships, these authors have evaluated the r.m.s. distances f o r a number of proteins. This i s fortunate since, as mentioned before, i t w i l l allow a comparison for the predicted values obtained from the properties of the e l a s t i n protein. However, i n order to ensure that t h i s comparison takes place between eguivalent variables, a number of corrections must be made to the published values. . 20 3 (b) Correction of visccsit_y values The eguation that describes the molecular weight dependence of the i n t r i n s i c v i s c o s i t y for l i n e a r polymers takes the form (Yang 1961) : £ n ] D =KM* 8.3 Where [ n ] Q i s the i n t r i n s i c v i s c o s i t y , K i s an empirical constant for a given polymer-sclvent system, M i s the molecular weight of the polymer (or the number of residues i n a protein), and X i s the exponent that describes the polymer conformation, having a value of 0.5 for random polymers in a theta solvent, and a value greater than 1 for rod-like proteins (Tanford 1961). In the case of proteins, i t i s convenient to deal with them i n terms of the number of residues as opposed to the actual mclecular weight, since d i f f e r e n t proteins have dif f e r e n t average residue weights. Doing t h i s , Tanford e t . a l . . (1966, 1967) assigned a values of 0.684 and 0.67 for K and X respectively. The value of 0.67 obtained for X indicates that guanidine hydrochloride i s a good solvent for proteins. This should result in the expansion of the randcm-coil domain and, hence, i t i s necessary that the expansion coeffecient, <Xs , be evaluated and corrected for before making the comparison to the predictions from the e l a s t i n network. I have chosen to do t h i s by manipulating the vi s c o s i t y data by an alternate analysis. The i n t r i n s i c v i s c o s i t y of random-coils can be described by (Kurata and Stockmayer 1963): 20 4 [n]=KMV 2<* 3 8.4 In t h i s formulation -» i s taken to be egual to 0.5, and any deviations due to solvent effects i s accounted for i n the expansion coeffecient °<- . This forms the basis for the evaluation of c< according to: o< 3 = [n]/[n]_ 8.5 Hence i f one can evaluate K, i t i s possible to obtain a t h e o r e t i c a l value for [ n ]_ by assuming <x =1. One can then u t i l i z e the actual value measured for the i n t r i n s i c v i s c o s i t y , £n], the calculated value for [ n ] , and equation 8.5, to get a value for <K . The evaluation of K for the Tanford data was done by the method of Stockmayer and Fixman (1963), which gave a value of 1.3 cc/gm for t h i s parameter (see appendix 3). The results of the subseguent calculations are presented i n table 8.1, along with the corrected values for the end-tc-end distance obtained by Tanford e t _ a l _ (1966, 1967). It should be mentioned that the high values obtained for the expansion coeffecient, <X , are consistent with 6molar GuHCl being a good solvent. Furthermore, the molecular weight dependence of o< i s also consistent with the results expected from the theore t i c a l treatment for random polymers (Flory 1953). 0". Predictions From The E l a s t i n Network _a_ Calculation of s Eguation 8.1 states that i f the number of random l i n k s , 205 Table. 8..1.: Predict ions for random-coil proteins. PREP1CTI0HS FOR THE PIKENS10HS OF RAWDOH-COIL PR0TE1WS. nusber of residues expansion coeffecient: cr corrected < r V ' V preaicwa-r b r">- ~A" c <r*> b 1 / 2 * r V / 2 o b s . c Insulin 26 44 0.97 45 38 41 .84 .92 rlbonuclease 124 101 1.03 98 83 90 .85 .92 hemoglobin 144 112 1.07 106 89 97 .84 .92 myoglobin 153 120 1.09 1.10 22 100 .84 .91 J-Iactoglobulln 162 126 1.11 113 95 103 .84 .91 chymotrypslnogen 242 148 1.10 134 116 126 .87 .94 aldolase 365 189 1.12 168 142 154 .85 .92 serum albumin 627 258 1.17 220 1S6 202 .85 .92 thyreoglobulin 1500 401 1.18 341 288 313 .85 .92 myosin 1790 443 1.19 372 314 342 .84 .92 "data from Tanford et . a l 1966, 1967. ''for 7aa/random li n k . c f o r 8aa/randoni l i n k . 206 and the length of the l i n k i s known, then, i t i s possible to predict a value for the end-to-end distance, <r 2> 1/ 2. The analysis of the properties of the e l a s t i n network indicated that between 7 and 8 amino acid residues are reguired to give a •functional' random l i n k . Hence, i t i s possible to obtain a value of s for any given protein by dividing the the number of residues i n the protein by the number of amino acid residues per l i n k ( i _ e _ 7 to 8). In trying to evaluate a value for the length of the l i n k , 1 , I have chosen a q u a l i t a t i v e approach which i s based on the t h e o r e t i c a l derivations for the dimensions cf random proteins. _b_ Calculation of 1 Flory and his co-workers have dealt with the t h e o r e t i c a l evaluation of random proteins and have evaluated the energetics of random protein dimensions, which usually resulted i n energy minima for bond angles at ^=270, (Ĵ  = 120 (Schimmel and Fiery 1968, M i l l e r ______ 1967, Miller and Goebel 1968). Although the actual bend angles present i n random polymers w i l l be distributed over a range of values, the value at the energetic minimum should r e f l e c t an 'average' for the d i s t r i b u t i o n . . I have therefore used this value (^=270, (1̂  = 120) to calculate the displacement along the random lin k using the relationships provided by Schellman and Schellman (1 963). Doing th i s I obtained values of 19.8A° and 23.4A<> for the length, 1, cf a random li n k made up of 7 and 8 amino acids respectively, which allowed me t c predict values 20 7 of r.m.s. distances for various proteins using eguation 8.1 and a value cf 1 f or c{ • This should permit a v a l i d comparison to the observed values (Tanford e t . a l . 1966, 1967) since they have been corrected fcr the expansion coeffecient as described above. |. Discussion and Conclusions The r e s u l t s , which are summarized i n table 8.1 and figure 8.3, indicate that the predictions obtained from the properties of the e l a s t i n network are in excellent agreement, within l i m i t s of the uncertainties inherent i n t h e i r c a l c u l a t i o n , with both the t h e o r e t i c a l expectations from the s t a t i s t i c a l mechanical treatment of peptide conformation (Miller and Goebel 1968) (figure 8.3,a) and the experimental observations (figure 8.3,b) on random coiled proteins. The fact that the predictions from the e l a s t i n data are lower than the expected values can be reasonably attributed to the high glycine content of t h i s protein. Glycine makes up almost a t h i r d of the amine acid content of e l a s t i n (Sandberg 1976), and one would expect t h i s to r e s u l t i n a reduction of the r e s t r i c t i o n s cn the protein chain mobilities i n the network. This would lower the number of amino acid residues reguired to give a random l i n k , as compared to Host proteins which contain lesser amounts of glycine. I t i s reasonable to expect that more than 8 amino acid residues would be reguired to give a random li n k f c r proteins which do not exhibit such extremes i n amino acid composition as e l a s t i n . This would have the e f f e c t 208 Figure.8.2: Predictions for the dimensions of randcm-coil protein, Boot-mean-sguare distance for various proteins as a function of the number of amine acid residues. (a) Theoretical predictions from M i l l e r and Gcebel 1968. (b) Experimentally obtained values from Tanford e t _ a l . 1966 and 1.967, corrected for non-ideality (see Table 8.1). (c) Predictions from the e l a s t i n system according to eguation 8.1: (solid line) for 7aa/random l i n k , (broken line) for 8aa/random l i n k . 209 210 of increasing the value of 1, which i s the length of the random l i n k , resulting i n even closer agreement between the predicted and observed values. 21 1 Chapter. IX. . CONCLUSIONS^ Unlike most cf the bio-polymers known to us, there are a number of proteins that f u l f i l l their b i o l o g i c a l roles by being random. The elastomers R e s i l i n , Abductin and as shown i n t h i s thesis, E l a s t i n , belong to t h i s group of biomolecules. The b i o l o g i c a l function performed by these e l a s t i c proteins i s to antagonize the movements of muscles. Muscles can only contract, and because of t h i s there i s a need for an opposing mechanism that can restore the muscle to i t ' s functional state aft e r each contraction cycle. This antagonism can be provided by other muscles as i n s k e l e t a l movements, hydrostatic systems as i n many marine organisms, and passive e l a s t i c elements. The e l a s t i n proteins probably evolved i n response to t h i s l a s t need. Furthermore, muscles are r e s t r i c t e d i n t h e i r performance by t h e i r metabolic needs, and are generally characterized by high turnover. On the other hand, elastomers being passive mechanical components, are not r e s t r i c t e d by metabolic needs, and due to their low turnover the only metabolic cost to the organism i s that of synthesizing i t in the f i r s t place. Hence, i t seems plausible that the advantages of passive e l a s t i c i t y are r e a l i z e d i n the metabolic savings to the animal,.This describes t h e i r function. But as stated in the introduction to th i s t h e s i s , simply stating that proteins l i k e e l a s t i n are rubber-like does not t e l l us about the functioning mechanism. Over the years, several theories of e l a s t i c i t y have been proposed in an attempt to characterize the the e l a s t i c mechanism but only one theory. The Kinetic Theory of Rubber E l a s t i c i t y , has been able to account for and, in most cases predict, the macroscopic properties of elastomeric materials. Like most th e o r e t i c a l frameworks that are based on mathematical derivations the kinetic theory makes some very convenient and c r u c i a l assumptions, the most important of which i s that of a random conformation. It i s t h i s assumption which has prevented i t ' s general acceptance by protein chemists. The current thought in biochemistry and biopolymer research i s summarized by the statement—-"if i t has a primary seguence i t must have a t e r t i a r y structure". Although t h i s statement might be v a l i d for proteins in general, i t does not account for the exceptional c l a s s of protein elastomers. On the basis of t h i s statement a number cf people have stated that the approach adopted by the kinetic theory i s unacceptable since, i n their minds i t invokes the concept of 'phantom' chains and 'ideal' l i n k s . This b i t t e r p i l l of random conformation might be easier to swallow i f the phantom chains are restated i n terms of r e a l polymers to whom a l l conformations ARE EQUALLY ACCESSIBLE (WHICH IS THE SAME AS SAYING THAI THERE IS NO ONE STABLE conformation). This allows the mathematics of random walks to be applied to r e a l polymer networks that are devoid of stable secondary structures. In using the k i n e t i c theory relationships to explain the macroscopic properties of single e l a s t i n f i b r e s , I have also had to assume a random network conformation for el a s t i n . . T h i s assumption was tested using d i f f e r e n t methods of investigation (see summary figu r e , 9 . 1 ) . Viscosity experiments were used tc 213 f i g a r e . 9 . 1: Summarj f i g u r e f o r the t h e s i s . 2 1 4 Figure.9. 1 SUMMARY. SOLUBLE PEPTIDE BIREFRINGENCE SCANNING ELECTRON STUDIES: a . v i s c o s i t y . b. n . m . r . STUDY OF MICROSCOPY. SINGLE ELASTIN FIBRES. / / / / / RANDOM-COIL CONFORMATION FOR THE ELASTIN NETWORK. KINETIC THEORY RELATIONSHIPS: a . mechan i c a l p r o p e r t i e s . b. p h o t o e l a s t i c p r o p e r t i e s . c. n on -Gau s s i a n p r o p e r t i e s . PREDICTIONS FOR RANDOM-COIL PROTEINS. 215 evaluate the shape cf the soluble e l a s t i n . These studies did not support the presence of any rod-like structures. Polarized microscopy was used to test for molecular organization. The r e s u l t s indicated a random conformation. Nuclear magnetic resonance studies were used to investigate the mobility of the e l a s t i n network". The analysis showed that the e l a s t i n network i s characterized by rapid motions. Assimilating these r e s u l t s , one should be able to state guite confidently that the assumption of a random network for e l a s t i n i s j u s t i f i e d . The fact that e l a s t i n can serve as a model f o r random-coil proteins offers further support i n favour of t h i s assumption. In conclusion i t seems that the kinetic theory of rubber e l a s t i c i t y provides a valid t h e o r e t i c a l framework for the analysis of the entropic elastomeric properties of the rubber- l i k e proteins which, in general are characterized by random conformations. 2 1 6 APPENDIX.I: Thermoglasticity ___ Ther adynamic Relationships Since an e l a s t i c system, in i t ' s functional state, i s involved in the storage and dissapation of mechanical energy, i t i s possible to evaluate the basis cf t h i s mechanism by using thermodynamic relationships. According to the f i r s t law of thermodynamics, energy, E, can be converted from one form to another, but i t cannot be created or destroyed. Hence when considering an e l a s t i c sample and i t ' s surroundings, for a given deformation, dE i s egual to zero. I f , however, one considers only the sample, for a reve r s i b l e process one can write: dE=dg +dw A. 1 . 1 where g represents the heat evolved by the process, and w represents the work. Both dg and dw are considered to be positive when heat flows from the sample to the surroundings and work i s done b_ the sample on the surroundings, respectively. Eor a reversible process, the second law of thermodynamics defines the change i n entropy, S, i n terms of the heat and the absolute temperature, T, according to: dS = dg/T A. 1 . 2 Solving f c r dg, and substituting into eguation A . 1 . 1 , one gets: dE=TdS+dw., A. 1 . 3 Using these equations i t i s possible define a state function. 217 r e f e r r e d t o a s t h e H e l m h c l t z f r e e e n e r g y , F , a s : F = E - T S A . 1.4 A t c o n s t a n t t e m p e r a t u r e , p r e s s u r e , a n d v o l u m e , t h i s e g u a t i o n t a k e s t h e f o r m : d F = d E - T d S A . 1. 5 S u b s t i t u t i n g e g u a t i o n A . 1 . 3 i n t o t h e a b o v e g i v e s : d F = d w A . 1. 6 U t i l i z i n g e g u a t i o n s A . 1 . 2 a n d A . 1 . 6 o n e can w r i t e e g u a t i o n A . 1 . 5 a s : d w = d E - d g A . 1.7 T h i s e g u a t i o n f o r m s t h e b a s i s o f t h e t h e r m o d y n a m i c a n a l y s i s o f r e v e r s i b l e s y s t e m s , a n d i t i s p o s s i b l e , b y m e a n s o f t h e r m o d y n a m i c b o o k - k e e p i n g , t o e v a l u a t e t h e v a r i o u s c o m p o n e n t s o f a g i v e n p r o c e s s . I n t h e p a r t i c u l a r c a s e o f a m a t e r i a l s m e c h a n i c a l r e s p o n s e , i t a l l o w s o n e t o d e t e r m i n e t h e m a g n i t u d e s o f t h e c o n t r i b u t i o n t c t h e t o t a l r e t r a c t i v e f o r c e , f , f r o m t h e e n e r g y , f e , a n d t h e e n t r o p y , f s , c o m p o n e n t s : f = f e + f s A . 1. 8 JB1 T h e T h e r m o d y n a m i c E x p e r i m e n t T h e t h e r m o d y n a m i c e x p e r i m e n t c a n b e c a r r i e d o u t i n t w o w a y s : J . . T h e s a m p l e c a n b e i m m e r s e d i n a d i l u e n t , and k e e p i n g i t a t a f i x e d e x t e n s i o n , o n e c a n f o l l o w t h e c h a n g e i n t h e r e t r a c t i v e f o r c e , f , a s a f u n c t i o n o f t e m p e r a t u r e . T h e d a t a c a n t h e n b e e v a l u a t e d a c c o r d i n g t o : 218 f=fe + T (df/dT) ........ A. 1.9 2± The sample, i n a diluent, i s extended at a constant temperature, and the heat released, along with the work ( i.e_. the area under the force extension graph) i s monitored. The energy component i s then evaluated according to eguation A. 1.7. For the sake of convenience, the two methods outlined above w i l l be referred to as constant s t r a i n and constant temperature thermoelastic experiments, respectively. (C) Thermoelasticity. of Kinetic Elastomers With reference to the e l a s t i c i t y of materials, i f the mechanism responsible for the r e t r a c t i v e force consists of a system that involves the deformation of a r i g i d l a t t i c e (as i n the case of metals, collagen, keratin, and s i l k ) , where most of the energy i s stored i n the a l t e r a t i o n of bond lengths and o r b i t a l s , the restoring force arises from a change i n the i n t e r n a l energy component as indicated by the magnitude of energy component, f e , obtained from a thermodynamic experiment (fe/f=1). In the case of natural rubbers, however, i t has been shown that the s t r a i n energy i s stored as a decrease in the conformational entropy of the system. These materials exh i b i t a f s / f r a t i o cf approximately one, with the fe term being small. Materials of t h i s type are thought to conform to the k i n e t i c theory of rubber e l a s t i c i t y , which reguires a random, k i n e t i c a l l y agitated conformation at the molecular l e v e l (Treloar 1 S75) . 220 ____________ ___________ OF SOLUBLE ELASTIN BY PHOTOLYSIS. A_ Introduction Recently there has been a l o t cf i n t e r e s t i n the physical properties and the primary structure of the precursor protein, tropoelastin. This research has created a need for a convenient method to i s o l a t e the tropoelastin molecule which, upto now, has been isolated from l a t h y r i t i c animals. The use of a b i o l o g i c a l source carries with i t the usual problems of high expense and lew productivity. A paper by Foster e t . a l . (1975), where they used 2000 chicks to get 55gms of wet tissue which in turn yielded 35mg of tropoelastin, i l l u s t r a t e s t h i s point guite c l e a r l y . Hence i t seemed valuable to develop a procedure to i s o l a t e the precursor protein, from the f r e e l y a v a i l a b l e , insoluble e l a s t i n by chemical means. This chapter deals with a method that u t i l i z e s the absorption c h a r a c t e r i s t i c s of the e l a s t i n cross-links to induce t h e i r l y s i s and the subseguent release of the soluble peptides. Since t h i s procedure i s a reversal of the b i o l o g i c a l pathway that results in formation of the insoluble e l a s t i n i n the f i r s t place (by the l i n k i n g of tropoelastin into an insoluble network), i t should hopefully y i e l d a monomeric peptide that represents the precurscr protein. B_ Methodology ja) Rationale 221 The rationale for the use cf photolysis to cleave the cross-links of e l a s t i n i s based on a paper by Joussot-Dubien and Houdard (1968) who studied the cleavage of pyridinium rings by u l t r a - v i o l e t r a d i a t i o n . The relevance to the e l a s t i n c ross-links l i e s i n the f a c t that the (iso)desmosines are tetra-substituted pyridinium rings, as demonstrated by Thomas et_.a 1... (1963). This aspect of e l a s t i n chemistry led Baurain et.ah ( 1976, 19 77) to attempt the use of photolysis for the purpose of s o l u b i l i z i n g fibrous e l a s t i n . These authors were able to demonstrate a cleavage of the (iso)desmosine cross- l i n k s , but they cculd not i s o l a t e any soluble peptides from the i r preparations. The methology presented i n t h i s chapter u t i l i z e s an additional step which re s u l t s i n the release of soluble peptides from the photolysed e l a s t i n . The hypothetical reaction pathway for t h i s method i s shown i n figure A.2.1 (as based on the the work of Joussot- Dubien 1968 and Baurain 1976). The i r r a d i a t i o n of the cross- l i n k s with u l t r a - v i o l e t l i g h t (275-285nm) re s u l t s i n the cleavage of the single bond between the nitrogen and carbon 6 (figure A. 2.1). This product i s unstable at low ph and/or high temperature and can be broken down (figure A.2.1). The next step involves the cleavage of the unsaturated carbon bonds, using standard oxidative technigues, to give soluble peptides. This pathway i s e s s e n t i a l l y the same for both the desmosine and iso-desmosine as outlined i n figure A.2.1. The obvious guestion to ask at t h i s time i s : why not just oxidize the unsaturated cross-links without having to bother 222 Figure.A.2. 1: Photolysis of ________ (I) oxidation pathway for e l a s t i n . (II) photolysis of e l a s t i n followed by oxidation. (A: pathway for desmosines, E: pathway for isodesmosines.) 2 2 3 . II 224 with the photolysis? The reason for t h i s i s demonstrated by the pathway outlined in figure A.2 .1 . Seduction of the desmosine by dihydroborate followed by potassium permanganate oxidation r e s u l t s in the separation of the two cross-linked peptides (figure A. 2 . 1 ) . The same procedure, i f u t i l i z e d on the iso-desmosines, does not give sat i s f a c t o r y r e s u l t s , since the movement of the cross-l i n k i n g position from carbon* to carbon 6 prevents the release of the two peptides. As the tropoelastin molecule i s linked at about ten points along i t ' s length (Gray et_al_. 1 9 7 3 ) , i f any of these cross-links were to be an isc-desmosine, the peptides would be retained i n the insoluble network. It i s t h i s problem which i s cicumvented by the photolysis, allowing the combination of the two procedures to give soluble peptides. Jb_ Procedure E l a s t i n from ligament nuchae was pu r i f i e d by extraction with 0 .1 N NaOH at 100°C for 45 minutes (Lansing 1 9 5 2 ) . The resu l t i n g material was dried and ground to a f i n e powder and washed with copious guantaties of b o i l i n g d i s t i l l e d water,. 50Omg of t h i s powdered e l a s t i n was hydrated overnight i n 200ml of d i s t i l l e d water that had been adjusted to ph4.4 with 2N HC1. This solution was photolysed using an immersion type u l t r a v i o l e t lamp (Hanovia 450W, medium pressure, u.v. Model). A Corex f i l t e r was used to remove wavelenghts below 270nm to prevent the photolysis of peptide bends and, at the same time, provide adeguate i r r a d i a t i o n for the cleavage of the cross- 225 l i n k s which absorb at 275-285nm (Thcmas et. al._. 1963) . The samples were photolysed for 6 hours, at the end of which the s o l i d e l a s t i n was collected by centrifugation. I t was then s t i r r e d into a periodate-permanganate oxidation solution (Lemieux 1 955) at 48<>C for 24 hours. The mixture was centrifuged and the supernatant was car e f u l l y aspirated. The pre c i p i t a t e , which contained mostly insoluble e l a s t i n was discarded. The supernatant was dialyzed against running tap water for 24 hours, followed by d i a l y s i s against 4L of d i s t i l l e d water (1L X 24 hrs.) and ly o p h i l i z e d . The re s u l t i n g peptides had a clean white appearance. A control consisting of the oxidation step without photolysis was also conducted. C_. Results and Discussion Ja)_ Yield The amount of peptide recovered after the l a s t l y o p h i l i z a t i c n step was about 40mg/gm of o r i g i n a l dry weight. Although t h i s i s a substantial y i e l d as compared to the b i o l o g i c a l sources (Sykes and Partridge 1974), i t i s probably not the optimum y i e l d for t h i s procedure. Examination of the oxidative step (in retrospect) reveals a very c r u c i a l blunder. S p e c i f i c a l l y , soluble e l a s t i n peptides such as tropoelastin and alpha-elastin, undergo an inverse temperature t r a n s i t i o n at 3 7 0 C , which upon standing f o r 12 or more hours forms an i r r e v e r s i b l e p r e c i p i t a t e . As i s clear from the methods section, the oxidative step was carried out at 45°C f o r 24 226 hours, and i t i s possible that a major part of the soluble e l a s t i n s prepared i n t h i s manner precipitated out and were l o s t during the seperation of the insoluble e l a s t i n from the supernatant, which contained the remainder of the soluble proteins. Oxidation of the photclysed e l a s t i n at a lower temperature f o r longer periods should r e s u l t i n better y i e l d s . The oxidation of unphctolysed e l a s t i n gave no detectable r e s u l t s . Jb_ Characterization of the soluble peptides Molecular weight determination of the peptides was performed on a 15% pclyacrylimide-SDS gel. The results are presented in figure A.2.2. Much to my surprise, and delight, there appeared to be only one protein band at 66,000 molecular weight. This could mean that (a) the procedure yi e l d s a homogeneous peptide or (b) that there are other peptides released i n small guanities that could not be detected by the crude procedures used i n t h i s i n i t i a l characterization. This aspect has not been investigated f e l l y . Although the amino acid analyses has not been done, i t i s possible to make some predictions based on the work of D a v r i l e t . a l . , (1 577). It i s expected that the soluble protein should have a higher lysine content as compared tc insoluble e l a s t i n , due to the l i b e r a t i o n of a free lysine after the photolysis of the c r o s s - l i n k s . .In addition to t h i s , one would expect to have a lower content of tyrosine due to t h e i r destruction at the wavelength used for the photolysis, which i s i n the range of 2 2 7 the a b s o r p t i o n peak f c r t h i s amino a c i d . The r e s t of the amino a c i d s are not expected to be a l t e r e d . The branching c h a r a c t e r i s t i c s c f t h i s peptide have not been s t u d i e d as y e t , but t h i s deserves seme mention here. I f the p e p t i d e t u r n s out to be a branched polymer, e i t h e r due to incomplete p h o t o l y s i s or o x i d a t i o n , then t h i s method i s not u s e f u l f o r the production of p e p t i d e s , that can be more e a s i l y a t t a i n e d by other methods. But i f t h i s peptide t u r n s out to be an unbranched l i n e a r polymer i t might be i n t e r e s t i n g t o s p e c u l a t e on the i m p l i c a t i o n s . F i r s t , i t c o u l d be p o s s i b l e that t h i s p e p t i d e r e p r e s e n t s the p r e c u r s o r component of the i n s o l u b l e e l a s t i n p r o t e i n . Second, s i n c e the molecular weight cf t r o p o e l a s t i n i s about 7 2 , 0 0 0 as compared to the value Of 6 6 , 0 0 0 obtained f o r the p e p t i d e i n t h i s study, i t i m p l i e s a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of the t r o p o e l a s t i n p r o t e i n before i t ' s i n c o r p o r a t i o n i n t o the i n s o l u b l e s t a t e . T h i s i s supported by s t u d i e s which r e p o r t t h a t enzyme i n h i b i t o r s are necessary i n the b i o l o g i c a l i s o l a t i o n procedure to o b t a i n a 7 2 , 0 0 0 weight t r o p o e l a s t i n molecule (Foster e t . a l , 1 9 7 5 ) . In the absence of these i n h i b i t o r s the r e s u l t a n t product has a molecular weight of 6 6 , 0 0 0 (Sandberg 1 9 6 9 ) and has been shown to be missing a N-terminal peptide. In c o n c l u s i o n , the high y i e l d cf s o l u b l e peptides and the p o s s i b i l i t y that t h e y / i t might t u r n out to be the p r e c u r s o r p r o t e i n argues f o r the f u r t h e r development of t h i s technique f o r the i s o l a t i o n of e l a s t i n fraqments, to be followed up with 228 P i ^ f l E i - A ° 2.2: Molecular ______ cf __________ peptides. SDS-polyacrylimide gel electrophoresis results for the photolysis products: (o) molecular weight markers: Albumin (66,000), Ovalbumin (45,000), Trypsincgen (26,000), Beta- lactoglobulin (18,000), Lysozyme (14,000). (•) photolysis peptide.  2 3 0 farther characterization of the soluble products. 231 ' AEjoendix^IIIj. PEECICTIONS FOR TEOPGELASTIN VISCOSITY 7W The Relevant Equation The molecular weight dependence for the i n t r i n s i c v i s c o s i t y , £n], of unlranched, random pointers, can be stated in terms of an empirical constant, K, and the number of residues, M, as (Stockmayer and Kurata 1963): [n]=KMV 2 A.3.1 o This eguation applies to i d e a l polyners and polymers i n theta- solvent systems. A more useful derivation of t h i s eguation provided by Stockmayer and Fixman (1963) allows the characterization of the vi s c o s i t y i n any solvent, and takes the form: [n ]= KM V 2 + 0. 51<[BM A. 3.2 where i s a universal constant with a value of 2.1 X 10 23 (c.g.s.) and E i s defined according to: B=v2 (1-2XJ/VN A.3. 3 where v, i s the p a r t i a l s p e c i f i c volume of the polymer, V i s the molar volume of the solvent, N i s Avcgadro's number, and X is the Flory interaction parameter (Flory 1953). .implication to Tropoelastin According to equation A.3.2, a plot of [n]/M 1/ 2 versus M1/2, should give a straight l i n e with an intercept equal to K. This has been done for the data obtained for proteins (in a random c o i l conformation) by Tanford e t , a l . .  (1966, 1967) and was calculated to have a value of 1.3 cc/gm (figure A.3.1). 232 F__!LT2________ Viscosity of _ _ _ _ c j _ _ c c i l proteins. Plot of data frcm Tanford e t . a l . 1966 , according eguation A. 3. 2. . 2 3 3 F i g e r e . A . 3 . 1. 2 3 4 . T a b l e . A . 3, 1: P e e d i c t i o n f o r t r o p o e l a s t i n v i s c o s i t y . PREDICTIONS FOR TROPOELASTIN VISC0.T TY. T°C * V n B (10" 2 6) I n] cc/g 0 .C99 -2. 66 35 .48 10 .786 -3. 83 34. .42 20 .875 -5. 02 33. 33 30 .943 -5. 93 32. 50 40 1.01 -6. 80 31. 71 i 1977. 235 What makes t h i s manipulation useful i s that i t i s possible to obtain X values for the e l a s t i n protein from studies of network swelling (Gosline 1978). E l a s t i n decreases i t ' s volume with an increase in temperature and according to eguation A.3.2 and A.3.3,'this should lead tc a decrease i n B and a subsequent decrease in the i n t r i n s i c v i s c o s i t y . These eguations allow the prediction of the magnitude of t h i s decrease i n [n]. The re s u l t s cf the calculations for the i n t r i n s i c v i s c o s i t y of tropoelastin i n water, as a function of temperature, are presented in table A.3.1, using M egual to 850 (Sandberg 1976) , X values taken frcm Gosline (1977) and v equal to .725 (Partridge e t . a l . 1955). I t should be in t e r e s t i n g to compare the actual experimental r e s u l t s to these t h e o r e t i c a l predictions in order to support a random c o i l conformation for the tropoelastin molecule. 236 ____________ _V_L___I__ OF JROTEIN CONFORMATION In chapter 8 I have presented seme values for the parameters that characterize random proteins. Eeing encouraged by t h e i r apparent generality, I have ventured to devise an empirical method for the evaluation of protein conformation. Using the values of s and 1 (as presented in chapter 8) and eguation 8.1 i t should be possible to predict the end-to- end distance for any given protein made up of a given number of. residues. From this value one can calculate the radius of gyration, Eg (figure A.4.1), for the random molecule according to (Tanford 1961): Rg2 = <r>2/6 A.4.1 If the density of the protein i s known, one can calculate the volume, v, of the single molecule: v= (molecular weight)/(density) 6.02 X 10 2 3 A. 4. 2 (although t h i s analysis assumes that there i s no hydration of the molecule, the hydrodynamic volume could be used in place of the volume given by eguation A. 4.2) . Using t h i s value of volume one can calculate the dimensions and the radius of gyration for a number of shapes as follows. For rods of length 1 , the radius of gyration i s given by (Tanford 1961): Eg 2=l2/12 A. 4. 3 The length can be calculated from the volume for d i f f e r e n t diameter reds according to: l=v/( TTr 2) A. 4. 4 For spheres of radius, r, the radius of gyration i s given 2 3 7 Figure.A.4. 1: Radius of gyration for various shapes. Radius of gyration for rods, random-coils, and spheres, from Tanford 1961. 2 3 8 - 2 3 9 Figure. A . 4 . 2 : Evalaation of protein conf ormati_gn_. Plots of radius of gyration Eg, versus number residues for d i f f e r e n t shapes: (A) lew molecular weight range. (B) high molecular weight range. 2 4 0 24 1 by (Tanford 196 1) : Eg2= (3/5)r 2 A. 4.5 and s i m i l a r l y r could be calculated from the volume and the following eguation: r 3= (v) (3/4 T) A. 4. 6 These abcve eguaticns can be used to generate graphical relationships for the d i f f e r e n t shapes. This has been done i n figure A.4.2a for the dependence of the radius of gyration on the number cf residues. Given a protein of known molecular weight and composition, a comparison of Rg values obtained from experiments such as l i g h t scatterring (Timasheff and Townend 1970) to the standard curves should allow a rapid evaluation of the proteins conformational state. As i s evident from figure A,4.2a, the uncertainties at the lower l i m i t of molecular weight are high, but t h i s type cf analysis should provide reasonable answers for proteins of high molecular weight (figure A. 4. 2b). 242 A££endix___V_: DETEBMINATION OF SOLVENT EFFECTS ON RANDOM COILS A similar approach as the one presented above, could be used to evaluate the effects of solvents cn proteins that are .known to be i n the random c o i l conformation. One can predict Bg as a function of the number of residues from eguation 8.1 and A.4.1, and generate standard curves for different values of the expansion factor, <K : Bg2= <<2Bgo2 A. 5.1 This has been done i n figure A.5 . 1 . Again, a comparison of Bg values obtained from ether sources w i l l allow a graphical evaluation of c< , for any given protein. It might also be feasible tc incorporate the relationships provided Zimm and Stockmayer (1949), which would allow t h i s method to be extended to the analysis of branched polymers. As a f i n a l comment, i t should be pointed out that the entire basis for the arguments presented in t h i s , and the previous, appendix assumes that the absolute numbers obtained for the composition of a random l i n k (8.4aa/link) and the length of the l i n k , 1 , apply tc proteins in general. This i s probably an unreasonable assumption f c r proteins that show extremes in the i r amino acid composition. Also the comparison of Figures A.4.2a and A.5.1, indicates that i t would be impossible tc distinguish between reds and random c o i l s at low molecular weights, where the interference e f f e c t s , from the solvent interactions with the random c o i l s , would tend to mask the shape anisotropy of the reds. This problem should be reduced at the higher molecular weights. 243 F i g u r e . A . 5 . 1: Evaluation of solvent effects on ______ ______ Plots of radius of gyration Eg, versus number of residues for random-coils for different values of the expansion coeffecient . 2 4 4 Figure. JW5. 1.. 500 1000 ^residues 2 4 5 ____________ AMINO ACID COMPOSITION AND ELASTIN EVOLUTION In a recent publication Sage and Gray (1979) have presented data for the amino acid composition of e l a s t i n from a wide variety cf sources. In t h e i r data their i s a substantial amount of information for the e l a s t i n composition of the bony f i s h e s , and I have attempted to use the 'likeness' of the e l a s t i n from the d i f f e r e n t sources to develop an evolutionary succession for the e l a s t i n ox" these fishes. The numerical analysis i t s e l f was carried out by the following expression (Matsumura e t . § 1 . 1 579) : h DIJK = 5> | AA,j -AAJL<| A. 6.1 where AAjj i s the number of residues per thousand of the amine acid i , in a protein specified by j , and AA1K i s the number for the same amino acid in the protein specified by k. Similar expressions to t h i s have been u t i l i z e d by a number of workers to evaluate the relatedness between proteins and, then, to extrapolate to the relatedness of the source species (Fondy and Holonan 1971, Metzger ______ 1968, Harris and T e l l e r 1973). .Equation A.6.1 was evaluated cn a computer with a Fortran program, for 13 groups of fis h e s , and t h i s allowed me to compile a difference index matrix for their e l a s t i n composition (table A.6.1).. Based on t h i s r e s u l t an evolutionary scheme was derived as shown in figure A.6,1. No attempt was made to analyze the re s u l t s beyond t h i s point. 246 Table.B,6. 1: Difference index for e l a s t i n composition T O r-» u*> •—• r-. rtca — — <3 ft £ « o; — — — — ' 3 5 8 N - i fy, _, CO ~H i - l m —H c_ <n t-n »n *r *r csi ^ o; ^ o in *r CO — • " - « .-4 f—• i- p. oi cn « L Ql c r» - H « s s _ I to m ro r*. V c c TD-- r •-•_*»- _i Q. * — 3 (U O O "3 o> o LO C> --« CL «— cn o O U U U C ' f •Q <Q C (J «— <— -r~ n3 ^> __ f — Q. 2 4 7 Fig are. A. 6. 1_: E l a s t i n evolution. Evolutionary seguence for e l a s t i n of higher fishes based on table A.6. 1. 2 4 8 F i g u r e * A . 6 . 1 . Elast in Evolution 13 10 9 \ 12 3 249 I.ITEBATOBE CITED Aaron, B.E., and Gosline, J.M. 1980. Optical properties of single e l a s t i n f i b r e s indicate a random conformation. Nature in press. Aaron, B.B., and Gcsline, J.M. 1980. E l a s t i n as a random network elastomer: a mechanical and o p t i c a l analysis of single e l a s t i n f i b r e s . 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