UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Adsorption of bovine serum albumin to polyethylene tubing reversibility and pH-dependence Needham, Judy 1988

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1988_A6_7 N43.pdf [ 4.95MB ]
Metadata
JSON: 831-1.0046947.json
JSON-LD: 831-1.0046947-ld.json
RDF/XML (Pretty): 831-1.0046947-rdf.xml
RDF/JSON: 831-1.0046947-rdf.json
Turtle: 831-1.0046947-turtle.txt
N-Triples: 831-1.0046947-rdf-ntriples.txt
Original Record: 831-1.0046947-source.json
Full Text
831-1.0046947-fulltext.txt
Citation
831-1.0046947.ris

Full Text

ADSORPTION OF BOVINE SERUM ALBUMIN TO POLYETHYLENE TUBING REVERSIBILITY AND pH-DEPENDENCE By JUDY NEEDHAM B.Sc, The University of Leeds, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1988 0 JUDY NEEDHAM, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^ T « - V The University of British Columbia Vancouver, Canada Date QctoVaer; \^>g^ DE-6 (2/88) ABSTRACT This thesis i s concerned with the adsorption of bovine serum albumin to polyethylene tubing. A method using radioiodinated protein was developed to measure the surface concentration taking into account the d i l u t i o n e f f e c t f o r miscible displacement i n a c a p i l l a r y . A steady-state surface concentration was established within 2 hours. Adsorption did not depend on the r a t i o of r a d i o l a b e l l e d to unlabel led protein. The adsorption isotherm was Langmuir -1ike with a plateau concentration of approximately 2 0.2 ug/cm . Two methods were used to c a l c u l a t e the surface concentration i n the desorption study. The surface concentration calculated by depletion of the t o t a l r a d i o a c t i v i t y was always higher than that calculated from assaying the r a d i o a c t i v i t y associated with the tubing. Desorption of at least 5% of the loosely bound protein occurs. The surface concentration-pH data show two maxima. The f i r s t i s at the i s o e l e c t r i c point of the albumin while the second i s at pH 9.5-10. The second maximum seems to be due to p r e f e r e n t i a l adsorption of the higher molecular weight oligomers i n the protein sample. TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i LIST OF APPENDICES ix ACKNOWLEDGEMENTS x CHAPTER 1 INTRODUCTION 1 1.1 Protein structure 3 1.1.1 Bovine serum albumin i n s o l u t i o n 4 1.1.2 Albumin oligomers 5 1.2 Driving forces f o r adsorption 6 1.3 Mechanisms f o r adsorption 8 1.4 C h a r a c t e r i s t i c s of protein adsorption 11 1.4.1 Protein desorption and exchange 17 1.5 Objectives and methods 21 CHAPTER 2 EXPERIMENTAL 24 2.1 Ra d i o l a b e l l i n g 24 2.1.1 TCA p r e c i p i t a t i o n assay 25 2. 1.2 Gel f i l t r a t i o n 25 2.1.3 Thin-layer chromatography 26 2.2 Protein electrophoresis 26 2.1.1 Materials 27 2.2.2 Sample preparation 27 2.2.3 Method 28 2.2.4 Gel s t a i n i n g 30 2.3 Protein concentration 30 2.4 Adsorption experiments 31 2.4.1 Methods and materials 31 2.4.2 Surface concentration as a function of time 32 2.4.3 Surface concentration as a function of the r a t i o of r a d i o l a b e l l e d to unlabel led BSA 33 Page 2.4.4 Adsorption isotherm 33 2.4.5 Surface concentration as a function of pH 33 CHAPTER 3 RESULTS AND DISCUSSION 34 3.1 R a d i o l a b e l l i n g 34 3.1.1 Degree of r a d i o l a b e l l i n g 34 3.2 E x t i n c t i o n c o e f f i c i e n t 36 3.3 SDS-PAGE 38 3.4 Adsorption experiments 49 3.4.1 Surface concentration as a function of the r a t i o of r a d i o l a b e l l e d to unlabel led BSA 49 3.4.2 Surface soncentration as a function of time 50 3.4.3 Adsorption isotherm 52 3.4.4 R e v e r s i b i l i t y of the adsorbed BSA 59 3.4.5 E f f e c t of pH on BSA adsorption 66 CHAPTER 4 CONCLUSIONS 72 REFERENCES 75 iv LIST OF TABLES Table Page 1.1 The adsorption of human serum albumin (HSA) on various surfaces. 13 2.1 Composition of buffers and 3.75% gels f o r SDS-PAGE. 29 3. 1 Molecular weight assignments f or the protein standards used on the 3.75% SDS-PAGE gels. 39 3.2 The e f f e c t of pH on the amount of BSA polymer present i n the stock solutions. The amount of polymer i s represented as a percentage of the t o t a l p r otein ± error. The errorwas c a l c u l a t e d from the a c t i v i t y ; see Appendix 2. 43 3.3 The amount of BSA polymer present i n various samples represented as a percentage of the t o t a l protein ± error. 46 3.4 The e f f e c t of l a b e l l e d BSA content on the adsorption to polyethylene from a 0.5 mg/ml solution, (see Appendix 2 fo r the c a l c u l a t i o n of AD. 50 3.5 Dimensions of bovine serum albumin. 58 2 3.6 Comparison of the surface concentration (ug/cm ) of BSA at pH 7.4 c a l c u l a t e d from cutt i n g up the tubing, T (tube), and from by depletion of the t o t a l counts, T (cpm); see Appendix 2 f o r error analysis. 62 3.7 Surface concentration ( D of BSA at pH 7.4 from a 0.5 mg/ml sol u t i o n . 67 3.8 Surface concentration ( D calculated from the ri n s e d tube and from depletion of t o t a l cpm. The pH was adjusted with borax or NaOH. 69 v LIST OF FIGURES Figure 1.1 A schematic view of a protein i n t e r a c t i n g with a well-characterized surface. The protein has a number of surface domains with hydrophobic, charged and polar character. The s o l i d may have a s i m i l a r domain-like character. (Taken from Andrade, 1985, p.4). 3.1 R a d i o a c t i v i t y vs f r a c t i o n number for the samples c o l l e c t e d from a Sephadex G-25 column following r a d i o l a b e l l i n g . 3.2 C a l i b r a t i o n curve of BSA. Optical density vs BSA concentration at 278 nm. 3.3 The molecular weight on a semi-log scale i s pl o t t e d against the r e l a t i v e mobility (R ) f o r a va r i e t y of SDS-protein complexes run on 3.75% gels. 3.4 SDS-PAGE rod gels stained with coomassie blue. A low molecular weight standards B high molecular weight C unlabel led stock BSA D r a d i o l a b e l l e d BSA 3.5 The mobility of BSA in 3.75% SDS-PAGE gels: ( ) densitometric scan of BSA. The protein was coomassie blue stained and scanned at 595 nm. TD = tracking dye. ( ) r e l a t i v e mobility vs cpm f o r r a d i o l a b e l l e d BSA. 3. 6a Relat ive mobi1ity vs r e l a t ive radioact i v i t y of BSA, pH 7. 4. 3. 6b Re1 at i ve mobi1ity vs r e l a t ive radioact i v i t y of BSA, pH 4.4. 3. 6c Re 1 at i ve mobi1ity vs r e l a t ive r a d i o a c t i v i t y of BSA, pH 9.4. 3. 6d Re 1 at i ve mobi1ity vs r e l a t ive radioact i v i t y of BSA, pH 12.0 3-7a Re 1 at i ve mobi1ity vs r e l a t ive r a d i o a c t i v i t y of sample ( i ) . 3. 7b Re 1 at i ve mobi1ity vs r e l a t i v e radioact i v i t y of sample ( i i ) . 3. 7c Re 1 at i ve mobi1ity vs r e l a t ive radioact i v i t y of sample ( i i i ) 3. 7d Re 1 at i ve mobi1ity vs r e l a t i v e r a d i o a c t i v i t y of sample ( i v ) . vi Figure Page 3.8 Time dependence f or the adsorption of BSA on polyethylene at a s o l u t i o n concentration of 0.19 mg/ml. 51 3.9 Adsorption isotherm f o r albumin on polyethylene at 23°C. 53 3. 10a Surface concentration vs tube section f o r BSA adsorbed from a 8 ug/ml solution. In a l l cases tube s e c t i o n 1 i s the f r a c t i o n c o l l e c t o r end i.e. , output end, and tube section 25 i s the syringe end i.e. , input end. 54 3.10b Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 17 fig/ml solution. 54 3. 10c Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 44 ug/ml solution. 55 3. lOd Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 67 ug/ml solution. 55 3. lOe Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 178 fug/ml solution. 56 3. lOf Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 326 fig/ml solution. 56 3. lOd Surface concentration vs tube section f o r BSA adsorbed from a 493 ug/ml solution. 57 3. lOe Surface concentration vs tube section f o r BSA adsorbed from a 2.694 mg/ml solution. 57 3.11 The concentration of the BSA s o l u t i o n displaced from an adsorption experiment vs the volume c o l l e c t e d . The i n i t i a l bulk concentrations of the BSA sol u t i o n s were (A) 274 and (B) 761 pg/ml. 63 3. 12 Adsorption isotherms f o r BSA on polyethylene. The surface concentration was calculated from the rinsed tubing (*) and by depletion of the t o t a l r a d i o a c t i v i t y (o). 64 3.13 Cb/r versus Cb f o r adsorption data c a l c u l a t e d from the rin s e d tubing (*) and by depletion of the t o t a l r a d i o a c t i v i t y (o). 65 3. 14 The surface concentration of BSA adsorbed to polyethylene tubing p l o t t e d against the pH. The bulk concentration i n each case was 0.5 mg/ml. 68 3.15 Adsorption of BSA on polyethylene as a function of pH from a 0.5 mg/ml solution. The surface concentrations were c a l c u l a t e d from the rinsed tubing (*) and from depletion of vi i Figure Page the t o t a l r a d i o a c t i v i t y ( o ) . 71 A3.1 C/C Q vs x f o r the miscible displacement of BSA with buffer i n a c a p i l l a r y tube. 93 v i i i LIST OF APPENDICES Page APPENDIX 1 ABBREVIATIONS 81 APPENDIX 2 CALCULATIONS AND ERROR ANNALYSIS 83 A2.1 Error analysis 83 A2.2 Imprecision i n the a c t i v i t y 85 A2.3 SDS-PAGE GELS, '/.monomer or polymer ± error 87 A2.4 Surface concentration from c u t t i n g up the tubing 88 A2.5 Surface concentration calculated from the t o t a l counts 88 APPENDIX 3 MISCIBLE DISPLACEMENT IN A CAPILLARY 90 APPENDIX 4 STATISTICAL ANALYSIS 95 A4.1 Testing the differ e n c e between the two regression l i n e s 98 A4.2 The paired-sample test f o r the adsorption isotherm data 99 A4.3 The paired-sample test f o r the pH data 100 ix ACKNOWLEDGEMENTS I would l i k e to thank Dr. D. E. Brooks, my supervisor, f o r his help and guidance throughout t h i s study; Johann Janzen f o r his help with some of the experimental work and invaluable discussion; Charles Ramey f o r h i s discussion and suggestions. A s p e c i a l thank-you goes to a l l the members of the Brooks' lab f o r a l l t h e i r help and support. F i n a l l y I wish to thank Terry J a r v i s and Tim Haddad f o r t h e i r patience during the preparation of t h i s thesis. x CHAPTER 1 INTRODUCTION Protein adsorption to s o l i d surfaces i s of great b i o l o g i c a l , medical and technological s i g n i f i c a n c e . The blood c o m p a t i b i l i t y or i n c o m p a t i b i l i t y with non-biological materials i s important e s p e c i a l l y with the increasing use of p r o s t h e t i c materials i n the body and t h i s i s generally considered to be r e l a t e d to protein adsorption. When a synthetic material i s introduced into the cardiovascular system the i n i t i a l event i n a complex seri e s of reactions i s the rapid adsorption of a proteinaceous layer (Baier and Dutton, 1969). Subsequent c e l l u l a r i n t e r a c t i o n s lead to thrombus formation, the entrapment of erthyrocytes and other formed blood elements i n a f i b r i n network, and coagulation as determined by the adsorbed proteins (Baier, 1977; Brash, 1981). It has been observed that d i f f e r e n t materials have d r a s t i c a l l y d i f f e r e n t thrombogenic a c t i v i t y . This suggests that the c h a r a c t e r i s t i c s of the p r o t e i n layers are d i f f e r e n t for d i f f e r e n t materials. It has been shown that by precoating surfaces with plasma proteins, p l a t e l e t adhesion, a p r e r e q u i s i t e to thrombus formation, i s g r e a t l y altered. An albumin coated surface reduces p l a t e l e t adhesion (Lyman et al., 1971; Packman et al., 1969), while fibrinogen greatly enhances i t and 9r-globulin a c t i v a t e s the release reaction (Packman et al., 1969; Jenkins et al., 1973). Protein adsorption i s a key event i n the blood-surface i n t e r a c t i o n 1 that .nust be understood before we understand the mechanisms of surface-induced thrombosis. Over the past 30 years many investigators have studied protein adsorption to a v a r i e t y of non-biological surfaces. Many techniques have been developed providing information regarding the absolute q u a n t i t i e s adsorbed from s i n g l e solutions (Bull, 1956; Brash and Uniyal, 1979), the r e l a t i v e q u a n t i t i e s adsorbed from complex solutions (Brash and Davidson, 1976; Lee et al., 1974), the number of surface attachments (Morrissey and Stromberg, 1974), desorption and exchange (Brash and Samak, 1978; Chuang et al., 1978), and measurements of the adsorbed layer thickness (Morrissey et al., 1976; Cuypers et al., 1977). There i s s t i l l a lot of controversy regarding protein adsorption at s o l i d / l i q u i d interfaces, p a r t i c u l a r l y concerning the r e v e r s i b i l i t y and the c o n f i g u r a t i o n of the adsorbed molecules. The present study was aimed at c l a r i f y i n g the question of r e v e r s i b i l i t y . Various techniques have been employed to study protein desorption. Solution-depletion methods followed by d i l u t i o n were used to study albumin desorption from glass (Bull, 1956), and from s i l i c a (MacRitchie, 1972). Radiolabelled proteins were used to give a d i r e c t measure of the amount of adsorbed protein. Desorption studies usually followed a r i n s i n g period (Brash et al. , 1974). In t h i s study a more d i r e c t technique using a r a d i o l a b e l l e d protein was developed to measure the amount of protein desorbed and information regarding albumin adsorption to polyethylene and r e v e r s i b i l i t y was obtained. Many f a c t o r s contribute to determining the c h a r a c t e r i s t i c s of protein 2 adsorption including the nature of the protein, the medium in which i t i s located and the nature of the s o l i d surface. These properties give an insight into the d r i v i n g forces and the mechanisms of adsorption. The information required to give a complete picture of protein adsorption includes the amount of protein adsorbed as a function of s o l u t i o n concentration and time (adsorption k i n e t i c s ) . The o r i e n t a t i o n and conformation of the protein upon adsorption i s important since a conformational change, known as denaturation, a l t e r s the properties of the protein. Other desired information includes the capacity of the protein i n s o l u t i o n to compete f o r the surface, and the a b i l i t y of the protein to desorb or exchange. 1.1 Protein structure Protein structure i s largely determined by the interactions among the amino acids which comprise i t , and between the protein molecule and the environment. Because of the structure-function r e l a t i o n s h i p s of proteins t h e i r three-dimensional structure i s of interest. Proteins are high molecular weight polyamides b u i l t up by the s p e c i f i c copolymerization of amino acids f o r p a r t i c u l a r functions. Each protein has a unique amino a c i d sequence known as the primary structure. The secondary structures, ordered three-dimensional regions, are the a-helix and (3-sheet r e s u l t i n g from hydrogen bonding in the protein backbone. The protein's t e r t i a r y structure i s the complete three-dimensional structure and i s the r e s u l t of intramolecular interactions such as i o n i c or e l e c t r o s t a t i c i n t e r a c t i o n s , hydrogen bonding, hydrophobic interactions, s a l t bridges and covalent d i s u l f i d e bonds. The noncovalent a s s o c i a t i o n of independent 3 t e r t i a r y structure gives the quarternary structure. 1.1.1 Bovine serum albumin i n s o l u t i o n Serum albumin i s the protein that i s present in the largest amount in blood plasma, where approximately h a l f of the protein i s albumin. It i s one of the most intensely studied proteins. Globular proteins i n aqueous solutions near t h e i r i s o e l e c t r i c point have compact configurations with low permeability to water. The nonpolar groups are l a r g e l y excluded from the surface of the protein while the polar and charged amino acids are at the surface and interact strongly with water. S h i f t i n g the pH of a protein s o l u t i o n away from the i s o e l e c t r i c point can decrease the s t a b i l i t y of the protein. The protein unfolds exposing the inner part of the molecule. Bovine serum albumin (BSA) expands very r e a d i l y below the i s o e l e c t r i c point of pH 4.9. This expansion at pH 4.5-3.5 depends on the fact that the carboxyl groups are s u c c e s s i v e l y transformed into the uncharged form, leaving the p o s i t i v e l y charged groups i n excess. The repulsion between various segments of the peptide chains increases. This conformational change i s usually c a l l e d the N-F transformation (Foster, 1960). BSA i s somewhat more stable on the a l k a l i n e side of the i s o e l e c t r i c point. Expansion s i m i l a r to the conformational change i n acid takes place i n a l k a l i n e pH, but the a l t e r a t i o n i n s i z e and shape does not begin to occur u n t i l pH 10.3 (Tanford et al., 1955). A small conformational change also has been shown to occur in the pH i n t e r v a l 7-8 (Leonard et al., 1963; Harmsen et al., 1971). The s o l u b i l i t y of BSA i s a function of pH. As the pH moves away from the i s o e l e c t r i c point the net charge on the protein increases thus 4 increasing the s o l u b i l i t y . 1.1.2 Albumin oligomers Albumin samples are heterogeneous in the sense that they contain mercaptalbumin, the f r a c t i o n having a f r e e l y reactive s u l f h y d r y l group, and nonmercaptalbumin, the f r a c t i o n showing no sulfhy d r y l a c t i v i t y . Most c a r e f u l l y prepared albumin preparations have a sulfhy d r y l content of 0.65-0.70 su l f h y d r y l groups per albumin molecule. In most regards mercaptalbumin and nonmercaptalbumin are remarkably s i m i l a r . Physical chemical studies on whole serum albumin y i e l d r e s u l t s i n d i s t i n g u i s h a b l e from those on mercaptalbumin. In v i r t u a l l y a l l samples of BSA, dimers and higher oligomers, aggregates of monomers, ex i s t . It has been shown that such dimers and oligomers a r i s e as a r t i f a c t s during and a f t e r i s o l a t i o n and are not present i n the bloodstream (Andersson, 1966). Albumin preparations contain variable amounts of dimers and higher polymers depending on the source of plasma ( F r i e d l i and K i s t l e r , 1970), the f r a c t i o n a t i o n procedure (Smith et al., 1972; S o l l i and B e r t o l i n i , 1977), the storage conditions (Finlayson et al., 1960) and the length of time of storage (Finlayson et al., 1960; Finlayson, 1965). The most l i k e l y source of dimerization would be the d i r e c t formation of d i s u l f i d e linkages through oxidation reactions involving the s u l f h y d r y l residues of two mercaptalbumin monomers. If t h i s i s true then a t h i o l reagent would break the d i s u l f i d e linkage, but i t i s well known that t y p i c a l albumin samples contain a portion of dimeric forms which are not broken down by reduction with t h i o l reagents (Hartley et al., 1962; 5 Janatova et al., 1968). Andersson showed that dimer i s o l a t e d from serum albumin was heterogeneous (Andersson, 1966). Approximately one-third of the dimer was s p l i t into monomer by mercaptoethanol, a reducing agent, at pH 8 or by standing i n a l k a l i n e solution, pH 11.4, f o r 2 days. The part of the dimer not s p l i t by mercaptoethanol was r e l a t i v e l y stable. A small degree of cleavage res u l t e d when the dimer was treated with dioxan or a detergent s o l u t i o n i n d i c a t i n g that hydrophobic bonding i s of l i m i t e d importance i n holding the dimer together. The s t a b i l i t y of the mercaptoethanol r e s i s t a n t dimer at low and high pH values indicates that e l e c t r o s t a t i c bonds cannot be the explanation. Andersson suggested that both types of dimers are held together by d i s u l f i d e bonds. In one dimer the d i s u l f i d e bond i s sit u a t e d i n the i n t e r i o r of the molecule and therefore not accessible to react with mercaptoethanol. It was also proposed that hydrogen bonding may be responsible f o r the s t a b i l i t y of the dimer not s p l i t by mercaptoethanol (Andersson, 1966). 1.2 Driving forces f o r adsorption Before looking at the mechanism of protein adsorption one should look at the d r i v i n g forces f o r the process. For protein adsorption to be spontaneous the change i n free energy AG = AH - TAS , must be negative, where AH i s the enthalpy of ads ads ads ads adsorption, T the absolute temperature and AS i s the entropy of ads adsorption. Calorimetric measurements (Norde and Lyklema, 1978; Nyilas et al., 1974), give a d i r e c t measure of enthalpy and the data show that both e n t h a l p i c a l l y and e n t r o p i c a l l y driven adsorption occur since enthalpy changes ranged from p o s i t i v e to negative depending on pH f o r albumin 6 adsoroed on negatively charged polystyrene. Interactions such as covalent, e l e c t r o s t a t i c and hydrogen bonding between the pr o t e i n and the surface are l i k e l y to be exothermic while changes i n hydrophobic interactions, which are a r e s u l t of the ordering of water molecules near the surface of the protein or adsorbent, contribute to the changes i n entropy. Proteins have low s o l u b i l i t i e s which r a r e l y exceed 1% by weight due to high molecular weights. It has been shown (B u l l , 1956), that at the i s o e l e c t r i c point proteins usually display minimum s o l u b i l i t y and maximum adsorption. The s o l u b i l i t y of a protein i s determined by the balance of the a t t r a c t i o n of the protein molecules f o r each other, which tends to prevent s o l u t i o n and the a t t r a c t i o n of the solvent molecules f o r the protein, which tends to promote solution. At the i s o e l e c t r i c point the protein has a net neutral charge and the a t t r a c t i o n of the protein molecules f o r each other i s maximal. When the pH i s s h i f t e d away from the i s o e l e c t r i c point the protein molecule becomes charged. This decreases the a t t r a c t i o n of the pr o t e i n molecules f o r one another and leads to an increase i n s o l u b i l i t y since the proteins charged groups are more solvated. The s o l u b i l i t y and adsorption of proteins i s analogous to some aspects of synthetic polymer adsorption. An increase i n adsorption with decreasing s o l u b i l i t y has been shown f o r various polymers on glass (Rowland and E i r i c h , 1966). For synthetic polymers the amount adsorbed per unit area increases with increasing molecular weight ( G i l l i l a n d and Gutoff, 1960). From a mixture of polymers of varying molecular weight the larger molecules are adsorbed p r e f e r e n t i a l l y since they can form more bonds per molecule with the surface. 7 Proteins adsorb to non-biological surfaces due to t h e i r amphipathic nature, high molecular weight, limited s o l u b i l i t y and a b i l i t y to change con f i g u r a t i o n at an interface. The decrease i n free energy may re s u l t from a gain i n entropy due to the disorder of water released from the surface or prot e i n but i t may also be due to exothermic events. 1. 3 Mechanisms f o r adsorption Adsorption from an aqueous s o l u t i o n i s a competitive process, since, when p r o t e i n molecules are adsorbed solvent molecules are displaced. When determining the mode of adsorption a l l the in t e r a c t i o n s i n the system must be taken into account. When a protein s o l u t i o n flows past a s o l i d surface the protein reaches the surface by a diffusion-convection process, then binds. The i n i t i a l rate of adsorption depends on the transport and binding. Once protein has been adsorbed onto the surface the surface a v a i l a b i l i t y becomes the dominant f a c t o r and therefore r a t e - c o n t r o l l i n g and now protein-protein i n t e r a c t i o n s may become important. Proteins may bind to the surface v i a ion i c or e l e c t r o s t a t i c i n t e r a c t i o n s , hydrophobic interactions, hydrogen bonding and by charge-transfer or p a r t i a l donor-acceptor interactions. Covalent bonding does not r e s u l t from adsorption under b i o l o g i c a l conditions. Figure 1.1 depicts the d i f f e r e n t types of regions of a protein molecule that may be involved i n the adsorption process. Ionic or e l e c t r o s t a t i c interactions, due to the a t t r a c t i o n or repu l s i o n of two or more groups carrying a net charge, are important i n many systems. Proteins may bind to an oppositely charged surface v i a e l e c t r o s t a t i c bonds. However, a t t r a c t i v e e l e c t r o s t a t i c bonds may also be 8 formed between a protein carrying a net charge equal to that of the surface. For instance, i t has been shown that the adsorption of negatively charged proteins on a negative polystyrene latex occurs spontaneously and exothermically (Norde and Lyklema, 1978). SOLID—SOLUTION INTERFACE Fig. 1. 1. A schematic view of a protein interacting with a well-characterized surface. The protein has a number of surface domains with hydrophobic, charged and polar character. The solid may have a similar domain-like character. (Taken from Andrade, 1985, p.4). Charges on a protein surface are surrounded by unlike charges in a 9 d i f f u s e double layer rather than being only solvated by water (Wada and Nakamora, 1981). The s t a b i l i t y of a protein depends in some cases on intramolecular e l e c t r o s t a t i c i nteractions (Perutz, 1978). If a charged group important f o r pr o t e i n s t a b i l i t y i nteracts with a charged surface a conformational change may r e s u l t due to a change i n the e l e c t r o s t a t i c i n t e r a c t i o n s i n the protein. Proteins adsorbed e l e c t r o s t a t i c a l l y should be s e n s i t i v e to changes i n the i o n i c composition and pH. One would expect r e v e r s i b l e adsorption since continuous exchange of the protein with other ions i n the blood ought to occur. Protein adsorption i s thought to be i r r e v e r s i b l e or p a r t i a l l y r e v e r s i b l e on many surfaces, however, i n d i c a t i n g that e l e c t r o s t a t i c i n t e r a c t i o n s are only of minor importance. Another protein-surface i n t e r a c t i o n i s the hydrogen bond, a predominantly e l e c t r o s t a t i c i n t e r a c t i o n . The dipole/dipole i n t e r a c t i o n s may, i n an extreme case, give r i s e to i n t e r a c t i o n energies s i m i l a r to weak covalent bonds and due to the small s i z e of the hydrogen atom a small binding distance r e s u l t s . However, in proteins the binding energies are much smaller and the binding distance larger. Hydrogen bonds are important i n proteins and they contribute to the s t a b i l i t y of the inter n a l structure and s t a b i l i z a t i o n of the a-helix and /3-sheet structures. Again, competition from surface hydrogen bonding groups can cause conformational changes on binding. The hydrophobic patches on a protein can interact with hydrophobic polymer surfaces such as polyethylene or Teflon. The hydrophobic i n t e r a c t i o n i s an e n t r o p i c a l l y driven i n t e r a c t i o n r e s u l t i n g from a gain i n free energy caused by the loss of structured water at the hydrophobic 10 i n t e r f a c e when two such surfaces come together. This i s of great importance i n protein adsorption since the s t a b i l i t y and i n t e r a c t i o n s of proteins depend on the o v e r a l l free energy. The ordering of water at a i r or apolar i n t e r f a c e s i s e n t r o p i c a l l y undesirable. To keep these i n t e r f a c i a l areas at a minimum the hydrophobic amino a c i d side chains are excluded from the protein surface. Globular proteins have a hydrophobic core and a r e l a t i v e l y h ydrophilic s h e l l i n aqueous solutions but complete burying of the hydrophobic regions i s generally not possible. Intramolecular hydrophobic bonding i n dissol v e d proteins may a f f e c t p r o t e i n adsorption e s p e c i a l l y when intramolecular hydrophobic bonding i s required f o r the s t a b i l i z a t i o n of the protein structure. Rearrangement of str u c t u r e upon adsorption i s now probable ( B i r d i , 1973). Charge t r a n s f e r i n t e r a c t i o n s i n aqueous solutions are due to n-n e l e c t r o n e f f e c t s and these are important i n protein s t a b i l i z a t i o n and surface i n t e r a c t i o n . Excess e l e c t r o n density can be donated to an e l e c t r o p h i 1 i c species or e l e c t r o n density can serve as an acceptor f o r p o s i t i v e charge. 1.4 C h a r a c t e r i s t i c s of protein adsorption Many in v e s t i g a t o r s have found that the adsorption of proteins from s o l u t i o n to non-biological surfaces i s apparently of the Langmuir type ( B u l l , 1956; Oreskes and Singer, 1961; Cheng et al., 1978; Young et al.. 1988). The surface concentration increases asymptotically with an increase i n s o l u t i o n concentration u n t i l a steady state, plateau value i s reached. This i s assumed to be associated with the formation of a complete monolayer. The amount adsorbed i s not s i g n i f i c a n t l y d i f f e r e n t from that 11 expec';ed f o r a close-packed monolayer of native protein i n a side-on or end-on conformation depending on the system and conditions. Even though the adsorption isotherm i s of the Langmuir type there i s no reason to accept the a p p l i c a b i l i t y of t h i s model of adsorption because many of the Langmuir assumptions are not s a t i s f i e d , e.g., adsorption does not take place on s i t e s ; adsorption i s not f u l l y reversible. M u l t i l a y e r adsorption has been demonstrated (Oreskes and Singer, 1961; P i t t and Cooper, 1986; Young et al., 1988). Adsorption experiments were c a r r i e d out on various surfaces and the data plotted using modified versions of the Langmuir equation (Oreskes and Singer, 1961; Young et al., 1988). M u l t i l a y e r adsorption was suggested since the data were f i t t e d by two or more regions of d i f f e r e n t slopes, each slope representing a d i f f e r e n t binding constant. The f i r s t steeper slope was interpreted as representing the i n i t i a l p r o tein layer bound to the polymer. The second l i n e can be interpreted as a second layer of protein due to prot e i n - p r o t e i n int e r a c t i o n s or to a reorganization of the monolayer from a side-on to an end-on d i s p o s i t i o n thereby increasing the amount adsorbed. Protein adsorption has been shown to be pH dependent ( B u l l , 1956, Morrissey and Stromberg, 1974); see e a r l i e r discussion (Section 1.2). Maximum adsorption occurs near the i s o e l e c t r i c point of the protein. A predominantly nonionic, hydrophobic mechanism i s suggested since the prot e i n has no net charge at t h i s pH. Protein adsorption to various surfaces has been shown to depend on the surface. Table 1.1 shows the plateau values, following r i n s i n g with buffer, f o r the adsorption of albumin onto various hydrophobic and o hydrophilic surfaces from a 1 mg/ml solution, pH 7.4 at 23 C. 12 Table 1. 1 The adsorption of human serum albumin (HSA) on various surfaces. Surface Plateau surface concentration (pg/cm ) Ref. Polyurethane 1540 (hydrophilic) 0. 02 1 Polyurethane 600 (hydrophilic) 0. 04 1 Glass 0. 04 2 Si 1ica 0. t 09 3 Collagen-coated glass 0. 09 1 P o l y ( v i n y l chloride) 0. 17 4 S i l i c o n i z e d glass 0. 18 1 Polyethylene 0. 18 2 Polystyrene 0. 20 1 Polyurethane (hydrophobic) 0. 57 1 t surface concentration f o r bovine serum albumin Key to references: 1 Brash and Uniyal, 1979. 2 Brash and Davidson, 1976. 3 Morrissey and Stromberg, 1974. 4 Young et al., 1988. The h y d r o p h i l i c surfaces show low surface concentrations. On these surfaces desorption occurs, therefore there may be some uncertainty with respect to the surface concentration because r i n s i n g may remove adsorbed pr o t e i n (Chan and Brash, 1981; MacRitchie, 1972). The hydrophobic surfaces show a varying range of plateau values. Polyethylene, polystyrene and s i l i c o n i z e d glass show a steady state surface concentration of 13 approximately 0.2 ug/cm . These are a l l e f f e c t i v e l y hydrocarbon polymers. The hydrophobic polyurethane shows a higher plateau surface concentration. This polymer contains a high proportion of ether oxygen and urethane f u n c t i o n a l i t i e s as well as hydrocarbon. The surface has been shown to posses domains i.e. , polyether-rich and urethane-rich regions, which may be responsible f o r the high albumin adsorption. Many adsorption studies have been c a r r i e d out on tubing . Infrared i n t e r n a l r e f l e c t i o n techniques were used by Lee and Kim to study the e f f e c t of time and flow rate on the adsorption of serum albumin, y- g l o b u l i n and prothrombin to s i l i c o n e rubber (SR), f l u o r i n a t e d ethylene-propylene copolymer (FEP) and segmented polyether urethane (PEUU) (Lee and Kim, 1974). The adsorption was rapid and dependent on the substrate not on the protein. The plateau value concentration was shown to depend on the flow rate with SR but not with PEUU. Increasing the flow rate can delay the plateau time because of shear forces opposing the d i f f u s i o n of protein molecules to the surface. There was a s i x f o l d increase i n the plateau concentration f o r albumin on SR when the flow rate was increased from 0 to 12 ml/sec. This was explained i n terms of surface roughness. A rough surface such as SR f o r example, has a greater surface area, therefore more anchoring s i t e s are av a i l a b l e f o r the greater number of protein molecules i n the v i c i n i t y with an increased flow rate. Perhaps t h i s may be due to the formation of a thick adsorption-entanglement layer along the wall analogous to the ones observed i n flowing high-molecular weight polymer so l u t i o n s (Hikmet et al., 1985). FTIR-ATR ( f o u r i e r transform infrared spectroscopy coupled with attenuated t o t a l reflectance optics) studies on albumin adsorbed onto 14 polyeinaneurea showed that the adsorption k i n e t i c s were not s i g n i f i c a n t l y a f f e c t e d by the shear rate over a range 0-1800 s V However, protein adsorbed at 0 to 200 s 1 desorbed more r a p i d l y than that adsorbed at 1800 s 1. This suggests that the protein adsorbed under higher shear rates may bind more t i g h t l y ( P i t t and Cooper, 1986). The conformation and conformational changes upon the adsorption of plasma proteins may be a way of p r e d i c t i n g the e f f e c t of i n t e r a c t i o n s with the surface and have received considerable attention. Polymers are adsorbed to surfaces by the attachment of various segments along the chain which may occur s i n g l y or i n runs which have loops extending from the surface into the solution. S t a t i s t i c a l mechanics has been applied to independent polymer molecules adsorbed to planar surfaces and one can predict the f r a c t i o n of bound segments, the d i s t r i b u t i o n of the segments normal to the adsorbing surface and the average number of loops (Cohen Stuart et al., 1986). However, f o r adsorbed proteins such p r e d i c t i o n s are not possible due to the numerous intermolecular i n t e r a c t i o n s described e a r l i e r . Morrissey and Stromberg used in f r a r e d d i f f e r e n c e spectroscopy to study p r o t e i n adsorption on s i l i c a p a r t i c l e s . By observing a s h i f t of 20 cm 1 i n the amide I band upon adsorption, the f r a c t i o n of adsorbed protein carbonyl groups bound to the surface could be measured and used to c a l c u l a t e the number of surface attachments (Morrissey and Stromberg, 1974). The conformation of serum albumin, fibrinogen and prothrombin was studied as a f u n c t i o n of surface concentration, time of adsorption, pD and ioni c strength. There was no change i n the bound f r a c t i o n of albumin or prothrombin along the isotherm. Both these proteins have an average bound 15 f r a c t i o n of 0.11 i n d i c a t i n g about 80 carbonyl attachments to the surface but most of the molecule i s i n s o l u t i o n and away from the s o l i d surface. The bound f r a c t i o n does not change with the time of adsorption. This data suggests that conformational changes do not occur or are minimal upon adsorption, therefore i t may be s a i d that the internal bonding i n these proteins i s s u f f i c i e n t to prevent s t r u c t u r a l changes. The bound f r a c t i o n of fibrinogen was found to increase with increasing adsorption which may suggest i n t e r f a c i a l aggregation. Studies on cross-linked and denatured albumin showed that c r o s s - l i n k e d albumin gave a bound f r a c t i o n s i m i l a r to that of the native protein, while unfolded albumin gave an increase of 55 contacts and aggregated albumin re s u l t e d i n a decrease of 50 surface contacts. It i s concluded that no aggregation or conformational changes occur upon adsorption of the native serum albumin. Other investigators have also concluded by i n f r a r e d spectroscopic techniques that in general plasma proteins were not dimensionally denatured, i.e . , no change i n conformation occurred on adsorption to the surfaces studied (Brash and Lyman, 1969). It has been shown that adsorbed fibrinogen and j - g l o b u l i n are required f o r p l a t e l e t adhesion and are therefore important for surface induced thrombosis (Zucker and Vroman, 1969; Kim et al., 1974). A study of these from a protein mixture or plasma may help predict biocompatibi1ity. Radiolabelled proteins have to be used since spectroscopic techniques do not discriminate between d i f f e r e n t proteins. Lee and coworkers studied the competitive e f f e c t s of plasma proteins adsorbed to hydrophobic polymer surfaces (Lee et al. , 1974). The rates of 16 adsorption of I l a b e l l e d albumin, y-globulin and fibrinogen were measured separately and from a mixed solution. The composition of the adsorbed layer, following r i n s i n g , at the plateau value was determined. The amount of each protein adsorbed from a mixed s o l u t i o n was less than that compared to the adsorption from a s i n g l e protein solution. The time to reach the plateau concentration f o r albumin was doubled while that f o r f i b r i n o g e n and y- g l o b u l i n decreased by a f a c t o r of ten. Lyman and coworkers t r i e d to e s t a b l i s h a r e l a t i o n s h i p between the adsorbed protein composition and the extent of surface-induced thrombosis on PEUU, SR and FEP (Lyman et al., 1974). R e c i r c u l a t i o n tubes were implanted i n dogs f o r varying periods of time. The tubes were rinsed and then soaked i n a detergent to remove the adsorbed protein. The amount of albumin, ^ - g l o b u l i n and other globulins was determined using acrylamide gel electrophoresis. The r e s u l t s indicated that low thrombogenic surfaces adsorbed mainly albumin while thrombogenic materials adsorbed l a r g e l y globulins. 1.4.1 Protein desorption and exchange The r e v e r s i b i l i t y of protein adsorbed has to be established i n order to j u s t i f y using thermodynamic equations to describe the adsorbed phase. It i s also of in t e r e s t to e s t a b l i s h any changes in the composition of the pr o t e i n f i l m s adsorbed from mixtures. If protein adsorption i s assumed to occur at multiple s i t e s on the protein, desorption r e s u l t s only when a l l these s i t e s simultaneously detach. This would be expected to be a very improbable event and therefore one would expect s i g n i f i c a n t desorption not to occur. 17 The r e v e r s i b i l i t y of protein adsorption i s somewhat c o n t r o v e r s i a l . Adsorption data has frequently been found to be in good agreement with the Langmuir model ( B u l l , 1956; Brash and Lyman, 1969; Chuang et al., 1978; Young et al., 1988). This might be interpreted as supporting r e v e r s i b i l i t y i n terms of a dynamic equilibrium. However, there i s l i t t l e evidence that supports the Langmuir assumptions of r e v e r s i b l e binding to s i n g l e s i t e s per protein. Generally i t has been observed that s i g n i f i c a n t desorption from a hydrophobic surface does not occur, while f o r hydrophilic surfaces both r e v e r s i b i l i t y and i r r e v e r s i b i l i t y of the adsorbed protein has been found. Various methods have been employed in order to study the desorption of proteins from s o l i d surfaces and care must be taken to ensure that one measures the amount of protein a c t u a l l y desorbed. Bul l used s o l u t i o n - d e p l e t i o n methods to study the adsorption of bovine serum albumin on glass ( B u l l , 1956). The surface concentration i s determined by monitoring a change in the bulk s o l u t i o n concentration. In t h i s way the adsorption at equilibrium i s measured. The absorbance near 278 nm i s measured following e q u i l i b r a t i o n and c e n t r i f u g a t i o n of the suspended s o l i d . A large surface area to s o l u t i o n volume r a t i o was required to produce a s i g n i f i c a n t decrease in s o l u t i o n concentration. Desorption was studied by allowing two samples of pyrex glass to adsorb pr o t e i n at pH 5.05. The amount of protein adsorbed from a 0.0140% protein s o l u t i o n was 0.78 mg per gram of glass. One of the samples was then d i l u t e d with buffer to give an equilibrium protein concentration of 0.0065% and the amount of p r o t e i n adsorbed was found to be 0.77 mg per gram of glass. The amount of p r o t e i n adsorbed i n each case was found to be approximately the same. It was concluded that no protein was removed from 18 the glass surface by d i l u t i n g the protein solution. B u l l c a r r i e d out an experiment to determine i f protein could be removed from the surface by extensive washing. Glass powder was suspended in a protein s o l u t i o n and allowed to eq u i l i b r a t e . The pro t e i n s o l u t i o n was removed and the glass was resuspended i n buffer at selected pH values. The resuspension procedure was repeated f i v e times. The protein was removed from the glass by suspending i t in one molar sodium acetate and i t s concentration determined. The r e s u l t s show that a considerable amount of the adsorbed p r o t e i n i s removed by extensive washing. The removal i s pH dependent and i t i s more d i f f i c u l t to remove the protein at pH values near the i s o e l e c t r i c point of albumin. MacRitchie used s i m i l a r solution-depletion techniques and d i l u t i o n with buffer to study the desorption of bovine serum albumin from hydrophobic and hydr o p h i l i c s i l i c a p a r t i c l e s (MacRitchie, 1972). It was shown that at pH 7.5 albumin adsorption to hydrophilic s i l i c a was completely r e v e r s i b l e , but at the albumin i s o e l e c t r i c point, pH 4.9, adsorption was not r e v e r s i b l e . R e v e r s i b i l i t y was not observed with the hydrophobic s i l i c a surface. Brash and his coworkers studied the desorption and exchange of serum albumin on polyethylene and cuprophane (Brash et al, 1974). The polymers were i n the form of tubes. Radiolabelled serum albumin was pumped through the tubes. Af t e r a 24 hour r i n s i n g period the tube was assayed v i a gamma counting (gamma counted) to give the surface concentration. The adsorption and exchange of albumin adsorbed from a 0.1 mg/ml s o l u t i o n on polyethylene was studied. No desorption into water was detected and exchange of the r a d i o l a b e l l e d with nonlabelled albumin at 0.1 mg/ml s o l u t i o n concentration 19 d i d rot occur under s t a t i c conditions. Rapid desorption would not be detected i n t h i s way since the tubing i s washed before the "equilibrium" adsorption i s determined by gamma counting. Exchange of r a d i o l a b e l l e d p r o t e i n was detected at a so l u t i o n concentration of 3.7 mg/ml. The turnover was 10% i n the f i r s t hour and 85% i n 220 hours. Grant et al. and Stromberg et al. used rapid r i n s i n g techniques to study desorption (Grant et al., 1977; Stromberg et al., 1975). Adsorption was c a r r i e d out using r a d i o l a b e l l e d albumin. The substrate was removed and immersed i n a rin s e vessel and continuously washed with water. Desorption from polyethylene was not detected. Albumin adsorbed on chromium showed r e v e r s i b i l i t y with up to 25% of the protein being removed i n the f i r s t minute. Chuang et al. used polymer discs to study desorption and exchange (Chuang et al., 1978). Cuprophane and po l y ( v i n y l c h l o r i d e ) , (PVC), discs 125 were precoated with I-protein by incubating the disc i n a protein s o l u t i o n f o r 30 minutes. The discs were washed by dipping i n Tyrode's buffer. Desorption and exchange studies were c a r r i e d out by incubating the precoated d i s c s i n Tyrode's buffer or in homologous unlabel led proteins f o r 24 hours at room temperature. The residual r a d i o a c t i v i t y was gamma counted. It was demonstrated that desorption and exchange was dependent on both the s p e c i f i c p r o t e i n species and the type of polymer surface. Albumin adsorbed to cuprophane did not desorb into Tyrode's buffer but 38% of the r a d i o l a b e l l e d albumin was found to exchange with unlabel led albumin from a 1 mg/ml so l u t i o n . For fibrinogen adsorbed on cuprophane 23% desorbed into Tyrode's buffer and 42% exchanged with unlabelled fibrinogen at a f a s t e r 20 rate than f o r albumin under s i m i l a r conditions. I-IgG adsorbed on cuprophane showed a 19% desorption and gave an exchange of 30% with 125 unlabel led IgG, whereas f o r I _IgG adsorbed on PVC the desorption was 4% and exchange was 6%, both ocurring at a much slower rate. Double isotope l a b e l l i n g experiments have shown exchange of the adsorbed p r o t e i n with the protein i n s o l u t i o n even though the quantity of p r o t e i n adsorbed remains the same (Brash and Samak, 1978; Chan and Brash, 1981). In these experiments the polymer surface was rinsed with buffer before exchange runs were c a r r i e d out. The r e s u l t s indicate a constant exchange of p r o t e i n between the surface and s o l u t i o n . The l e v e l l i n g o f f o the p r o t e i n loss and gain curves suggest that there i s a f r a c t i o n of the adsorbed p r o t e i n that i s exchangeable and a f r a c t i o n that i s not and that t h i s v aries with conditions. The rates and extent of exchange have been shown to be greater f o r glass and hydrophilic surfaces (Chan and Brash, 1981) than f o r hydrophobic surfaces (Brash and Samak, 1978; Cheng et al., 1987). This would suggest stronger binding f o r hydrophobic surfaces. 1. 5 Objectives and methods In many studies on protein adsorption, and in a l l studies on adsorption to tubing, the protein layer i s washed before determining the "equilibrium" adsorbed protein concentration. Desorption studies are then c a r r i e d out on the remaining adsorbed layer. The problem with t h i s protocol i s that any weakly bound protein w i l l be washed off. It i s t h i s weakly bound p r o t e i n with which t h i s thesis i s concerned. Such material i of i n t e r e s t because weakly adsorbed macromolecules are known to play a 21 c e n t r a l r o l e i n some blood c e l l adherence phenomena (Brooks et al., 1980). The tubing form for the substrate i s important because i t i s t h i s form that i s u t i l i z e d i n many blood c o m p a t i b i l i t y applications. Desorption studies have also been c a r r i e d out using s o l u t i o n - d e p l e t i o n techniques. A s o l i d i s placed i n a protein s o l u t i o n and a protein layer i s adsorbed to the surface. The protein s o l u t i o n i s then d i l u t e d . A decrease i n surface concentration following d i l u t i o n would indicate desorption. Solution-depletion techniques have had lim i t e d a p p l i c a t i o n since a f i n e l y d i vided substrate has to be used and there may be some uncertainty i n determining the av a i l a b l e surface area per gram of material. Also, the geometry of the substrate has been shown to af f e c t the amount of pro t e i n adsorbed (Oreskes and Singer, 1961) and i t i s not possible, i n general, to produce dispersions and tubular geometries of the same material with the same surface properties. In the adsorption experiments of t h i s study, BSA was adsorbed to a length of polyethylene tubing. Following e q u i l i b r a t i o n of the r a d i o l a b e l l e d BSA with the surface the protein s o l u t i o n was displaced with buffer. A l l the f r a c t i o n s c o l l e c t e d were gamma counted. In an experiment of t h i s type the surface concentration can be calculated by a v a r i e t y of methods. F i r s t l y , a minimum surface concentration i s determined by c u t t i n g up the polyethylene tubing following r i n s i n g and gamma counting. In t h i s case any r e v e r s i b l y adsorbed protein w i l l have been washed o f f . The amount of p r o t e i n displaced from the tube, taking into account the d i l u t i o n e f f e c t , can be calculated from the a c t i v i t y of the c o l l e c t e d samples. From the t o t a l a c t i v i t y added and the a c t i v i t y of the protein displaced the surface concentration i n the tube can be calculated. This s o l u t i o n 22 depletion value gives the maximum amount of protein adsorbed at e q u i l i b r i u m and the difference between these two values, i f any, represents the loosely bound protein. 23 CHAPTER 2 EXPERIMENTAL 2. 1 Rad i o1abe11i ng R a d i o l a b e l l i n g of bovine serum albumin (BSA) was c a r r i e d out using iodo-beads, a commercial s o l i d state reagent (Markwell, 1982). The iodo-beads have N-chloro-benzenesulfonamide, an oxidant, Immobilized on 2.8 mm diameter non-porous polystyrene spheres. The i o d i n a t i o n involves the oxidation of the radioiodide which then reacts with tyrosine (4-hydroxyphenylalanine) residues of the protein by the e l e c t r o p h i 1 i c s u b s t i t u t i o n of the ortho hydrogens on the phenolic r i n g (Regoeczi, 1984). Bovine serum albumin (Fraction V, code no. 81-003, Miles S c i e n t i f i c , Rexdale, Ont.), the protein to be iodinated, was dissolved i n phosphate buffered s a l i n e (PBS)/azide, pH 7.4, to give a concentration of 1 to 4 mg/ml. Isotonic PBS/azide pH 7.4 consisted of Na2HP0^ 2.367 g/1, NaH2P0Ii 0.400 g/1, NaN3 0.195 g/1 and NaCl 7.621 g/1. The iodo-beads (Pierce Chemical Company, Rockford, 111.) with an oxidative capacity of 0.45 umol/bead f o r tyrosine-containing peptides, were washed twice i n PBS/azide and blotted dry on f i l t e r paper. To a 1.5 ml Eppendorf micro test tube 2-4 iodo-beads and 0.5 ml of the protein s o l u t i o n were added. The r e a c t i o n was 125 i n i t i a t e d by the add i t i o n of 10-20 ul (200-400 pCi) of c a r r i e r free Na I (Amersham, Arli n g t o n Heights, 111.). The capped tube was rotated f o r 30 O minutes at room temperature (19 C) a f t e r which the reaction was monitered by a t r i c h l o r o a c e t i c a c i d (TCA) p r e c i p i t a t i o n assay to determine the amount 24 of free label hence the completeness of the reaction. 2.1.1 TCA p r e c i p i t a t i o n assay A small sample, 1 u l , of the reaction mixture was added to 1 ml of a 1 mg/ml BSA s o l u t i o n i n a polypropylene test tube. To t h i s 1 ml of 0.5 M t r i c h l o r o a c e t i c a c i d (TCA) s o l u t i o n was added to p r e c i p i t a t e the protein. The sample was centrifuged at 4500 x g f o r 10 minutes and 1 ml of the supernatant was pipetted into a second tube and both samples were counted i n a LKB-Wallac 1282 Compu Gamma gamma counter. The r a d i o l a b e l l e d protein 125 p e l l e t e d with the albumin while the free I was d i s t r i b u t e d evenly between the p e l l e t and the supernatant. The percent of pro t e i n bound was calc u l a t e d using the following equation P e l l e t - Supernatant % Bound = Pel l e t + Supernatant 2.1.2 Gel f i l t r a t i o n Following i o d i n a t i o n the free label was separated from the ra d i o l a b e l l e d p r o t e i n by gel f i l t r a t i o n . A Bio-Rad column (1 x 20 cm) was packed with Sephadex G-25 (Pharmacia, Uppsala, Sweeden) and e q u i l i b r a t e d with PBS/azide at pH 7.4 . Radiolabel free BSA, (2 ml at 1 mg/ml) was put on the column f i r s t to reduce binding of the l a b e l l e d protein to the gel. The r a d i o l a b e l l e d protein was loaded onto the column and eluted with PBS/azide buffer pH 7.4 and the eluate was c o l l e c t e d i n 20, 25 or 30 drop f r a c t i o n s . The f r a c t i o n s were sampled with 1 ul Drummond micro c a p i l l a r i e s (Fisher S c i e n t i f i c ) and gamma counted. The appropriate f r a c t i o n s were 25 pooled and before use the samples were eit h e r put through a second Sephadex G-25 column or dialysed against PBS/azide using an u l t r a f i l t r a t i o n unit (molecular weight cut o f f = 10,000, M i l l i p o r e Ltd., Mississauga, Ont.) to remove more free label. The sample was s p l i t into convenient aliquots and o stored at -20 C. The amount of free label was checked by TCA p r e c i p i t a t i o n and instant t h i n layer chromatography (TLC). 2.1.3 Thin-layer chromatography A small amount, 1 u l , of the r a d i o l a b e l l e d protein was added near the base of a 10 x 1.5 cm s t r i p of p o l y s i l i c i c a c i d gel chromatography media (Gelman Sciences Inc., Ann Arbor, Mi.). This was put into a chamber and developed with 1:1 (v/v) acetone:methanol. The s t r i p was a i r - d r i e d , cut up into 1 cm sections perpendicular to the d i r e c t i o n of migration and placed i n gamma tubes containing 2 ml of 10 mM NaOH and counted. The amount of free label can be calculated since the free -label migrates up the s t r i p and the r a d i o l a b e l l e d protein remains where spotted. 2.2 Protein Electrophoresis The p u r i t y of the r a d i o l a b e l l e d BSA was determined using polyacrylamide gel electrophoresis i n sodium dodecyl s u l f a t e (SDS-PAGE), (Ornstein 1964, Davis 1964). The mobility of a protein i n a polyacrylamide gel i s governed mainly by the protein molecular weight (Sharpiro et al., 1967; Weber and Osborn, 1969). In the presence of SDS, a l l proteins whatever t h e i r o r i g i n a l charge, are converted to complexes having strong negative charges. This causes them to behave as rods of constant diameter. Electrophoresis c a r r i e d out in gels with pores small enough to r e s t r i c t 26 m o b i l i t y shows that the observed mobility i s r e l a t e d very nearly l i n e a r l y to the log of the molecular weight of the protein. This i s unaffected by the proteins o r i g i n a l charge. The p r o t e i n sample i s layered on the polyacrylamide gel and a voltage gradient i s applied. The macromolecules migrate at d i f f e r e n t (constant) rates i n the gel and t h e i r location i n the gel i s determined a f t e r the experiment by s t a i n i n g with Coomassie Blue, a c a t i o n i c dye that binds mainly to amines (Fazekas et a l . , 1963), or by gel s l i c i n g and counting the s l i c e s f o r gamma radiation. 2.2.1 Materials The following were of electrophoresis p u r i t y from Bio-Rad laboratories, Richmond, CA.: acrylamide, N,N-methylene-bis-acrylamide (BIS), sodium dodecyl sulphate (SDS), N,N,N',N'-tetramethylenediamine (TEMED) and ammonium persulphate. The disodium ethylenediamine-tetraacetate (EDTA) was from Fisher S c i e n t i f i c Company, F a i r Lawn, N.J.. Tris(hydroxymethyl)aminomethane (tris-base) and Tris(hydroxymethyl)aminomethane hydrochloride ( t r i s - H C l ) were obtained from Sigma Chemical Company, St.Louis Mo.. Sucrose was from Baker and Adamson, Morristown, N.J.. Pyronin Y (C.I. 45005) was from J.T. Baker Chemical Company, P h i l l i p s b u r g N.J.. Coomassie B r i l l i a n t Blue G-250 (42655) and Photo-Flo 200 s o l u t i o n were obtained from Eastman Kodak Co. Rochester N.Y.. 2.2.2 Sample preparation The samples for electrophoresis contained 1 mg/ml of protein or had approximately 2000 cpm/ul. 27 following an adsorption experiment the tubing was placed in a hot water bath (80 C) f o r 5 minutes. The s o l u t i o n in the tube was displaced with a hot a l k a l i n e s o l u t i o n of SDS (4%). The hot SDS s o l u t i o n was allowed to s i t f o r 5 minutes and was then displaced. This procedure was repeated u n t i l the counts coming o f f the tube were n e g l i g i b l e . The displaced so l u t i o n s were pooled and concentrated using a M i l l i p o r e u l t r a f i l t r a t i o n unit ( M i l l i p o r e Corporation, Bedford, Mass.). The u l t r a f i l t r a t i o n unit (10,000 molecular weight cut o f f ) connected v i a s i l i c o n e tubing to a 50 ml syringe was lowered into the sample to be concentrated in a 15 ml polypropylene tube. A vacuum was applied using the syringe which allowed the f i l t r a t e to pass through the membrane into the syringe. The r a d i o l a b e l l e d BSA remained in the tube.The sample was then used f o r SDS-PAGE. 2.2.3 Method The procedure was a modification of that used by Fairbanks (Fairbanks et a l . , 1971). The solutions and concentrated stock solutions were mixed i n the order and proportions given i n Table 2.1. The 3.75% gel system was prepared and cast as rod gels in acid cleaned 125 x 7 mm inside diameter glass tubes, 2 ml gel s o l u t i o n was used per tube. The gels were overlayed with buffer to produce a f l a t surface on the gel on which to layer the sample. When polymerization was complete, 40 minutes at room temperature or overnight, the gels were mounted in a Bio-Rad model IS0A electrophoresis chamber and the overlay s o l u t i o n was flushed away.with f r e s h r e s e r v o i r buffer. The samples were mixed 1:1 (v/v) with the sample reagent and 20 ul 28 Table 2.1 Composition of buffers and 3.75% gels f o r SDS-PAGE. 10 X Acrvlamide-Bis Acrylamide 40.0 g Bis 1.5 g Water to 100 ml Fairbanks Reservoir Buffer 10 X Buffer 200 ml 4 % SDS 100 ml Water 1700 ml Fairbanks Gel System Fairbanks Sample Reagent 10 X Acrylamide-bis 10 X Buffer Water 4 % SDS 0.5 % TEMED 1.5 % Ammonium persulphate 3.0 ml 3.0 ml 18.0 ml 1.5 ml 1.5 ml 3.0 ml Tris-HCl EDTA SDS Sucrose Pyronin Y Water to 50 ml 0.1211 g 0.0372 g 1.0 g 7.0 g Overlay Solution The same as the gel s o l u t i o n with the acrylamide-bis replaced with water Fairbanks 10 X Buffer Tris-base 24.23 g Sodium acetate 13.61 g EDTA 3. 72 g Water to 500 ml pH to 7.4 with g l a c i a l a c e t i c a c i d a l i q u o t s were layered on the gel surface with the displacement pipettor. For the protein washed o f f the tubing 60 ul-was used to provide s u f f i c i e n t r a d i o a c t i v i t y . Avoltage was applied across the gels at a constant current of 0.5 mA per tube u n t i l the sample entered the gel and then the current o was increased to 6 mA per tube. The gels were run at 4 C f o r approximately 2 hours u n t i l the tracer dye was near the bottom of the gels. A syringe was f i l l e d with water and a few drops of g l y c e r i n and a hypodermic needle was attached. The gels were removed from the glass tubes which were rotated as the hypodermic needle was pushed between the tube and the gel while expressing a f i n e stream of the g l y c e r i n solution. The 29 p o s i t i o n of the tracking dye was marked in each gel by p r i c k i n g i t with a needle dipped i n Indian ink. 2.2.4 Gel s t a i n i n g The gels were stained f o r protein with coomassie blue f o r 1-2 hours. Coomassie blue s t a i n consisted of coomassie blue G-250 0.20 g, methanol 28 ml, g l a c i a l a c e t i c a c i d 5 ml, perc h l o r i c acid (70%) 25 ml, water to 500 ml. Destained with methanol f i x (methanol 300 ml, g l a c i a l a c e t i c a c i d 50 ml, water 650 ml) f o r one hour and the f i n a l c l e a r i n g i n 7% g l a c i a l a c e t i c a c i d overnight u n t i l the background was clear. The s t a i n i n g was repeated i f necessary. Densitometry was performed with a Auto Scanner F l u r - V i s equipped with a 595 nm f i l t e r and a zig-zag time base integrator. Gels run with r a d i o l a b e l l e d samples were s l i c e d using a Bio-Rad model 195 e l e c t r i c gel s l i c e r with a 1 mm blade-to-blade separation. The s l i c e s were t r a n s f e r r e d to gamma tubes and counted f o r radi a t i o n . 2.3 Protein Concentration Proteins show strong absorption at a wavelength of approximately 280 nm due to the residues of phenylalanine, tyrosine and tryptophane. The prot e i n concentration can be determined by measuring the o p t i c a l density (OD) at about 280 nm i f the molar e x t i n c t i o n c o e f f i c i e n t i s known. The e x t i n c t i o n c o e f f i c i e n t of the BSA sample used at 278 nm was determined. BSA (5 g) was freeze dried f o r 24 hours and then dri e d over phosporous pentoxide f o r 3 days u n t i l no change in weight was observed. A 10 mg/ml stock s o l u t i o n of BSA i n PBS/azide was prepared. A s e r i e s of s e r i a l d i l u t i o n s were c a r r i e d out and the 0D of the solutions at 278 nm 30 were .neasured and the e x t i n c t i o n c o e f f i c i e n t calculated. The concentrations of r a d i o l a b e l l e d protein solutions were determined by measuring the OD at 278 nm. This was found to be more r e l i a b l e and consistent than the colourimetric method used by Smith et al.,1985. 2.4. Adsorption Experiments The aim of the work f o r t h i s thesis was to develop a technique that would detect, i f any, the r e v e r s i b l y adsorbed protein bound to a p l a s t i c surface. The technique developed was to use a long tube of small diameter that would give a large surface area. The r a d i o l a b e l l e d protein was pumped into the tube, l e f t to e q u i l i b r a t e and pumped out. By c o l l e c t i n g a l l f r a c t i o n s , the amount of pr o t e i n i s calculated. The experiments were performed to obtain the surface concentration as a function of time of contact, s p e c i f i c a c t i v i t y , pH and concentration. 2.4.1 Methods and Materials 125 The BSA was l a b e l l e d with I using iodo-beads; the amount of free label was less than 1% as checked by TLC. The average degree of i o d i n a t i o n 125 was less than one I atom per molecule of protein and t h i s degree of s u b s t i t u t i o n has been shown to leave the protein properties b i o l o g i c a l l y unaltered, (McFarlane, 1963; Harwig et al., 1975). The polyethylene tubing of inside diameter 0.038 cm was obtained from Intramedic. This material i s intended f or c l i n i c a l use and i s made from low density polyethylene. It alle g e d l y contains no additives or p l a s t i c i z e r s . The tubing was prepared f o r adsorption experiments by 31 pumping methanol and then d i s t i l l e d water through the tubing. Solutions of BSA were made up in PBS/azide, pH 7.4, to the desired s p e c i f i c a c t i v i t y and concentration. Protein concentrations were ca l c u l a t e d by measuring the OD at 278 nm or from a known s p e c i f i c a c t i v i t y . A 5 m length of tubing was connected v i a a three-way valve to a syringe on a Harvard pump. The valve allowed the removal of a i r bubbles through the side arm before introducing protein s o l u t i o n or buffer. Adsorption runs were c a r r i e d out by f i l l i n g the e n t i r e system with PBS/azide buffer, d i s p l a c i n g with protein s o l u t i o n and c o l l e c t i n g a l l f r a c t i o n s using a Gil s o n micro-fraction c o l l e c t o r . The protein s o l u t i o n O was l e f t to e q u i l i b r a t e f o r 4 hours at room temperature (23 C), except i n the case of the time dependence experiment. The contents of the tube were displaced and the tube rinsed with PBS/azide and 3-drop f r a c t i o n s were c o l l e c t e d . A chart recorder was connected to the f r a c t i o n c o l l e c t o r to provide a time base and enable the volume of the samples to be determined. The tubing was f i n a l l y cut into 20 cm segments. A l l the f r a c t i o n s and tubing were counted f o r gamma radiation. 2.4.2 Surface concentration as a function of time To determine the time f o r the system to reach steady state the e q u i l i b r a t i o n time was varied from 0.5 to 24 hours. One stock protein s o l u t i o n was used f o r the serie s of experiments to ensure a constant concentrat ion. 32 2.4.3 Surface concentration as a function of the r a t i o of r a d i o l a b e l l e d to unlabel led BSA The e f f e c t of the r a t i o of radiolabelled:unlabel led protein on the surface concentration was determined. Using a saturating concentration of 12S BSA, 500 ug/ml, the r a t i o of I-BSA to unlabel led BSA was varied, a se r i e s of experiments were c a r r i e d out and the surface concentrations calculated. 2.4.4 Adsorption Isotherm Adsorption was studied as a function of concentration. Protein s o l u t i o n s were prepared at various concentrations with a s p e c i f i c a c t i v i t y 4 of approximately 2x10 cpm/ug. Protein concentrations were determined from the OD or from a known s p e c i f i c a c t i v i t y . 2.4.5 Surface concentration as a function of pH A s e r i e s of protein solutions was prepared at d i f f e r e n t pH values by mixing r a d i o l a b e l l e d and unlabelled BSA with NaOH or HC1 at a f i n a l p r otein concentration of 500 ug/ml. A stock s o l u t i o n of BSA was adjusted to the appropriate pH using a pHM63 D i g i t a l pH meter. Approximately 1.5 ml of the stock BSA was weighed and a small amount of r a d i o l a b e l l e d BSA was added. The pH was checked using pH paper since the s o l u t i o n contained r a d i o l a b e l l e d BSA. 33 CHAPTER 3 RESULTS AND DISCUSSION 3. 1 Radiolabel1ing A plot of the r a d i o a c t i v i t y (counts per minute (cpm)) from a 1 ul sample of each f r a c t i o n against the f r a c t i o n c o l l e c t e d from a Sephadex G-25 column (June 23, 1987) following radiolabel1ing i s shown i n Figure 3.1. The f i r s t peak corresponds to the r a d i o l a b e l l e d protein. The plot shows that the r a d i o l a b e l l e d BSA and the free iodide f r a c t i o n s are well separated. In t h i s example a TCA p r e c i p i t a t i o n assay of the pooled f r a c t i o n s (12 to 14) showed that 6% of the a c t i v i t y was due to the free l a b e l . The amount of free label was reduced to less than 1%, as tested by TLC, by a second column or by d i a l y s i n g against PBS/azide when a more concentrated s o l u t i o n was required. 3.1.1 Degree of Radiolabel1ing The degree of radiolabel1ing was found to be less than one molecule of 125 I per molecule of BSA and t h i s has been reported to have no e f f e c t on the b i o l o g i c a l a c t i v i t y , (McFarlane, 1963; Harwig et al. , 1975). For a t y p i c a l l a b e l l i n g experiment the degree of r a d i o l a b e l 1 i n g i s 125 c a l c u l a t e d as follows. The Na I on June 1, 1987 had an a c t i v i t y of 16.6 125 mCi/ug of iodide. The atomic weight of I i s 126.9 g/mole and 9 125 18 1 mCi = 2.2 x 10 dpm, therefore the a c t i v i t y of the I was 4.63 x 10 125 dpm/mole. The dpm of the I on June 24, 1987 i s calculated from the 34 20 Fraction number Fig. 3.1. Radioactivity vs fraction number for the samples collected from a Sephadex G—25 column following radiolabelling. following equation A = A 0e (3.1) where A 0 and A are the a c t i v i t i e s at times t 0 and t r e s p e c t i v e l y and A i s the decay constant which i s given by A = l n 2 ( t / 2 ) _ 1 (3.2) where t/2 i s the h a l f - l i f e . Substituting into equations 3.2 and 3.3 using 1 PS 12B 60 days as the ha l f l i f e of I, on June 24, 1987 the a c t i v i t y of I was 18 3.55 x 10 dpm/mole. The a c t i v i t y of the r a d i o l a b e l l e d BSA on June 24, 1987 was 196,000 cpm/pg or assuming 77% e f f i c i e n c y of the gamma counter (Janzen, 1985) 255,000 dpm/ug. Using 66,000 as the molecular weight f o r BSA i t s a c t i v i t y 16 was about 1.68 x 10 dpm/mole. Dividing the two a c t i v i t i e s gives a degree 125 of r a d i o l a b e l l i n g of 211 moles of BSA per mole of I. 3.2 E x t i n c t i o n c o e f f i c i e n t The o p t i c a l d e n s i t i e s of a s e r i e s of solutions of known BSA concentration were measured. A plot of BSA concentration against 0D at 278 nm i s given i n Figure 3.2. The plot indicates a l i n e a r r e l a t i o n s h i p between the 0D and a concentration of 0-1.63 mg/ml. The e x t i n c t i o n c o e f f i c i e n t i s given by the slope and i s calculated to be 0.641 ± 0.002. This value i s s i m i l a r to the BSA e x t i n c t i o n c o e f f i c i e n t of 0.66 given i n the l i t e r a t u r e (Cohn et al., 1947). 36 Concentration (mg/ml) Fig. 3.2. Calibration curve of BSA. Optical density vs BSA concentration at 278nm. 3.3 SDS-PAGE The p u r i t y of the BSA used i n the adsorption experiments was determined using SDS-PAGE. The gels were c a l i b r a t e d using Pharmacia elect r o p h o r e s i s c a l i b r a t i o n k i t s . In both the high molecular weight k i t (HMWK) and the low molecular weight k i t (LMWK) several SDS-denatured proteins were run on the same gel. The r e l a t i v e mobility (FLJ of a protein i s c a l c u l a t e d as distance of protein migration Relative mobility = distance t r a v e l l e d by tracking dye The R^ , values f o r the proteins of known molecular weights are given i n Table 3.1. The r e l a t i v e m o b i l i t i e s were plotted against the known molecular weights expressed on a semi-logarithmic scale. The plot i n Figure 3.3 indicates a l i n e a r r e l a t i o n s h i p and provides the molecular weight c a l i b r a t i o n f o r the gels. The SDS-PAGE rod gels of the stock unlabel led BSA and r a d i o l a b e l l e d BSA along with the molecular weight standards are shown i n Figure 3.4. A densitometric scan of the unlabel led BSA and the r e l a t i v e mobility versus cpm f o r r a d i o l a b e l l e d BSA are given i n Figure 3.5. The unlabel led BSA showed bands with apparent molecular weights of 60,000, 133,000 and 214,000 corresponding to R^ , values of 0.540, 0.371 and 0.270. The R^ , values of the r a d i o l a b e l l e d BSA at 0.539, 0.371 and 0.264 likewise imply molecular weights of approximately 60,000, 134,000 and 220,000. The more intense band with a molecular weight of approximately 60,000 corresponds to the BSA monomer while the less intense bands at 134K and 220K presumably correspond 38 Table 3.1 Molecular weight assignments f o r the protein standards used on the 3.75% SDS-PAGE gels. P r o t e i n Molecular weight R f Thyroglobulin 330,000 0. 165 F e r r i t i n (half unit) 220,000 0. 311 Phosphorylase b 94,000 0. 420 Albumin (BSA, HMWK) 67,000 0. 495 Albumin (BSA, LMWK) 67,000 0. 506 Catalase 60,000 0. 540 Ovalbumin 43,000 0. 607 Lactate dehydrogenase 36,000 0. 653 Carbonic anhydrase 30,000 0. 702 Trypsin i n h i b i t o r 20,000 0. 771 F e r r i t i n 18,500 0. 786 oc-Lactalbumin 14,400 0. 831 to the dimer and trimer. The 220K band i s less intense than the 134K band. The BSA molecular weight determined from the gels i s lower than the actual value of 66,000. This may have been due to the wide bands of stained p r o t e i n i n the standards. Since the R^ . values f o r both the l a b e l l e d and the r a d i o l a b e l l e d BSA are s i m i l a r i t i s concluded that r a d i o l a b e l 1 i n g does not e f f e c t the mobility of the protein. The s p e c i f i c a c t i v i t y f o r the oligomeric species have been ca l c u l a t e d to be approximately the same as the monomer. The nature of the BSA dissolved i n solutions at a v a r i e t y of pH values 39 100 1 -I 1 1 1 1 1 1 1 1 1 1 0.0 0.2 0.4 0.6 0.8 1.0 Rf Fig. 3.3. The molecular weight on a semi—log scale is plotted against the relative mobility (Rf) for a variety of SDS—protein complexes run on 3.75* gels. 40 Molecular we i ght 94,000 67,000 14,400 A B C D Fig. 3.4. SDS-PAGE rod gels stained with coomassie blue A low molecular weight standards B high molecular weight standards C unlabel led stock BSA D r a d i o l a b e l l e d BSA 41 1.0 E c 0.8 -LO cn LO -•+-> O C D 0.6 -O C o — _Q t O 0.4 -to D -> -\—> O 0.2 -C D 0.0 I 1 r I1 I1 I1 I1 1 Ii , TD \ • , , i 0.0 0.2 0.4 0.6 0.8 Relative mobility 1.0 M . 5 T 2.5 2.0 h0.5 0.0 X E Q_ O Fig. 3.5. The mobility of BSA in 3.75* SDS-PAGE gels: ( ) densitometric scan of BSA. The protein was coomassie blue stained and scanned at 595 nm. TD = tracking dye. ( ) relative mobility vs cpm for radiolabelled BSA. 42 was investigated by electrophoresis. The samples of the gels p l o t t e d i n Figures 3.6a-d were from the stock solutions in the adsorption experiments. The amounts of monomer and polymer i n the BSA samples were c a l c u l a t e d from the r a d i o a c t i v i t y of the gel s l i c e s and the data i s presented i n Table 3.2. It i s apparent from the plo t s that as the pH increases there i s a s i g n i f i c a n t decrease i n the amount of monomer present. Table 3.2 The e f f e c t of pH on the amount of BSA polymer present i n the stock solutions. The amount of polymer i s represented as a percentage of the t o t a l p r o t e i n ± error. The error was calculated from the a c t i v i t y ; see Appendix 2. pH % monomer % dimer % trimer % tetramer % pentamer 4. 4 83. 36 + 0. 72 11. 42 + 0. 21 2. 68 + 0. 13 1. 31 + 0. 08 7. 4 83. 16 + 0. 48 11. 87 + 0. 14 2. 93 + 0. 08 1. 54 + 0. 08 9. 4 71. 61 + 0. 45 19. 42 + 0. 20 5. 17 + 0. 10 1. 94 + 0. 06 1. 05 ± 0. 06 12. 0 52. 96 + 0. 31 25. 53 + 0. 22 10. 92 + 0. 14 4. 95 + 0. 10 2. 01 ± 0. 05 It i s of in t e r e s t to e s t a b l i s h i f the BSA i s a l t e r e d upon adsorption to the polyethylene tubing. An adsorption experiment was run at pH 7.4 and SDS-PAGE was c a r r i e d out on, ( i ) the stock r a d i o l a b e l l e d BSA i . e . , the r a d i o l a b e l l e d BSA before adsorption, ( i i ) the r a d i o l a b e l l e d BSA pumped out of the tubing during input of the BSA, ( i i i ) the BSA pumped out of the tubing a f t e r the four hour e q u i l i b r a t i o n time and (iv) the BSA washed o f f the tubing using hot basic SDS. Plots of the gels are shown i n Figures 43 0.4 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.6a. Relative mobility vs relative radioactivity of BSA, pH 7.4. 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.6b. Relative mobility vs relative radioactivity of BSA, pH 4.4. 44 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.6c. Relative mobility vs relative radioactivity of BSA, pH 9.4. 0.15 Relative mobility Fig. 3.6d. Relative mobility vs relative radioactivity of BSA, pH 12.0. 45 3.7a-d. The amount of monomer and polymer i n each sample i s given i n Table 3.3. Table 3.3 The amount of BSA polymer present i n various samples represented as a percentage of the t o t a l protein ± error. Sample % monomer % dimer % trimer % tetramer ( i ) 77. 91 + 0. 54 14. 83 + 0. 19 3. 47 + 0. 10 1. 86 + 0. 08 ( i i ) 78. 21 + 0. 66 14. 21 + 0. 23 3. 51 + 0. 12 1. 54 + 0. 10 ( i i i ) 79. 87 + 0. 48 14. 15 + 0. 16 3. 34 + 0. 08 1. 41 + 0. 06 (iv) 65. 91 + 0. 60 17. 68 + 0. 26 9. 45 + 0. 19 The data i n Table 3.3 shows that there i s no difference between samples i , i i and i i i but that the BSA washed o f f the tubing (iv) contains less monomer. The bands i n Figure 3.7d are wider since a larger sample volume (60 ul) was used to obtain a s i g n i f i c a n t level of r a d i o a c t i v i t y . 20 u l samples were loaded on the other gels. Assuming that the BSA washed o f f the tubing i s representative of the BSA adsorbed then one may conclude that less monomer i s adsorbed. It i s seen that more of the higher molecular weight dimer and trimer are p r e f e r e n t i a l l y bound. The r a d i o a c t i v i t y at higher R^ , values may be due to low molecular weight fragments r e s u l t i n g from the harsh treatment with hot basic SDS. It was possible to remove more than 90% of the adsorbed BSA with hot basic SDS as indicated by counting the tubing f o r r a d i o a c t i v i t y following the washing procedure. 46 0.6 0.5 H > 0.4 H •4-» o I 0.3 H o -*-» I 0.1 H 0.0 A 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.7a. Relative mobility vs relative radioactivity of sample (i). 0.4 ^0.3 H *> o 1 0.2 H 0> > 0.1 H or 0.0 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.7b. Relative mobility vs relative radioactivity of sample (ii). 47 0.4 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.7c. Relative mobility vs relative radioactivity of sample (iii). 0.20 *> '•+-> o I 0.10 D > or: 0.00 T 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility Fig. 3.7d. Relative mobility vs relative radioactivity of sample (iv). 48 3. 4 Adsorption Experiments The surface concentration (weight/unit area) of albumin was ca lcula ted by comparing the s p e c i f i c a c t i v i t y (cpm/ug) of BSA with the a c t i v i t y of a known surface area and i s computed using equation 3.3 SA 4 (3.3) where 2 T = surface concentration (ug/cm ) C = counts per minute from the tubing (cpm) SA = s p e c i f i c a c t i v i t y (cpm/ug) 2 4 = surface area (cm ) 3.4.1 Surface concentration as a function of the r a t i o of r a d i o l a b e l l e d to  unlabel led BSA As a preliminary to studying adsorption i t was necessary to e s t a b l i s h whether the r e l a t i v e amounts of l a b e l l e d and unlabelled p r o t e i n had any e f f e c t on the adsorption. The adsorption experiments were c a r r i e d out using a s e r i e s of s o l u t i o n s with a concentration of 0.5 mg/ml but the r a t i o of l a b e l l e d to unlabelled protein was varied. The e q u i l i b r a t i o n time was 4 o hours at 23 C. The r e s u l t s are given i n Table 3.4, (assuming the molecular weight of BSA to be 66,000). The r e s u l t s indicate that the surface concentration i s e s s e n t i a l l y independent of the r a t i o of l a b e l l e d to unlabelled protein. Therefore one can conclude that i o d i n a t i o n at a tracer 125 l e v e l of less than one I atom per protein molecule does not a f f e c t the proteins' a f f i n i t y f o r the polyethylene surface. 49 TABLE 3.4 The e f f e c t of l a b e l l e d BSA content on the adsorption to polyethylene from a 0.5 mg/ml so l u t i o n , (see Appendix 2 f o r the c a l c u l a t i o n of AD. Moles of unlabel led BSA Surface concentration ( D 125 2 per mole of I-BSA (ug/cm ± AD 668 0.197 ± 0.019 804 0.223 ± 0.021 1352 0.215 ± 0.022 1682 0.215 ± 0.022 1721 0.196 ± 0.023 2350 0.192 ± 0.018 2470 0.202 ± 0.020 mean ± error 0.206 ± 0.050 3.4.2 Surface concentration as a function of time It was important to determine the time f o r the surface concentration to reach a steady state so that a su i t a b l e time could be chosen f o r the adsorption study. Using the technique from the l i t e r a t u r e of counting the tubing f o l l o w i n g washout of protein by buffer, adsorption runs were c a r r i e d out using various e q u i l i b r a t i o n times. The time curve f o r a 0.19 mg/ml BSA s o l u t i o n at 23°C i s given i n Figure 3.8. The k i n e t i c s of adsorption was in agreement with e a r l i e r reports (Brash and Davidson, 1976). It was found 2 that the surface concentration reaches a value of approximately 0. 14 fxg/cm within 2 hours and remains constant over a period of up to 8 hours. 50 0.16 en CM 0.14 H E P 0.12 cn =1 0.10 c o £ 0.08 H -4-< c c 0.06 o o g 0.04 o t co 0.02 0.00 i — i — i — i — | — i — i — i — i — | — i — i — i — i — | — r 0 5 10 15 Time (hours) 1 1 1 1 1 1 1 r -20 25 Fig. 3.9. Time dependence for the adsorption of BSA on polyethylene at a solution concentration of 0.19 mg/ml. 3.4.3 Adsorption isotherm The BSA-polyethylene system was investigated by determining the quantity of protein adsorbed as a function of s o l u t i o n concentration. The surface concentration was c a l c u l a t e d d i r e c t l y by c u t t i n g up the tubing and gamma counting. Since the tubing had been rinsed p r i o r to counting, t h i s value must represent a minimum amount adsorbed. Any loosely bound pr o t e i n would have been removed during the r i n s i n g period. The adsorption isotherm o f o r albumin at 23 C over a concentration range of 0 to 2.7 mg/ml i s shown i n Figure 3.9, the upper l i m i t being about 6% of the value in blood. The e r r o r i s c a l c u l a t e d from the various components in equation 3.3, see Appendix 2. The surface concentration increases with concentration asymptotically 2 — 12 2 u n t i l a plateau value of approximately 0.20 fig/cm , (3 x 10 M/cm ). The surface concentration i s s i m i l a r to the values reported previously (Brash and Davidson, 1976; Morrisey and Stromberg, 1974). This behaviour indicates a l i m i t e d capacity of the surface f o r adsorption. This general behaviour has been observed with other types of macromolecules at a s o l i d - s o l u t i o n interface (Silberberg, 1962) and i s in general t y p i c a l of a l l p r o t e i n - p l a s t i c systems. The surface concentration quoted i s an average taken from the 5 m tube length. The p l o t s i n Figures 3.lOa-h show the surface concentration against tube s e c t i o n f o r BSA adsorbed from r a d i o l a b e l l e d BSA solutions of varying concentrations. Tube se c t i o n 1 i s the end of the tube at the f r a c t i o n c o l l e c t o r while tube s e c t i o n 25 i s the end of the tubing connected to the syringe. Adsorption from a s o l u t i o n concentration of 8 ug/ml i s not uniform along the tube. This may be due to a concentration gradient along 52 0.25 CO E 0.20 o cn c 0.15 o § 0.10 c o o I 0.05 D CO H * 0.00 0.0 i—i—i—i—|—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—|—i—r 0.5 1.0 1.5 2.0 BSA concentration (mg/ml) 2.5 Fig. 3.9. Adsorption isotherm for albumin on polyethylene at 23°C. o \ 00 a. G O {ti L C to o c o u <D U Cti e« C 3 C/2 Tube section Fig. 3.10a. Surface concentration vs tube section f o r BSA adsorbed from a 8 ug/ml s o l u t i o n . In a l l cases tube section 1 i s the f r a c t i o n c o l l e c t o r end i.e. output end, and tube section 25 i s the syringe end i.e. input end. 0.12 o \ 60 a. c o (ti -u C <D U c o o V o cti t « u 3 cn 19 20 21 22 Tube section Fig. 3.10b. Surface concentration vs tube section f o r BSA adsorbed from a 17 ug/ml solu t i o n . 54 £ U \ M a. c o -P c 0) u c o o u RS u 3 Tube se c t i o n Fig. 3.10c. Surface concentration vs tube section f o r BSA adsorbed from a 44 ng/ml s o l u t i o n . £ O \ M C o L. C <ti o c o o <D o t r j 3 CO Tube section Fig. 3.lOd. Surface concentration vs tube section f o r BSA adsorbed from a 67 ug/ml solu t i o n . 55 0.17 O \ 60 a. c o «s c c <D O C o u <u o cd <H L. cn 10 11 12 13 U 15 Tube s e c t i o n Fig. 3.lOe. Surface concentration vs tube section f o r BSA adsorbed from a 178 ug/ml so l u t i o n . Q0 a. c o t, c d> o c o o <D O Ki c« U D cn V. / I n I / I K 4 M rf 1 / / \ / / 2 I ft "A /. / \ 'A / / /, 3 9 10 11 12 13 U 15 16 17 18 19 20 21 22 23 2* 25 Tube s e c t i o n Fig. 3.lOf. Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 326 ug/ml so l u t i o n . 56 £ U \ 00 a. c o c U c o V CD U Cti tw U 3 o.i* o.oi -vjk: 0.00 »0 YY ///Y "A Y Y / / / / / / / / A J A Y Y 1 KJ A YA yy yy, yy yy 'Y I ^y yy / / / A 'y, / / / / / / yy / / / /yy yy / / / Yy yy / / / / ( / / A A/ A / / Y y Y A 'Y Y r y / p yy "y, m y PHI/ Y, / YA / I M 3 i 5 3 7 8 9 10 11 12 13 U 15 1S Tube s e c t i o n 18 19 20 21 22 23 24 25 Fig. 3.lOg. Surface concentration vs tube s e c t i o n f o r BSA adsorbed from a 493 ug/ml so l u t i o n . 0.2+ £ U \ M a. c o £-C 0 o c o u <D O CM U 3 cn y Y Y Ay A/ % A/ // A/ Y Y / / t I 0^ / / / yy yy / / / / / / / / / / / / / / / / / / /y / yy, Yy AM/ i i i • "i 10 11 12 13 A/y yy yy, / / / / / / / / / / A y / A / yy Yv Mi 1 L A A 2 / / y y, % y A /. / A n YY, A, / Y^ YY % A / A/. m AAA T 1* 15 IS 17 18 19 20 21 22 23 2+ 25 Tube s e c t i o n Fig. 3.lOh. Surface concentration vs tube section f o r BSA adsorbed from a 2.694 mg/ml so l u t i o n . 57 the tube and to BSA being adsorbed. The BSA molecules f i r s t entering the tube w i l l be adsorbed thus decreasing the concentration of the s o l u t i o n as i t flows through the tube. This w i l l be s i g n i f i c a n t at low BSA concentrations since v i r t u a l l y a l l of the protein i s adsorbed. If most of the p r o t e i n i s adsorbed before the s o l u t i o n reaches the end of the tube the concentration at the end i s reduced s i g n i f i c a n t l y . Adsorption from a s o l u t i o n of bulk concentration 8 pg/ml shows a large change i n surface concentration over the length of the tube, probably due to the above e f f e c t . A uniform surface concentration i s obtained when BSA i s adsorbed from solutions with a concentration of 17 ug/ml or greater. The layer thickness and the average area per BSA molecule were 2 c a l c u l a t e d using a surface concentration of 0.20 pg/cm and assuming the 3 p r o t e i n density to be 1.3 g/cm and a protein molecular weight of 66,000. The c a l c u l a t e d values are given i n Table 3.5 along with the values f o r an end-on and a side-on molecule using reported dimensions of the native globular protein (Squire et al., 1968). Table 3.5 Dimensions of bovine serum albumin. Molecular weight 66,000 Overall dimensions 40 x 140 A Average area per molecule, side-on 5600 A Average area per molecule, end-on 1260 A2 Average area per molecule f o r BSA surface concentration of 0.2 ug/cm 5480 A Calculated layer thickness 15 A 58 The c a l c u l a t e d average area per molecule indicates that a surface 2 —12 2 concentration of 0.20 ug/cm (3x10 M/cm ) i s at the lower end of the range f o r a close packed monolayer configuration suggesting the molecules may be i n a side-on configuration. A close packed monomolecular layer of 2 BSA i s c a l c u l a t e d to have a surface concentration of 0.2 - 0.7 ug/cm . Assuming a uniform layer, the experimental layer thickness i s less than the diameter of the protein molecule, perhaps i n d i c a t i n g the adsorbed layer c o n s i s t s of a s i n g l e layer of s l i g h t l y uncoiled protein strongly bound to the surface. The molecular weight i s probably more accurate than the assumption that the protein density i s the same as i n the c r y s t a l l i n e form. The layer density at the surface would probably be less than that of the c r y s t a l and the layer thickness would be greater, thus, corresponding to a side-on configuration as suggested from the area. However, i t i s probable that the BSA does not form a complete monolayer and that gaps e x i s t between the adsorbed molecules so l i t t l e can be s a i d about the geometry of the layer from these measurements. 3.4.4 R e v e r s i b i l i t y of the adsorbed BSA One object of t h i s work was to look at the r e v e r s i b i l i t y of BSA adsorption, to determine i f there i s any loosely bound protein or desorption of BSA, i . e . , the movement of protein from the surface into pure buffer during the i n i t i a l r i n s i n g of the tube. This means that the r i n s i n g procedure f o r the removal of the radioactive protein s o l u t i o n p r i o r to counting the tubing i s c r i t i c a l . Hence, a l l the f r a c t i o n s coming from the tubing were c o l l e c t e d and counted. The surface concentration was c a l c u l a t e d two ways, f i r s t d i r e c t l y by c u t t i n g up the tubing a f t e r washing, 59 Figure 3.9. Secondly i t was calculated by depletion of the t o t a l a c t i v i t y using the following equation (3.4) SA d where T i s the surface concentration, C i s the t o t a l counts i n the tube T during e q u i l i b r a t i o n , C q the counts output during the c o l l e c t i o n of the f i r s t f r a c t i o n s a f t e r e q u i l i b r a t i o n , SA i s the s p e c i f i c a c t i v i t y and A the surface area. It was possible to c a l c u l a t e C D by Taylor's analysis; see Appendix 3. The output concentration from the tube stays constant near the bulk concentration f o r the f i r s t f r a c t i o n s coming out of the tube and then decreases r a p i d l y to nearly zero. An example of the concentration change i s given i n Figure 3.11. The data i n Table 3.6 shows the calculated surface concentrations and the % protein adsorbed from solution. At a s o l u t i o n concentration of 8 ug/ml a l l the p r o t e i n i s adsorbed from s o l u t i o n and there i s no detectable desorption on washing the tube with PBS/azide, pH 7.4. At higher concentrations less of the protein was adsorbed and a suggestion of desorption was observed. The adsorption isotherm f o r the surface concentration c a l c u l a t e d from the tubing i s given i n Figure 3.9 and by depletion from the tubing and t o t a l counts i n Figure 3.12. The isotherms show that the depletion values are always higher than those determined from the rinsed tubing. However, the data points are close enough, and the uncertainties large enough, to require a s t a s t i c a l a n alysis of the r e s u l t s to determine t h e i r probable s i g n i f i c a n c e . 60 A s t a t i s t i c a l comparison between the two sets of data f o r the adsorption isotherm, was c a r r i e d out to determine i f there was any s i g n i f i c a n t d i f f e r e n c e between them. There i s no unequivocal way to test the s i g n i f i c a n c e of the difference between two non-linear plots. Therefore, the data was l i n e a r i z e d by applying an equation with the form of a Langmuir isotherm. The Langmuir isotherm i s given by _ _ MW d N S A KC b 1 + KC b (3.5; where T = weight of protein adsorbed per unit area of surface MW = molecular weight of the adsorbing protein d^ = surface area per s i t e N = Avogadro's number A K = adsorption constant C^  = bulk p r o t e i n concentration At the plateau surface concentration the monolayer concentration i s T = WA/d N . Rearranging equation 3.5 gives m S A C . C 1 + * (3.6] Kr m P l o t t i n g C / r against C gives a straight l i n e i f K and V are constant. b b m The slope i s equal to 1/r and the adsorption constant i s given by the m slope/intercept. A plot of C /r versus C f o r the isotherm data i s given i n Figure b b 3.13. Each set of data appears to be f i t by a s t r a i g h t l i n e , the least squares regression l i n e s are shown. 61 Table 3.6 Comparison of the surface concentration (ug/cm ) of BSA at pH 7.4 ca l c u l a t e d from c u t t i n g up the rinsed tubing, T (tube), and by depletion of the t o t a l counts, T (cpm); see Appendix 2 f o r error analysis. Concentration (C ) T (tube) T (cpm) % Protein adsorbed b 2 2 (mg/ml) (ug/cm ) (ug/cm ) from s o l u t i o n 0. 008 0. 076 + 0. 010 0. 076 + 0. 001 100 0. 017 0. 102 + 0. 010 0. 102 + 0. 001 63 0. 035 0. 105 + 0. 011 0. 106 + 0. 001 32 0. 044 0. 108 + 0. 011 0. 108 + 0. 002 26 0. 067 0. 111 + 0. 012 0. 112 + 0. 002 17 0. 068 0. 150 + 0. 018 0. 151 + 0. 003 23 0. 080 0. 131 + 0. 013 0. 131 + 0. 002 17 0. 167 o 0. 097 + 0. 013 0. 104 + 0. 003 6 0. 178 0. 158 + 0. 015 0. 159 + 0. 003 9 0. 210 0. 183 + 0. 027 0. 216 + 0. 003 10 0. 253 0. 150 + 0. 017 0. 151 + 0. 003 6 0. 274 0. 129 + 0. 015 0. 131 + 0. 003 5 0. 326 0. 160 + 0. 022 0. 160 + 0. 006 5 0. 493 0. 197 + 0. 017 0. 201 + 0. 003 4 0. 516 0. 195 + 0. 019 0. 199 + 0. 004 4 0. 531 0. 215 + 0. 022 0. 220 + 0. 004 4 0. 623 0. 169 + 0. ,016 0. 175 + 0. 004 3 0. 761 0. 173 + 0. 020 0. 178 + 0. 005 2 0. 851 0. 196 + 0. ,020 0. 201 + 0. 005 2 1. 255 0. 178 + 0. ,015 0. 193 + 0. 007 1 1. 916 0. 192 + 0. ,018 0. 202 + 0. 007 1 2. 067 0. 202 + 0. ,020 0. 216 + 0. ,008 1 2. 694 0. . 204 + 0. ,021 0. 214 + 0. 008 1 62 300 Volume (/xl) 800 0 200 400 600 800 Volume (y^ l) Fig. 3.11. The concentration of the BSA solution displaced from an adsorption experiment vs the volume collected. The initial bulk concentrations of the BSA solutions were (A) 274 and (B) 761 ^ g / m l . 63 0 . 2 5 0) CM E 0 . 2 0 o c 0 . 1 5 o § 0 . 1 0 c o o | 0 . 0 5 13 CO 0 . 0 0 - | — i — i — i — I — I — i — i — i — i — l — i — i — i — i — l — i — i — i — i — I — i — i — i — i — I — i — r 0 . 0 0 . 5 1.0 1.5 2 . 0 2 . 5 BSA concentration (mg/ml) Fig. 3.10. Adsorption isotherm for albumin on polyethylene at 23°C. C b x 10" 3 (yug/cm 3) Fig. 3.13. C b/r versus C b for adsorption data calculated from the rinsed tubing (*) and by depletion of the total radioactivity (o). S t a t i s t i c a l a n a l y s i s was c a r r i e d out to determine i f there i s any s i g n i f i c a n t d i f f e r e n c e between the regression lines; see Appendix 4. The student's t test was used. A t value i s calculated which i s the di f f e r e n c e between the slopes divided by the standard error of the di f f e r e n c e between the slopes. If the value f o r t i s below a c r i t i c a l value the n u l l hypothesis, a statement of no difference, i s assumed i . e . , there i s no s i g n i f i c a n t d i f f e r e n c e between the slopes. From the ana l y s i s i t was concluded that there was a s i g n i f i c a n t difference between the regression l i n e s . This implies that a small amount of desorption occurs. S t a t i s t i c a l a n a l y s i s was also c a r r i e d out using a paired-sample t test; see Appendix 4. This test i s used when two sets of r e s u l t s are obtained under the same conditions. The test uses the d i f f e r e n c e between the two surface concentrations calculated by the two methods to determine the mean, variance, standard deviation, the standard er r o r and a t value. Again the ana l y s i s shows that there i s a s i g n i f i c a n t d i f f e r e n c e between the surface concentration c a l c u l a t e d from the rinsed tubing and from the depletion of t o t a l r a d i o a c t i v i t y . 3.4.5 E f f e c t of pH on BSA adsorption The s o l u b i l i t y and s t r u c t u r a l s t a b i l i t y of protein macromolecules are a r e s u l t of many interactions. If the e l e c t r o s t a t i c i n t e r a c t i o n s are modified by a change i n the pH a conformational change may be anticipated, p o t e n t i a l l y r e s u l t i n g i n a change i n the surface concentration of the adsorbed protein. A s e r i e s of adsorption runs was c a r r i e d out at various pH values and a concentration of 0.5 mg/ml. The r e s u l t s from counting the rinsed tube 66 are p l o t t e d i n Figure 3.14. The plot shows that as the pH increases the surface concentration increases to a maximum at around the i s o e l e c t r i c point of BSA, pH 4.9, and then decreases to a minimum going through pH 7.4. As the pH becomes more basic the surface concentration increases again and then decreases under very basic conditions. This behaviour d i f f e r s from the adsorption of BSA on s i l i c a with pH, (Morrisey and Stromberg, 1974), where there i s a maximum around the i s o e l e c t r i c point but then the adsorption decreases. Polyethylene i s an example of an inert hydrophobic surface i n that i t does not contain any rea c t i v e groups and i s capable of binding proteins only by d i s p e r s i o n forces and hydrophobic interactions. Surface contaminants may allow other types of bonding, however. It i s important to determine whether the a l k a l i n e s o l u t i o n had any e f f e c t on the surface. The tube was f i l l e d with PBS/azide, pH 9.0, l e f t f o r 4 hours and then an adsorption experiment was conducted at pH 7.4. The surface concentration along with others at pH 7.4 are given i n Table 3.7.The surface Table 3.7 Surface concentration ( D of BSA at pH 7.4 from a 0.5 mg/ml s o l u t i o n T tube ± s.d. 0.215 ± 0.022 0.223 ± 0.022 0.223 ± 0.023* t the tube was exposed to pH 9.0 before the adsorption run 67 Fig. 3.14. The surface concentration of BSA adsorbed to polyethylene tubing plotted against the pH. The bulk concentration in each case was 0.5 mg/ml. concentrations show that there i s no change on exposing the tubing to pH 9.0 before an adsorption run therefore one can conclude that the pH does not i r r e v e r s i b l y e f f e c t the s o l i d polyethylene surface. Reversible changes could not be detected by t h i s approach, however. To test f o r desorption a concentration of 100 pg/ml was used and adsorption experiments were c a r r i e d out. The displacement of the protein s o l u t i o n was with PBS at the same pH as the protein so l u t i o n . The r e s u l t s are given i n Table 3.8. Table 3.8 Surface concentrtations ( D calculated from the rinsed tube and from 1 2 depletion of t o t a l cpm. The pH was adjusted with borax or NaOH . pH Concentration T tube ± s.d. V cpm ± s.d. (Ug/ml) (ug/cm ) (ug/cm ) 5. 3 89 0. , 172 + 0. .017 0. , 176 + 0. , 002 8. 8 1 95 0. 267 + 0. 025 0. 270 + 0. 003 7. 4 80 0. , 131 + 0. .018 0. , 135 + 0. . 002 5. 0 488 0. , 293 + 0. .029 0. , 306 + 0. . 005 5. 3 511 0. .263 + 0. .025 0. . 319 + 0. , 004 8. 6 1 452 0. 269 + 0. 031 0. 301 + 0. 004 9. o 2 517 0. , 318 + 0. .031 0. , 320 + 0. , 005 7. 4 516 0. , 195 + 0. .019 0. , 200 + 0. . 004 At a low protein concentration the surface concentrations c a l c u l a t e d from the r i n s e d tube and from the depletion of t o t a l counts are s i m i l a r i n d i c a t i n g that n e g l i g i b l e or no desorption i s detected on washing the tubing. The amount of BSA adsorbed at pH 5.3 and pH 8.8 i s increased from 69 that at a pH of 7.4 at t h i s low concentration as well. To determine i f the NaOH had any e f f e c t on the adsorption experimental runs were c a r r i e d out using borax to a l t e r the pH. The r e s u l t s given in Table 3.8 show that the adsorption increases at pH 9 did not depend on the agent used to make the prot e i n s o l u t i o n a l k a l i n e . Again, the equilibrium values (depletion) are s l i g h t l y higher than the values following r i n s i n g , Figure 3.15. S t a t i s t i c a l analysis was c a r r i e d out using the paired-sample t test on the surface concentration-pH data; see Appendix 4. Again i t was concluded that the surface concentrations c a l c u l a t e d by the depletion of the t o t a l r a d i o a c t i v i t y were higher than those calculated from the rinsed tubing, suggesting protein i s desorbed. 70 CN E o o - M c <D O c o o <D o o t : CO 0.4 -CD 5 0.3 c o 0.2 0.1 -0.0 Fig. 3.15. Adsorption of BSA on polyethylene as a function of pH from a 0.5 mg/ml solution. The surface concentrations were calculated from the rinsed tubing (*) and from depletion of the total radioactivity (o). CHAPTER 4 CONCLUSIONS Measurements of adsorption of r a d i o l a b e l l e d BSA on hydrophobic o polyethylene tubing at 23 C showed that a steady-state surface concentration was established i n 2 hours and remained constant over a period of 8 hours. The adsorption isotherm was apparently Langmuir-1ike even though the Langmuir assumptions are not obeyed. The question of r e v e r s i b i l i t y was investigated i n t h i s thesis. The s t a t i s t i c a l a n a l y s i s showed a s i g n i f i c a n t difference between the surface concentration c a l c u l a t e d from the rinsed tubing and that from depletion of the t o t a l r a d i o a c t i v i t y . This suggests a small amount of desorption occurs on washing the tubing with buffer, estimated from Figure 3.13 to be approximately 5%. This i s a lower l i m i t to the amount desorbing, however, since the surface concentration calculated by the depletion method i s probably lower than the actual value due to the loosely bound pr o t e i n being released as the pro t e i n concentration decreases during displacement by buffer. Hence C D i s l i k e l y higher than the equilibrium value, implying that the surface concentration i s underestimated. The surface concentration-pH curve shows two maxima. The maximum at pH 5 occurs at the i s o e l e c t r i c point of the protein, t h i s has been observed by other workers; ( B u l l , 1956; Morrissey and Stromberg, 1974). This shows the importance of protein-protein e l e c t r o s t s t i c interactions. At the i s o e l e c t r i c point the protein has a net zero charge and maximum adsorption 72 occurs. Away from the i s o e l e c t r i c point the charge on the p r o t e i n increases, causing repulsion between protein molecules and the surface concentration decreases. The second maximum at pH 9.5-10 can be explained by an increase i n oligomer adsorption. From the SDS-PAGE ana l y s i s i t was shown that as the pH increases a greater percentage of BSA oligomers i s present in s o l u t i o n . The increase in surface concentration can be explained by an increase i n BSA dimer and higher oligomeric molecules being adsorbed. It was shown by releasing the adsorbed protein from the tube with hot SDS and running a gel that the larger molecular weight oligomers were adsorbed p r e f e r e n t i a l l y . P r e f e r e n t i a l adsorption of larger molecular weight species has been reported by other workers ( G i H i land and Guttoff, 1960). As the pH increases the BSA molecule uncoils and an expansion s i m i l a r to the one under a c i d i c conditions occurs around pH 10.3 (Tanford et a l . , 1955). A large increase in the net negative charge on the BSA molecules could therefore account for the decrease i n surface concentration seen above pH 9.5 due to e l e c t r o s t a t i c repulsion. S t a t i s t i c a l a n a l y s i s c a r r i e d out on the surface concentration-pH data showed a s i g n i f i c a n t difference between the surface concentration c a l c u l a t e d from c u t t i n g up the tubing and that c a l c u l a t e d by the depletion of the t o t a l r a d i o a c t i v i t y and desorption was concluded. The r e v e r s i b i l i t y of protein adsorption i s important i n developing thromoresistant materials. It has been shown that by precoating a surface with albumin the thrombogenic character of the material i s increased, since p l a t e l e t adhesion i s reduced (Lyman et al. , 1971). If albumin adsorption i s r e v e r s i b l e , as indicated here, an i n i t i a l l y thromboresistant albumin precoated surface would be expected to become less so with time. Hence, 73 the r e s u l t s obtained here bear relevance to the development of non-thrombogenic surfaces. 74 REFERENCES Andersson, L. 0. (1966). The heterogeneity of bovine serum albumin. Biochim. Biophys. Acta 117, 115-133. Andrade J. D. (1985). P r i n c i p l e s of protein adsorption. In Surfaces and interfacial aspects of biomedical polymers. Vol. 2. Protein adsorption; Andrade J. D., Ed; Plenium Press: New York, 1985; Baier, R.E. (1977). The organization of blood components near interfaces. Ann. N.Y. Acad. Sci. 283, 17-36. Baier, R. E. and Dutton, R. C. (1969). I n i t i a l events i n interactions of blood with a fo r e i g n surface. J. Biomed. Mater. Res. 3, 191-206. B i r d i , K. S. (1973). Spread monolayer f i l m s of protein at the air-water interf a c e . J. Colloid Interface Sci. 43, 545-547. Brash, J. L. (1981). Protein interactions with a r t i f i c i a l surfaces. In Interact ions of the blood with natural and artificial surfaces; Salzman, E. W., Ed; Marcel Dekker: New York, 1981; pp 37-60. Brash, J. L . and Davidson, V. J. (1976). Adsorption on glass and polyethylene from solutions of fibrinogen and albumin. Thromb. Res. 9, 249-259. Brash, J. L. and Lyman, D. J. (1969). Adsorption of plasma proteins i n s o l u t i o n to uncharged hydrophobic polymer surfaces. J. Biomed. Mater. Res. 3, 175-189. Brash, J. L. and Samak, Q. M. (1978). Dynamics of interactions between human albumin and polyethylene surface. J. Colloid Interface Sci. 65, 495-504. Brash, J. L. and Uniyal, S. (1979). Dependence of albumin-fibrinogen simple and competitive adsorption on surface properties of biomaterials. J. Polym. Sci., Polym. Symp. 66, 377-389. Brash, J. L., Uniyal, S. and Samak, Q. (1974). Exchange of albumin on polymer surfaces. Trans. Am. Soc. Artif. Intern. Organs 20, 69-76. Brooks, D. E. , Grieg, R. G. and Janzen, J. (1980). Mechanisms of erythrocyte aggregation. In Erythrocyte mechanisms and blood flow; Cokelet, G. R. , Meiselman, H. J. and Brooks, D. E., Eds.; Alan R. L i s s Inc., New York, 1980; pp 119-140.. B u l l , H. B. (1956). Adsorption of bovine serum albumin on glass. Biochem. Biophys. Acta 19, 464-471. 75 Chan, B. M. C. and Brash, J. L. (1981). Adsorption of fibrinogen on glass: R e v e r s i b i l i t y aspects. J. Colloid Interface Sci. 82, 217-225. Cheng, Y. C., Darst, S.A. and Robertson, C. R. (1987). Bovine serum albumin adsorption and desorption rates on s o l i d surfaces with varying surface properties. J. Colloid Interface Sci. 118. 212-213. Chuang, H. Y. K. , King, W. F. and Mason, R. G. (1978). Interactions of plasma proteins with a r t i f i c i a l surfaces: protein adsorption isotherms. J. Lab. Clin. Med. 92, 483-496. Cohen Stuart, M. A., Cosgrove, T. and Vincent, B. (1986). Experimental aspects of polymer adsorption at s o l i d / s o l u t i o n interfaces. Ad. colloid Interface Sci. 24, 143-239. Cohn, E. J., Hughes, W. L. Jr. and Wear, J. H. (1969). Preparation of serum and plasma proteins. XIII. C r y s t a l l i z a t i o n of serum albumin from ethanol-water mixtures. J. Am. Chem. Soc. 69, 1753-1764. Cuypers, P. A., Hermens, W. T. and Hemker, H. C. (1977). El 1ipsometric study of protein f i l m on chromium. Ann. N.Y. Acad. Sci. 283, 77-85. Davis, B. J. (1964). Disc electrophoresis. II. Method and a p p l i c a t i o n to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971). E l e c t r o p h o r e t i c a n a l y s i s of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617. Fazekas de St. Groth, S., Webster, R. G. and Datyner, A. (1963). Two new s t a i n i n g procedures f o r quantitative estimation of proteins on elec t r o p h o r e t i c s t r i p s . Biochim. Biophys. Acta. 71, 377-391. Finlayson, J. S. (1965). E f f e c t s of long term storage on human serum albumin. II. Follow-up of chromatographically and u l t r a c e n t r i f u g a l l y detectable changes. J. Clin. Invest. 44. 1561-1565. Finlayson, J. S., Suchinsky, R. T. and Dayton, A. L. (1960). E f f e c t s of long-term storage on human serum albumin. I. Chromatographic and u l t r a centrifuge aspects. J. Clin. Invest. 39, 1837-1840. Foster, J. F. (1960). Plasma albumin. In The Plasma Proteins, Putman F. W., Ed.; Academic Press, N.Y., 1960. F r i e d l i , H. and K i s t l e r , P. (1970). Polymers i n preparations of human serum albumin. Vox Sang. 18, 542-546. G i l l i l a n d , E. R. and Gutoff, E. B. (1960). R u b b e r - f i l l e r interactions: S olution adsorption studies. J. Appl. Polym. Sci. 3, 26-42. Grant, W. H., Smith, L. E. and Stromberg, R. R. (1977). Radiotracer techniques f o r protein adsorption measurements. J. Biomed. Mater. Res. Symp. 8, 33-38. 76 Harmsen, B. J. M., De Bruin, S. H., Janssen, L. H. M. , Rodrigues, J . F . and Van Os, G. A. J. (1971). pK change of imidazole groups i n bovine serum albumin due to conformational change at neutral pH. Biochemist ry 10. 3217-3221. Hartley, R. W. J r . , Peterson, E. A. and Sober, H. A. (1962). The r e l a t i o n of free s u l f h y d r y l groups to chromatographic heterogeneity and polymerization of bovine serum albumin. Biochemistry 1, 60-68. Harwig, S. S. L., Harwig, J. F., Coleman R. E. and Welch M. J. (1975). E f f e c t of i o d i n a t i o n l e v e l on the properties of r a d i o l a b e l l e d fibrinogen. Thromb. Res. 6, 375-386. Hikmet, R. A. M. , Narh, K. A., Barham, P. J. and Ke l l e r , A. (1985). Adsorption-entanglement layers i n flowing high molecular weight polymer solutions. Prog. Colloid Polym. Sci. 71., 32-43. Janatova, J., F u l l e r , K. and Hunter, M. J. (1968). The heterogeneity of bovine serum albumin with respect to s u l f h y d r y l and dimer content. J. Biol. Chem. 243, 3612-3622. Janzen, J. (1985). PhD. Thesis, U n i v e r s i t y of B r i t i s h Columbia, Canada. Jenkins, C. S. P., Packman, M. A., Guccione, M. A. and Mustard, J. F. (1973) . Modification of p l a t e l e t adherence to protein-coated surfaces. J. Lab. Clin. Med. 81, 280-290. Kim, S. W. , Lee, R. G. , Oster, H., Coleman, D. , Andrade, J. D., Lentz, D. J. and Olsen D. (1974). P l a t e l e t adhesion to polymer surfaces. Trans. Am. Soc. Art if. Intern. Organs. 20, 449-455. Lee, R. G., Adamson, C. and Kim, S. W. (1974). Competitive adsorption of plasma proteins onto polymer surfaces. Thromb. Res. 4, 485-494. Lee, R. G. and Kim, S. W. (1974). Adsorption of proteins onto hydrophobic polymer surfaces: Adsorption isotherms and k i n e t i c s . J. Biomed. Mater. Res. 8, 251-259. Leonard, W. J. J r . , V i j a i , K. K. and Foster, J. F. (1963). A s t r u c t u r a l transformation i n bovine and human plasma albumin i n a l k a l i n e s o l u t i o n as revealed by rotatory d i s p e r s i o n studies. J. Biol. Chem. 238. 1984-1988. Lyklema, J. and Norde, W. (1973). Biopolymer adsorption with s p e c i a l reference to the serum albumin-polystyrene latex system. Croatica Chemica Acta 45, 67-83. Lyman, D. J., Klein, K. G., Brash, J. L., F r i t z i n g e r , B. K. , Andrade, J.D. and Bonomo F. (1971). P l a t e l e t i n t e r a c t i o n with protein-coated surfaces: An approach to thromboresistant surfaces. Thromb. Diath. Haemorrh. Suppl. 42, 109-114. Lyman, D. J., Metcalf, L. C., Albo, D. J r . , Richards, K. F. and Lamb, J. (1974) . The e f f e c t on chemical structure and surface properties of synthetic polymers on the coagulation of blood. III. In vivo adsorption 77 of proteins on polymer surfaces. Trans. Am. Soc. Artif. Intern. Organs 20,'474-478. MacRitchie, F. (1972). The adsorption of proteins at the s o l i d / l i q u i d interface. J. Colloid Interface Sci. 38, 484-488. Markwell., M. A. K. (1982). A new s o l i d - s t a t e reagent to iodinate proteins. i4nal. Biochem. 125, 427-432. 131 McFarlane, A. S. (1963). In vivo behavior of I-fibrinogen. J. Clin. Invest. 42, 346-361. Morrissey, B. W., Smith, L. E. and Stromberg, R. R. (1976). E l 1ipsometric in v e s t i g a t i o n s of the e f f e c t of potential i n blood p r o t e i n conformation and adsorbance. J. Colloid Interface Sci. 56, 557-563. Morrissey, B. W. and Stromberg, R. R. (1974). The conformation of adsorbed blood proteins by infr a r e d bound f r a c t i o n measurements. J. Colloid Interface Sci. 46. 152-164. Norde, W. and Lyklema, J. (1978). The adsorption of human plasma albumin and bovine pancreas ribonuclease at negatively charged polystyrene surfaces. V. Microcalorimetry. J. Colloid Interface Sci. 66, 295-302. Nyilas, E., Chiu, T. H. and Herzlinger, G. A. (1974). Thermodynamics of native p r o t e i n / f o r e i g n surface interactions. I. Calorimetry of the human y-globulin/glass system. Trans. Am. Soc. Artif. Intern. Organs 20, 480-490.. Oreskes, I. and Singer, J. M. (1961). The mechanism of p a r t i c u l a t e c a r r i e r reactions. I. Adsorption of human y-globulin to polystyrene latex p a r t i c l e s . J. Immunol. 86, 338-344. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci. 121, 321-349. Packman, M. A., Evans, G., Glumm, M. F. and Mustard, J. F. (1969). The ef f e c t on plasma proteins on the int e r a c t i o n of p l a t e l e t s with glass surfaces. J. Lab. Clin. Ned. 73, 687-697. Perutz, M. F. (1978). E l e c t r o s t a t i c e f f e c t s i n proteins. Science 201, 1187-1191. P i t t , W. G. and Cooper, S. L. (1986). FTIR-ATR studies on the e f f e c t of shear rate upon albumin adsorption onto polyurethaneurea. Biomaterials 7 340-347. Regoeczi, E. (1984). Iodine-labeled plasma proteins. Volume 1. CRC Press, Inc. Boca Raton, F l o r i d a . Rowland, F. W. and E i r i c h , F. R. (1966). Flow rates of polymer s o l u t i o n s through porous disks as a function of solute. II. Thickness and structure of adsorbed polymer films. J. Polym. Sci. A-1 4, 2401-2421. 78 Shapiro, A. L. , Vinuela, E. and Maizel, J. V. (1967). Molecular weight estimation of polypeptide chains by electrophoresis i n SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. Shoemaker, D. P., and Garland, C. W. (1962). Experiments in physical chemistry, McGraw-Hill book company, Inc. . Silberberg, A. (1962). The adsorption of f l e x i b l e macromolecules. Part II. The shape of the adsorbed molecule; the adsorption isotherm surface tension and pressure. J. Phys. Chem. 66, 1884-1907. Smith, J. K., Walt, J. G., Watson, C. N. and Mastenbroek, G. G. A. (1972). A l t e r a t i o n s to freeze-drying f or removal of ethanol from plasma proteins. I. Vacuum d i s t i l l a t i o n of human albumin. Vox Sang. 22, 120-130. Smith, P. K., Krohn, R. I., Hermanson, G. T. , Mallia, A. K., Gartmer, F. H. , Provenzano, M. D. , Fujimoho, E. K., Goeke, N. M. , Olson, B. J. and Klenk, D. C. (1985). Measurement of p r o t e i n using b i u r i c h o n i n i c acid. Anal. Biochem. 150. 76-85. S o l l i , N. J. and,Be r t o l i n i , M. J. (1977). Polymer d i s t r i b u t i o n i n human serum albumin powders prepared by lyophi1ization or acetone drying. Vox Sang. 32, 239-241. Squire, P. G., Moser, P. andO'Konski, C. T. (1968). The hydrodynamic properties of bovine serum albumin. Biochemistry, 7, 4261-4272. Stromberg, R. R. , Morrissey, B. W., Smith, L. E. , Grant, W. H. and Fenstermaker, C. A. (1975). Interactions of blood proteins with s o l i d surfaces. PB 241-267 (Available from NTIS). Annual report prepared f o r the Biomaterials Program, National Heart and Lung I n s t i t u t e , NIH, Bethesada, Md. Tanford, C., Buzzell, J.G., Rends, D. G. and Swanson, S.A. (1955). The r e v e r s i b l e expansion of bovine serum albuminin a c i d solutions. J. Am. Chem. Soc. 77, 6421-6428. Taylor, G. (1953). Dispersion of soluble matter i n solvent flowing slowly through a tube. Proc. Roy. Soc. London A. 219, 186-203. Wada, A. and Nakamors, N. (1981). Nature of the charge d i s t r i b u t i o n i n proteins. Nature 293, 757-758. Wagner, M. L. and Scheraga, H. A. (1956). Gouy d i f f u s i o n studies of bovine serum albumin. J. Phys. Chem. 60, 1066-1076. Weber, K. and Osborn, M. (1969). The r e l i a b i l i t y of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244. 4406-4412. Young, B. R., P i t t , W. G. and Cooper, S. L. (1988). Protein adsorption on polymeric biomaterials. I. Adsorption isotherms. J. Colloid Interface Sci. 124, 28-43. 79 Zar, J. H. (1984). Biostatistical Analysis, 2nd Ed., Prentice-Hall, Inc., Englewood C l i f f s , N.J. . Zucker, M. B. and Vroman, L. (1969). P l a t e l e t adhesion induced by fi b r i n o g e n adsorbed onto glass. Proc. Soc. Exp. Biol. Med. 131. 318-320. 80 APPENDIX 1 ABBREVIATIONS A a c t i v i t y d surface area d surface area per s i t e s a equilibrium s o l u t i o n concentration BIS N,N-methylene-bis-acrylamide BSA bovine serum albumin C bulk protein concentration b C monolayer concentration m C weight of protein adsorbed per unit area of surface s cpm counts per minute dpm d i s i n t i g r a t i o n s per minute EDTA ethylenediamine tetraacetate FEP f l u o r i n a t e d ethylene-propylene copolymer FTIR-ATR f o u r i e r transform i n f r a r e d spectroscopy coupled with attenuated t o t a l reflectance optics HMWk high molecular weight k i t HSA human serum albumin K adsorption constant LMWK low molecular weight k i t MW molecular weight N Avogadro's number A OD o p t i c a l density 81 PAGE polyacrylamide gel electrophoresis PBS phosphate buffered s a l i n e PEUU polyether urethane PVC p o l y ( v i n y l chloride) R^ , r e l a t i v e mobility SA s p e c i f i c a c t i v i t y SDS sodium dodecyl sulphate SR s i l i c o n e rubber t t ime TCA t r i c h l o r o a c e t i c a c i d TD tracking dye TEMED N,N,N',N'-tetramethylenediamine TLC t h i n layer chromatography t r i s Tris(hydroxymethyl)aminomethane v/v volume per volume AG free energy of adsorption ads AH enthalpy of adsorption ads AS entropy of adsorption ads 2 T surface concentration (pg/cm ) A decay constant (s 1) 82 / APPENDIX 2 CALCULATIONS AND ERROR ANALYSIS A2. 1 Error analysis An equation can be represented by the formula F = f ( x , , x_, x ) (A2. 1 1 •* n The value of F i s ca l c u l a t e d by s u b s t i t u t i n g experimentally determined values of X [ into the formula ( f ) . An i n f i n i t e s i m a l change in F i s ca l c u l a t e d by considering the i n f i n i t i s i m a l change in dx ( and , r - 8F , 8F , 8F . , 0 i dF = -=— dx. + — 5 — dx, + + — dx (A2.2) dx, 1 3x, 2 Sx n For f i n i t e changes small enough not to e f f e c t the p a r t i a l d e r i v a t i v e . ^  3F . 3F . 3F . c A / - , o i AF = —=— Ax. + — Ax_ + + — Ax (A2.3J dx, 1 9x, 2 dx n 1 2 n This formula provides the most conservative estimate of the uncertainty i n F propogated by the uncertainties Ax^ i n the independent variables. However, i n r e a l i t y there i s a high p r o b a b i l i t y that some errors i n x^ w i l l cancel each other out. To allow f o r t h i s e f f e c t , square both side of equation A2.3: 83 , A r,,2 f 3F " I 2 , . ,2 ( 8F ' (AF) = — Ax.) + - 3 — ( A x , ) " + +2 [• 3F ] f_3F_" Ax ±Ax 2 + .... (A2.4; If the average i s taken over a l l values of Ax^ ^ and Ax 2, each Ax( has an average of zero and the cross terms vanish but the average of the squared terms are p o s i t i v e and remain. Taking the square root of each side the propogated uncertainty i n F i s given by (Shoemaker and Garland, 1962) AF = (Ax )' n 1/2 (A2.5) For the sum Y = A + B the imprecision in Y calculated from equation A2.5 i s given by AY = AA2 + AB 1/2 (A2.6] therefore AY = |^  AA2 + AB 2 j 1/2 (A2.7) AB For Y = — the error i n Y from equation A2.5 i s given by AY = -, 1/2 AC (A2.8] taking the square of each side (AY)' - (4-p * [4-] V • AC (A2.9] 84 m u l t i p l y i n g each side by 2 ' AB - 2 (A2.10] Taking the square root and multiplying by AB/C, (Y), gives the imprecision i n Y as AY = 1/2 (A2.11) The following error analysis w i l l use the equations i n the form of A2.7 and A2.11. A2.2 Imprecision i n the a c t i v i t y The net a c t i v i t y , (A ), (observed counts per unit time), of a N radioactive sample i s the difference between the t o t a l a c t i v i t y , (A) the background a c t i v i t y , (B). and A = A - B N (A2.12] When determining the imprecision i n the counts generally more than one a c t i v i t y i s added together. For more than one sample the net a c t i v i t y would be s. = I [ \ -B) = I \ IB (A2.13) where A^  i s the a c t i v i t y of the i t h sample and i i s the number of samples counted. The imprecision can be espressed as 85 AA = N i t * * , r + i( A B)' 1/2 (A2.14) since the a c t i v i t y i s the observed counts, (C), per unit time, ( t ) , A = l and B = the imprecision can now be written as AA = N I t l 2 V + i A t t B 1/2 (A2.15) and AA = A i AB = A i f c 1 r a c i i 2 + r At i i 2 -| t 1 -c t i j • r c i B r a c i B 2 + r At ] B 2 i t B J C L B t ^ B J -1/2 1/2 (A2.16) B (A2.17) from Poisson s t a t i s t i c s AX = v X s u b s t i t u t i n g equations A2.16 and A2.17 into equation A2.15 and assuming AA = N c 1 v 1 At, YA* + i B 2 At, 1/2 (A2.18) At, since i s very small the imprecision can be written as 86 AA = N 1/2 (A2.19) when, t = 1 minute, B = C and A = C B 1 i AA = N n 1/2 + iB (A2.20) A2.3 SDS-PAGE GELS. % monomer or polymer ± error The BSA monomer and polymers appear as separate bands on a SDS-PAGE gel. The amount of BSA in each band i s cal c u l a t e d by taking the sum of the a c t i v i t y i n a p a r t i c u l a r band (EA^) and d i v i d i n g by the t o t a l a c t i v i t y (A^). For example, the % monomer i n a sample i s given by I A % monomer (%M) = — — — x 100 (A2.21 and the erro r i s cal c u l a t e d from A°/=M = AA 2 n 1/2 %M (A2.22) using the imprecision i n the counts from equation A2.20 to ca l c u l a t e AZA. and AA . T 87 A2.4 Surface concentration from c u t t i n g up the tubing 1. The tubing was cut into twenty-five 20 cm sections and gamma counted. 2. The background count was subtracted from each section. 3. The surface concentration was ca l c u l a t e d f o r each section using the following equation where T = surface concentration C = tube s e c t i o n cpm d = surface area SA = s p e c i f i c a c t i v i t y 4. The err o r was c a l c u l a t e d using where AC was c a l c u l a t e d from equation 2.23 and ASA was obtained from the deviation i n determining the s p e c i f i c a c t i v i t y . 5. The value p l o t t e d i s an average of the twenty-five sections. A2.5 Surface concentration c a l c u l a t e d from the t o t a l counts The surface concentration was ca l c u l a t e d using the equation C r = (A2.23) SA d (A2.24) 88 r = c - c T 0 SA d (A2.25) where r = surface concentration d = surface area = tube cpm C q = output cpm from the displaced radiolabelled BSA and where C = C - 0 T (A2.26) C = t o t a l counts 0 = output counts during the input of labelled protein. The error i n calculating the surface concentration i s given by: Af = C - C T 0 c c T - 0 2 2 * + ASA 4. Ld SA d 1/2 f (A2.27) since A [ C - C 1 = [ AC 2 + AC 2 1 1/2 (A2.28) the imprecision can now be written as Af = AC 2 + AC 2 T 0 ( C T " C 0 ) - * ASA 2 + - • SA d 1/2 r (A2.29] 89 APPENDIX 3 MISCIBLE DISPLACEMENT IN A CAPILLARY Taylor's analysis was used to determine the length of the zone of mixing i . e . , the displacement front for miscible displacement i n a tube (Taylor, 1953). When a s i n g l e l i q u i d flows through a c y l i n d r i c a l tube, assuming laminar flow, the v e l o c i t y d i s t r i b u t i o n Is parabolic. The maximum v e l o c i t y at the axis of the tube i s twice the average v e l o c i t y . When a s o l u t i o n i s d i s p l a c i n g another of the same v i s c o s i t y and density the centre of the invading s o l u t i o n flows much fa s t e r than the s o l u t i o n near the edge of the tube. In the absence of r a d i a l d i f f u s i o n t h i s r e s u l t s i n an ever-lengthening needle of the invading s o l u t i o n down the tube. The t i p of t h i s needle w i l l reach the end of the tube when half of the s o l u t i o n i n the tube has been displaced. This i s the breakthrough point and always occurs when h a l f a tube volume has been injected. When displacement occurs the invading s o l u t i o n sets up a large r a d i a l concentration gradient. The two solutions w i l l i n t e r d i f f u s e r a d i a l l y thus blunting the needle-like p r o f i l e of the invading solution. If the invading s o l u t i o n i s spread over a length of tube L, the time required f o r convection to make an appreciable change i n the concentration i s of the order L/u 0, where u 0 i s the maximum ve l o c i t y . If the time f o r molecular d i f f u s i o n to minimize the r a d i a l concentration gradient i s much shorter than the time f o r an appreciable gradient to be established by the v e l o c i t y 90 d i s t r i b u t i o n no needle w i l l occur and u„ 3.82D (A3.1: where a i s the tube radius and D i s the relevant d i f f u s i o n c o e f f i c i e n t . The length of the front r e f e r s to the distance over which the concentration ranges from 0 to 100% of the invading solution. The p o s i t i o n of the front from the entrance of the tube i s a function of the tube diameter, the flow rate and the d i f f u s i o n c o e f f i c i e n t . S u b s t i t u t i n g into equation A3. 1 the relevant values from the -7 2 adsorption experiments; L = 500 cm, a = 0.038 cm, D = 5.9 x 10 cm /s (Wagner and Scheraga, 1956) and u Q = 0.933 cm/s 500 0.038 ° - 9 3 3 3.8 2 5.9X10"7 536 > 169 and r a d i a l d i f f u s i o n predominates. Taylor has obtained an approximate s o l u t i o n to the problem where longit u d i n a l molecular d i f f u s i o n has been neglected and where the r a d i a l d i f f u s i o n i s rapid. The longitudinal transfer i s due to convection. This w i l l be the case when a dissolved material of uniform concentration C 0 i s allowed to enter a pipe at a uniform rate. At time t = 0 the p o s i t i o n of the invading material i s given by x = 0, where x represents the distance from the entrance of the tube. The pipe i s f i l l e d with solvent only, concentration C = 0. The s o l u t i o n to t h i s problem i s given by 91 c/c„ = c/c„ = 1 • i e r f 2 2 1 - i erf 2 2 2v^kt x. 2/ kt ^ (x 1<0) (x 1>0) (A3.2) (A3.3) where erf z = 2 e dz (A3.4) x« = x - -u_t i 2 0 (A3.5) and k = 2 2 a u 0 192D (A3.6) In t h i s l i m i t the concentration i s constant across any cross s e c t i o n due to rapid r a d i a l d i f f u s i o n . Using Taylor's approximation i t was possible to c a l c u l a t e the length of the mixing zone f o r the displacement of a protein s o l u t i o n by a buffer i n a polyethylene tube. The relevant constants used i n the c a l c u l a t i o n were l i s t e d on page 91. The t h e o r e t i c a l d i s t r i b u t i o n of the concentration was ca l c u l a t e d f o r the displacement of BSA i n a polyethylene tube. The plot of C/C 0 against distance i s shown i n Figure A3.1. The plot shows that at C/C Q = 0.5, x = 500 cm, i . e . , the tube has been f i l l e d with one tube volume of the d i s p l a c i n g buffer. When x i s 700 cm, C/C D = 0.004 and 99.6% of the BSA has been displaced by the buffer. This length of 700 cm can be used to c a l c u l a t e the amount of protein displaced and f i n a l l y the amount of protein adsorbed. 92 250 350 450 550 650 750 x (cm) Fig. A3.1. C / C 0 vs x for the miscible displacement of BSA with buffer in a capillary tube. The volume contained i n a tube of diameter 0.038 cm and length 700 cm i. e . , volume used to displace 99.6% of the protein, i s 794 u l . From the displacement of the protein i n an adsorption experiment the number of counts, hence the amount of BSA, i n 794 ul i s determined. The surface concentration i s ca l c u l a t e d from the following equation SA d where T = surface concentration C t = t o t a l counts i n tube during the adsorption experiment C 0 = cpm output i n a volume of 794 pi SA = s p e c i f i c a c t i v i t y d = t o t a l surface area The surface concentration c a l c u l a t e d i n t h i s way w i l l represent the amount adsorbed i n equilibrium with the bathing solution. If anything i t w i l l underestimate the true equilibrium value since some rapodly desorbing material may appear in the displaced desorblng s o l u t i o n , thus increasing C 0 and reducing ( C - C 0 ) . 94 APPENDIX 4 STATISTICAL ANALYSIS A plot of C (bulk concentration) against C /T (bulk b b concentration/surface concentration per unit area,) appears to be best f i t by two li n e a r regions of d i f f e r e n t slopes. A li n e a r regression equation was calculated f o r each set of data. The question to ask i s , are the slopes of these l i n e s s i g n i f i c a n t l y d i f f e r e n t or are they estimating the same population? The n u l l hypothesis H0: 1 t = 1 2 w i l l be tested. The n u l l hypothesis i s a statement of no difference. In t h i s case we are t e s t i n g the e q u a l i t y of two l i n e a r regression l i n e s (Zar, 1984). For a simple l i n e a r regression Y = a + bX (A4.1) using the method of least squares the slope or regression c o e f f i c i e n t i s given by (A4.2) and a = Y - bX (A4.3) where the crossproducts sum of the deviations from the mean i s given as 95 Exy = E(X - X)(Y - Y) = ZXY - ££_±I (A4.4) X and Y are the mean values of X and Y respectively, and n i s the number of samples. x i s the de v i a t i o n of a X value from the mean of a l l X's and the sum of squares i s given as Ex 2 = S(X - X) = SX 2 - (A4.5) n The student's t test was used to test the eq u a l i t y of two regression l i n e s . The test s t a t i s t i c i s mean difference b t - b 2 t = = (A4. 6) standard error of mean difference s-b ± - b 2 If the test s t a t i s t i c , t, i s greater than some c r i t i c a l value the hypothesis H n i s rejected and the alternate hypothesis, H , 1, * 1_ accepted. The c r i t i c a l value of t depends on the degrees of freedom {v) and the le v e l of s i g n i f i c a n c e (a). For a two-tailed t test H 0 w i l l be rejected i f Itl > t (A4.7) 1 1 a(2),u The standard er r o r of the differe n c e between the regression c o e f f i c i e n t s of sample 1 and sample 2 i s 96 s- -b - b 1 2 1 Y - x J p + L Y x J p (A4.8: ( Z x 2 ) 1 ( Z x 2 ) 2 [ s 2 x] i s the pooled residual mean square and denotes the varience of the Y coordinate a f t e r taking into account the dependence of Y on the X coordinate. The pooled r e s i d u a l mean square i s given by (residual S S ) ± + (residual S S ) 2 [ s 2 ) = (A4.9) p (residual DF) ± + (residual DF) 2 where SS = sum of squares DF = degree of freedom 2 ( Z X y ; Z residual SS = Zy - (A4.10] 2 Zx residual DF = n - 2 (A4.ll) Zy 2 i s the sum of squares of the dif f e r n c e between Y and the mean Y, and i s given as Xy 2 = Z(Y - Y) = ZY 2 - i?XL (A4. 12) n In the comparison of the l i n e a r regression l i n e s sample 1 r e f e r s to the l i n e obtained from c u t t i n g up the tubing and sample 2 i s that c a l c u l a t e d from the depletion of t o t a l counts. 97 A4. 1 Testing the difference between the two regression l i n e s H0= 1± = 1 2 H : 1. * 1. For sample 1: For sample 2: ZX = 13433.03 ZX = 13433.03 ZY = 73031.48 ZY = 69946.35 The X and Y values were f i r s t divided by 1000. Zx 2 = 19.62382 2 Zy = 517.7767 Zxy = 100.4844 n = 23 a = 184.661 b = slope = 5.1205 re s i d u a l SS = 3.2426 re s i d u a l DF = 21 Zx = 19.62382 Zy 2 = 467.0201 Zxy = 95.40137 n = 23 a = 197.134 b = slope = 4.8614 residual SS = 3.2255 residual DF = 21 f s 2 ] = 0 . 1 5 4 0 s- - =0.1253 b - b 1 2 t = 2.0675 Reject H_ i f It I 2= t ° 0 1 1 Ct(2),l> The c r i t i c a l value of t f o r v = 42 and a s i g n i f i c a n c e l e v e l a = 0.05 ( i . e . , 5%) i s taken from tables and t = 2.018 0. 05(2) ,42 H 0 i s rejected, therefore one can conclude that there i s a s i g n i f i c a n t d i f f e r e n c e between the two regression l i n e s . 98 If there i s a c o r r e l a t i o n between sample 1 and sample 2 as in the case when the surface concentration i s ca l c u l a t e d by c u t t i n g up the tubing and by depletion of the t o t a l r a d i o a c t i v i t y , a paired-sample t test can be used. This two-tailed t test c a l c u l a t e s a t value by using the difference, d , between the two samples. The mean, variance, standard deviation and standard e r r o r are calculated using the differ e n c e betwwen the samples and n i s the number of differences. A4.2 The paired-sample test f o r the adsorption isotherm data H 0: p 1 = u 2 HA: a± * u 2 In t h i s case the alternate hypothesis i s given by H u±> u when sample 1 r e f e r s to the surface concentration (T) c a l c u l a t e d from the depletion of the t o t a l r a d i o a c t i v i t y and sample 2 to that from the rinsed tubing. From the r e s u l l t s i n Table 3.6 Zd = 0.125 Ed 2 = 1.911 x 10"3 n = 23 d = Zd/n = 5.343 x 10 - 3 v = 23 - 1 = 22 SS = I d 2 - = 1. 232 x 10"3 n 2 SS -s variance = s = = 5.60 x 10 d v standard d e v i a t i o n = s = <ls = 7.482 x 10 d d S -3 standard e r r o r = s- = = 1.560 x 10 d 4ir 99 t = — = 3.483 s-d A one t a i l d i s t r i b u t i o n i s used to determine the c r i t i c a l t value since we are t e s t i n g the difference i n one d i r e c t i o n i . e . , the surface concentration c a l c u l a t e d from the depletion of t o t a l r a d i o a c t i v i t y i s always higher than that c a l c u l a t e d by c u t t i n g up the tubing, t = 1.717 0. 0S(1 ) , 22 Therefore, r e j e c t HQ. The two sets of T are s i g n i f i c a n t l y d i f f e r e n t . A4.3 The paired-sample test f o r the pH data. H a: u ± = u 2 Ha: U l * u 2 Sample 1 Sample 2 PH r (cpm) r (tube) d 2.0 0. 221 0 .206 0. 015 3. 4 0. 225 0 . 220 0. 005 4. 7 0. 367 0 . 355 0. 012 5.0 0. 298 0 . 293 0. 005 5.3 0. 276 0 . 263 0. 013 5.6 0. 273 0 .270 0. 003 6.5 0. 226 0 . 216 0. 010 7. 4 0. 236 0 . 223 0. 013 7.4 0. 218 0 . 215 0. 003 7.4 0. 229 0 . 223 0. 006 8. 6 0. 290 0 .269 0. 021 8.8 0. 384 0 . 361 0. 023 9.0 0. 319 0 .318 0. 001 9. 1 0. 414 0 . 401 0. 013 9. 2 0. 319 0 .305 0. 014 9.4 0. 357 0 . 351 0. 006 10.5 0. 342 0 .331 0. 011 11.5 0. 254 0 . 237 0. 017 12.0 0. 201 0 . 197 0. 004 Zd = 0.195 Zd2= 2.709 x 10"3 n = 19 d = 1.026 x 10"2 v = 19 - 1 = 18 SS = 7.08 x 10"4 s 2 = 3.93 x 10"5 s = 6.270 x 10"3 s- = 1.438 x 10"3 d t = 7.135 t =1.734 0. 05(1) , 18 Therefore, reject HQ. The two sets of f are s i g n i f i c a n t l y d i f f e r e n t . 100 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0046947/manifest

Comment

Related Items