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Protein chemistry of triose phosphate isomerase Burgess, Helen Diana 1976

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PROTEIN CHEMISTRY OF TRIOSE PHOSPHATE ISOMERASE by HELEN DIANA BURGESS B.A., University of Lethbridge, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n The Department of Chemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF ERITI3H COLUMBIA June, 1976 In present ing th i s thes i s in p a r t i a l f u l f i lmen t o f the requirements for an advanced degree at the Un iver s i t y of B r i t i s h Columbia, I agree that the L i b ra ry sha l l make i t f r e e l y ava i l ab le for reference and study. I fu r ther agree that permission for extens ive copying of th i s thes i s fo r s cho la r l y purposes may be granted by the Head of my Department or by h i s representat ives . It i s understood that copying or pub l i c a t i on of th i s thes i s f o r f i n anc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of /Ul O^nj^M^ The Un iver s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS It I I . ABSTRACT t The protein, t r l o s e phosphate isomerase (TIM) has been i s o l a t e d from f r e s h chicken breast muscle and p u r i f i e d by an-ion exchange chromatography on DSAE Sephadex A50..column. Fur-ther p u r i f i c a t i o n proceeded v i a Biogel A DEAE r e s i n . The TIM f r a c t i o n s of both chromatographies were contained i n two adja-cent protein peaks, A and B. The separation of the two peaks was found to be based upon isozymic differences i n the TIM a c t i v e protein; I s o e l e c t r i c focusing, both column and g e l , showed one isozyme i n the Peak A protein with pl=7.65 while Peak B protein contained two Iso-zymes of p i 7.56 and 7.49. Focusing of old peak B protein yielded a fourth isozyme with pl=7»62. No Isozymic separation was observed with d i s c gel electrophoresis at pH 8.5. Amino ac i d analysis which was c a r r i e d out on p u r i f i e d Peak A protein showed sub s t a n t i a l deviations from l i t e r a t u r e values. The Peak A isozyme was modified v i a the reaction of the t h i o l of cysteine with the maleimide N-ethylmalelmide (NEM), or t r l f l u o r o N-ethylmaleimide (FEM), as well as with the d i s u l -f i d e s 4,4» d i t h i o p y r l d l n e (4--PDS) or 5*5'-dithiobis(2-nitroben-zoic acid) (DTNB)i Two equivalents of reagent per molecule re-acted but a k i n e t i c noh-eqiiiVaienee-of the'two s i t e s to modification 19 was observed. F NMR of the FEM labeled protein was performed. The f i n a l chapter of t h i s thesis deals with the k i n e t i c analysis at several temperatures of the modification of TIM by k-FD3 and DTNB both i n the presence and In the absence of sub-i i i . s t r a t e glyceraldehyde 3-phosphate. Blphasic Arrhenlus plots with a break at approximately 2 5 ° C were observed f o r the 4 -PDS modification; In the presence of substrate, the a c t i v a t i o n energy for T > 2 5 ° C was 7 » 2 kcal/mole while f o r T < J 2 5 ° C i t was 50.0 kcal/mole. In the absence of substrate, the a c t i v a t i o n energy f o r T > 2 5 ° C was 4 . 4 kcal/mole while f o r T < T 2 5 ° C , i t was 39.9 kcal/mole. L a s t l y , a segment discussing the importance of the r e s u l t s i-described i n t h i s t h esis, i n terms of the current T I M leterature . i s included. i v TABLE OF CONTENTS CBAPTER PAGE I. GENERAL INTRODUCTION• . 1 1.1 P u r i f i c a t i o n and Characterization. 3 1.2 Enzymatic I n h i b i t i o n and the C a t a l y t i c Process of TIM...................ii..........5 1*3 St r u c t u r a l Aspects .12 I I . ISOLATION AND PURIFICATION 2.1 Materials ; i 19 2.2 Methods i.i . . i i 20 2.3 Results....... i 26 T i l l . CHARACTERIZATION OF PEAK A AND . PEAK . B 3.1 Methods .; .39 3*2 Results t j r ? IV. PROTEIN MODIFICATION 4.1 Introduction., . i i i 75 4.2 Methods . i . . . . . 84 4.3 Results i i . . ....90 V. KINETICS OF DISULFIDE MODIFICATION OF TIM 5.1 Methods 105 5.2 Results. i 112 CONCLUSIONS 122 REFERENCES . • • . 125 V. LIST OF TABLES TABLE . PAGE L Determination of a c t i v i t y units followed throughout a t y p i c a l l i v e chicken prep-ara t i o n of chicken breast muscle...... 28 II Stock solutions and buffers for SDS ge l electrophoresis .41 III I s o e l e c t r i c focusing solutions for gels .52 IV Amino a c i d analysis of Peak A TI M . . . i i . . 73 V Modification with 4-PDS .......... 112 Modification with DTNB . 12° v i . LIST OF FIGURES FIGURE PAGE 1 U l t r a v i o l e t spectra or NAD+ and NADH.....* 24 2 I n i t i a l DEAE Sephadex A 5 0 Chromatography 27 3 I n i t i a l DEAE Sephadex A50 Chromatography of a Live Chicken Preparation .30 4 Rechromatograr>hy of TIM Fractions on DEAE Sephadex A50 33 5 Rechromatcgraphy of peak A and Peak B TIM Fractions on DEAE Sephadex kjO 35 6 Rechromatography of Peak A and Peak B TIM Fractions on DE52 Cellulose 36 7 Rechromatography of TIM Fractions on Biogel A DEAE 38 8 Electrophoresis Apparatus 40 9 SDS Gel Electrophoresis of Protein Standards 44 10 A Sketch of the I s o e l e c t r i c Focussing (IEF) Column 47 11 Disc Gel Electrophoresis Using Fresh Protein from Peak A (A) and Peak B (B), and Old Protein _ from peak B (B* ) ; 5 9 12 Column IEF of Rechromatographed Peak A..... 62 13 Column IEF of Rechromatographed Peak B... 64 14 Column IEF of Rechromatographed Peak A and Peak B 65 15 Gel i s o e l e c t r i c Focussing of Peak A and Peak B ...67 16 Sample Gels of Gel I s o e l e c t r i c Focussing of Peak A and Peak B 68 17 Gel I s o e l e c t r i c Focussing of Fresh Protein from Peak A and Old Protein from peak B 7 i 18 U l t r a v i o l e t Spectra of NEM and FEM 1 80 19 Pseudo F i r s t Order Analysis of the Modification of TIM with NEM 01 v i i . FIGURE PAGE 20 1 ^ F NMR of FEM Modified Triose Phosphate Isomerase. .95. 21 - Pseudo F i r s t Order Analysis of the Cyanide Displacement of TNB" from the DTNB Modified peak A Protein « ••100 22 A Ty p i c a l Example of Monophasic K i h e t i c s -4-PDS Modification of TIM at 22 CC i n the Absence of Glyceraldehyde 3-Phosphate. 109 23 A Ty p i c a l Example of Bi-phasic K i n e t i c s -4-PDS Modification of TIM at 25°C i n the Absence of Glyceraldehyde 3-Phosphate. 110 24 Arrhenius Plot for the 4--PDS Modification of TIM i n the Presence of Glyceraldehyde-3-Phosphate . 115 25 Arrhenius Plot f o r the 4-PDS Modification of TIM i n the Absence of Glyceraldehyde-3-Phosphate • 116 ACKNOWLEDGEMENTS I would l i k e to express my appreciation to Dr. D.G. Clark whose enthusiasm and knowledge greatly aided supervision of my work. I wouj.a xiKe to thank Josepn Durgo of the Biochemistry Department (UBC) for technical assistance with the amino acid analysis as well as Dr. A.G. Marshall for his assistance with 19 the F. NMR and Dr. R.E. Plncock for the use of his spectro-photometer. I would l i k e to thank L e s l i e deBruyn and Monica E. Rosen-berg for t h r i r help i n the production of t h i s t h e s i s . F i n a l l y , I am very g r a t e f u l for the generous support of a National Research Council Postgraduate Fellowship (1973-197*0 and a H.R. MacMillan Family Fellowship (1974-1975) to study at UBC. CHAPTER I GENERAL INTRODUCTION The purpose of t h i s introduction i s to provide, l ) the l i t -erature basis for the present understanding of the presence'of multi-enzyme forms of trlosephosphate isomerase (TIM) from d i f f e r e n t sources, 2 ) the mechanism of the c a t a l y t i c process, and 3) some chemical and s t r u c t u r a l aspects of the enzyme. The TIM used f o r : the work presented i n t h i s thesis was Isolated from chicken breast muscle; however much of the early investigations have involved enzyme from rabbit muscle ( 4 ) and yeast (5)« In addition d e f i n i -t i v e studies have been preformed on both human.(6) and b a c t e r i a l (7) t r i o s e phosphate isomerase. These studies from the various . sources, formed the background for the I s o l a t i o n and p u r i f i c a t i o n of the protein, as well as the characterization and the chemical mod-i f i c a t i o n s which were performed and reported i n t h i s t h e s i s . The enzyme, t r i o s e phosphate isomerase (TIM) plays a cen-t r a l r o l e l n g l y c o l y s i s , as well as l n glucneogenesis and has one Of the highest c a t a l y t i c rates of a l l the g l y c o l y t i c enymes. It catalyzes the reverse aldoseketose lsomerization of glyceralde-hyde 3-phosphate ( G 3 P ) and dlhydroxyacetone phosphate (DHAP). This b l f u n c t i o n a l protein has been considered to be an u n l i k e l y point f o r metabolic regulation since i t s i t s high c a t a l y t i c turnover rate a b i l i t y and i n vivo concentration has l e d to the concept that i t i s not metabollcally rate l i m i t i n g . However, there i s evidence ( l , 2 ) of g e n e t i c a l l y transmitted isomerase d e f i c i e n c i e s l n humans which leads to 5-20$ the normal l e v e l s and can r e s u l t 2 . i n a severe metabolic block, even though the anticipated l e v e l s of TIM would suggest that i t s t i l l should not be rate l i m i t i n g . The lsomerase has been shown by various groups to be composed of several isozymes (3)» A few examples (6) of the TIM isozymic content of various species are: Species Number of Isozymes Human Spleen Rhesus Bovine Porcine Dove T u r t l e Prog Ca t f i s h Erythrocytes S k e l e t a l muscle Brain Liver Cardiac muscle 3 3 3 3 3 3 3 3 3 3 3 3 Heart Crab Lobster Shrimp Beetle Cricket Grasshopper Clam S n a i l Squid A s c a r l s suum Sea Anemonae Euglena g r a c i l i s  Escherichia c o i i  B a c i l l u s s u l l t l l i s  pseudomanas aeruginosa  Staphylococcusaureus Muscle Li v e r Kidney Brain 3 3 3 3 3 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 3. 1*1 P u r i f i c a t i o n and Characterization In recent years Interest has centered on the TIM obtained from ra b b i t muscle and chicken muscle. Preparation procedures for the rabbit muscle enzyme have been described (9»10) as well as for the chicken TIM ( l i ) . Considerable evidence has been pre-sented to show that the native enzyme i s composed of two subunits (9-16). Values f o r the native dimer molecular weight of rabbit TIM obtained p r i o r to 1970, range from 4300u-6uuuu (summarized by Norton et a l (3)) although l a t e r molecular weights were calculated to be approximately 53*000 (3,11). Chicken muscle TIM was found to be 48,400 (11) but more recent amino acid sequence and x-ray; crystallographic r e s u l t s place i t nearer to that of the rabbit muscle with a molecular weight of 54,400 (17,18). I t has been demonstrated that there are no d i s u l f i d e bridges l i n k i n g the two subunits, or within a single subunit "(5). • The heterogeneity of t r i o s e phosphate isomerase has been reported f o r a wide variety of animal (19,20) and human tissues (3*4). Suggestions f o r the multiple nature of the enzyme have included i t being the r e s u l t of an ar t e f a c t of the i s o l a t i o n pro-cedure (23)> the presence of conformational isoenzymes (13) or the existence of non-Identical subunits (5,24). The isozymes to date have been i d e n t i f i e d through polyacrylamide (5»25) and starch gel electrophoresis (5»20). In addition, a chromatographic separation for the isozymes of r a b b i t muscle TIM has been reported by Krletsch et a l (16). A rechromatography of p u r i f i e d rabbit muscle TIM by a DEAE-cellu-lose column eluted by a very shallow gradient succeeded i n sep-arating the material into three major forms? ^ , p,. and (97%) and two minor components, d and £ ,(3%)* Hybridization studies showed the d and t Isozymes to he homodimers AA and BB respect-i v e l y and § the heterodimer AB. The heterogeneity of rabbit TIM was therefore found to be l a r g e l y due to the combination of two chemically non-identical subunits A and B. Hybridization of the 6 and £ forms was more complex with reassociatlon r e s u l t i n g i n the formation of a l l f i v e bands as well as two ( l n the case of £ ) or four ( i n the case of £ ) a d d i t i o n a l more electrophor-e t i c a l l y mobile bands. Amino a c i d analyses of the three major . isozymes were able to reveal small differences but the t i t r a -t i o n of t h i o l s with 5,5 ,-dithiobls-(2-nitrobenzoate) (DTNB) gave i d e n t i c a l numbers f c r the number of su l f h y d r y l (cysteine) residues. A degree of ambiguity concerning TIM isozymes has existed i n the l i t e r a t u r e f o r some time. For example Lee and Snyder separated commercial rabbit TIM into f i v e bands i n acrylamide electrophoresis (26) with a pattern of r e l a t i v e a c t i v i t i e s and protein concentrations of 7(d)i 15(p)s 6 ( 5 ) j 2(S)t 1(£) with 90$ of the a c t i v i t y and protein l n the f i r s t 3 bands o<, p , • and JC; however Burton and Waley (23) found only the three ^, j3 and bands with 85$ a c t i v i t y l n the slower migrating <A band and 15$ i n the second (p) and t h i r d (t) f a s t e r migrating forms, but Coulson and Knowles (25) could detect just one sharp zone i n the polyacrylamide electrophoresis of commercial enzyme. The exper-imental conditions of electrophoresis, most notably pH, were probably the c o n t r o l l i n g factors since Krietsch (16) observed that i n polyacrylamide electrophoresis with a 1 g e l at pH 9*5 i n the presence of mercaptoethanol TIM migrates as one single band, while at pH 6.6 the protein showed the same heterogeneity pattern as i n starch gel electrophoresis. Scopes (20) observed a similar phenomenon with chicken mus-cle TIM, namely a single band resulted upon acrylamide e l e c t r o -phoresis (pH 8.5) while a minor contaminating band was seen to separate out on starch gel electrophoresis. These r e s u l t s have encouraged the b e l i e f that the protein from chicken i s r e l a t i v e l y free from Isozymes. Thus, much of the most recent work, most notably x-ray c r y s t a l structure studies, has centered on the chicken muscle enzyme. The implications of t h i s i n l i g h t of the observations reported i n t h i s thesis, w i l l be discussed i n the main body of t h i s work. .A t h i r d source of TIM f o r which the enzyme heterogeneity has been characterized i s human erythrocytes (6). Three isozymes have been found, to e x i s t for the human TIM as a r e s u l t of an AA, AB and BB d i s t r i b u t i o n of dlmers. Amino acid analyses and pep-tide f i n g e r p r i n t s have been able to indicate that the two types of subunits are very s i m i l a r but contain several differences i n t h e i r primary structures. They show si m i l a r c a t a l y t i c properties and are found In a l l human t i s s u e s . 1.2 Enzymatic I n h i b i t i o n and the C a t a l y t i c Process of TIM Characterization of an enzyme's active s i t e may be aided by chemical modification i f the reagent's s p e c i f i c i t y allows i n t e r -6. pretation with respect to the reaction s p e c i f i c i t y (amino ac i d modified) and topographical s p e c i f i c i t y ; The problem of reac-t i o n s p e c i f i c i t y may be solved by taking advantage of known re-action types while the problem of topographical s p e c i f i c i t y may be resolved by the procedure of " a f f i n i t y l a b e l i n g * i n which a protein reagent i s designed to resemble the substrate and thus have an a f f i n i t y f o r the substrate binding s i t e . The i n a c t i v a -t i o n of t r i o s e phosphate lsomerase has been achieved by haloacetal phosphates and.epoxides ( g l y c i d o l phosphate) (25,27) which are reactive analogs of the substrate dihydroxyacetone phosphate and have been proven to be a successful example of a f f i n i t y l a b e l i n g . A highly s e l e c t i v e modification of only one residue was ob-served with l o s s of a c t i v i t y proceeding pseudo f i r s t order upon addition of high reagent to enzyme molar r a t i o s . I t has been found that ^-glycerophosphate, a competitive i n h i b i t o r of TIM, protects the enzyme against i n a c t i v a t i o n which suggests a compet-i t i o n of haloacetol phosphates and glycerophosphate f o r the same active s i t e . The inac t i v a t e d enzyme was found to contain 1 mole of co-valently bound reagent per mole of c a t a l y t i c subunit (15)» There-fore i t was possible f o r the f i r s t time to determine with some certainty the presence of 2 active s i t e s (one/monomer) f o r TIM. The I n i t i a l modification of the peptide chains was found to proceed v i a an e s t e r i f l c a t l o n of glutamic a c i d ; A hexapeptlde containing the haloacetal phosphate has been i s o l a t e d from both chicken (28) and rabbit muscle (52) with the sequence: Ala-Tyr-Glu*-Pro-Val-Trp 7. TIM from a wide variety of species has been found to be i n a c t i -vated by haloacetol phosphates, which Increases the l i k e l i h o o d that a common es s e n t i a l region i s the s i t e of modification. I f the glutamyl carboxylate i s func t i o n a l i n c a t a l y s i s , i t may be expected to be an Invariant feature among t r i o s e phosphate i s o -merases. The above mentioned hexapeptlde was found to be con-served during evolution (30) and i s therefore probably c r i t i c a l to function. A comparison of yeast and rabbit muscle TIM was chosen as an i n d i c a t i o n of the constancy of the active s i t e g lu-tamyl residue (and adjacent amino acid sequence) since there i s a wide evolutionary separation between the two organisms. The hexapeptlde containing the active s i t e glutamic a c i d was found to be i d e n t i c a l i n both species. The problem exists as to whether the enzymatic i n a c t i v a t i o n r e s u l t s from modifying a c a t a l y t i c a l l y f u n c t i o n a l residue or merely from preventing substrate binding. However, several ob-servations suggest that the glutamyl residue i s functional i n c a t a l y s i s . For example, the only functional group i n model com-pounds f o r proteins which reacts with haloacetol phosphates i s the su l f h y d r y l f u n c t i o n a l i t y (27)» but the group i n TIM that reacts i s a carboxyl. The rate of e s t e r i f i c a t i o n i s r a p i d . In addition, i f the e s t e r i f i e d glutamyl residue of rabbit TIM i s not c a t a l y t -i c a l l y e s s e n t i a l but merely located l n the area of the active s i t e , i t would be expected that i n some species, residues not susceptible to e s t e r i f i c a t i o n would occupy the corresponding pos-i t i o n . However, the findings that TIM from e v o l u t i o n a r l l y d i -fc$. verse species are inactivated by haloacetol phosphates with i n -a c t i v a t i o n proceeding at sim i l a r rates, i s consistent with mod-i f i c a t i o n of a c a t a l y t i c a l l y f u n c t i o n a l , invariant residue. The p o s s i b i l i t y of the c a t a l y t i c a l l y important residue be-ing a glutamate f i t t e d well with previous mechanism which were postulated as the chemical pathway fo r the aldol-keto isomerism. The mechanism which was f i r s t formulated by Rose {31,3'd) involved the abstraction of a proton from G-3 of dihydroxyacetone phosphate to give an enediolate anion which i s able to pick up a proton at C-2 to y i e l d glyceraldehyde-3 phosphates H r. H |,0H" H *' 0 H H N ^ 0 H B-H^j C-=0 C—0" " H — C —OH • r, 1 ^ • I ^ CH20 ® CH20 (?) CH20 Q) Any conjugate base, including a glutamyl ^-carboxylate, could function i n t h i s proton t r a n s f e r . I t has been established that carboxylates can promote enol i z a t i o n v i a general base c a t a l y s i s , s i m i l a r to the mechanism above ( 3 3 ) * Prom the pH dependence of the rate of Inactivation of yeast TIM, the apparent pKa of the ac-t i v e - s l t e carboxyl group was estimated by Hartman to be 3*9-0.1 ( 3 4 ) . A f f i n i t y l a b e l i n g of TIM was carr i e d out further by the prep-ara t i o n of a reagent designed to mimic the cis - e n e d i o l which i s the postulated Intermediate In the reaction catalyzed by t r i o s e phosphate isomerase. This reagent i s g l y c i d o l phosphate ( 2 , 3 -9. epoxypropanol) which was found to l a b e l the glutamyl group l n the Isolated hexapeptldet Ala-Tyr-Glu*-Pro-Val-Trp ( 3 5 ) . This i s the same residue that was labeled by the haloacetol phosphates. The l a b e l i n g of TIM by t h i s reagent strongly supports the single general base mechanism as proposed for the aldose-ketose isomer-i z a t i o n of TIM ( 3 6 ) . The most powerful competitive i n h i b i t o r of TIM i s 2-phos-phoglycollate whose structure i s given below: 0 II y°\ I 0 The i n h i b i t o r s s t r u c t u r a l s i m i l a r i t y to the ene-diol and i t s anionic centre i s important f o r t r a n s i t i o n state binding. The u l t r a v i o l e t spectrum of TIM i n the 280nm region may be changed by binding of 2-phosphoglycollate and 2-glycerophosphate (another reagent which i n h i b i t s due to i t s a b i l i t y to bind to the a c t i v e s i t e ) but not by the anionic i n h i b i t o r s s u l f a t e or inorganic phosphate ( 3 7 ) . The s h i f t , which was most marked i n the 280nm •'region which corresponded to tyrosine and tryptophan absoiXbtlons was re l a t e d to the presence of each of these amino acids i n the hexapeptlde which was found to be e s t e r i f i e d upon i n a c t i v a t i o n of TIM by g l y c l d o l phosphate ( 3 7 ) . In addition to the change i n the U.V. spectrum, there was observed to be a change i n the crys-t a l structure of the enzyme upon binding of g l y c l d o l phosphate 10. which was able to suggest that there are two or more important s i t e s of binding.. The binding of the i n h i b i t o r and t r a n s i t i o n state analogue 2-phosphoglycollate to the rabbit muscle enzyme was able to e f f e c t a s i m i l a r sort of change as observed by the h% contraction i n the c r y s t a l structure. At the same time, &$) 6% contraction has been noted for binding of either t r i o s e sub-strate to r a b b i t or chicken muscle TIM (37,38). I t appears l i k e -l y then that the magnitude of the change upon binding of inhib-i t o r , represents a s t r u c t u r a l change i n the protein conformation which mimics the conformational change which occurs upon binding of the two substrates dlhydroxyacetone phosphate and glyceralde-hyde 3-phosphate. The c a t a l y t i c r o l e of t h i s substrate-induced conformation change i s unknown and our understanding of t h i s phenomonen i s a long term goal of t h i s laboratory. The dependence of the rate of enzyme-catalyzed reactions on pH i s an important parameter that must be accommodated by any com-plete proposal of mechanism. There Is fundamental information to be gained from a knowledge of the pH-dependence of the i n d i v i d u a l k i n e t i c parameters. They must be consistent with the mechanistic proposals and be interpretable with respect to the knowledge of the three-dimensional structure of the enzyme and the functional c a t a l y t i c residues. The most comprehensive series of pH rate studies were per-formed by plaut and Knowles (39). One of the most usef u l studies was the determination of the s t a b i l i t y of the chicken muscle en-zyme as a function of pH. Incubation of enzyme i n buffers of 1 1 various pH's was car r i e d out at 38°C f o r 6 hours before determin-ation of the enzymatic a c t i v i t y . The r e s u l t s showed a plateau ( 1 0 0 $ a c t i v i t y ) between pH 6.5 and 7.5 with a f a i r l y rapid de-crease i n s t a b i l i t y at lower or higher pH's. An i n t e r e s t i n g contrast to t h i s was observed i n the TIM Isolated from a psychro-p h i l i c organism (optimum growth below 20°C), C l o s t r i d i a , in'which a 70% reduction In enzyme a c t i v i t y with a half hour heat t r e a t -ment of c e l l - f r e e extracts at 32*C was seen (pH 7-3) (40). This can probably be explained by the evolutionary adaptation of t h i s microorganism. Other pH studies showed a pH dependence of k„ ./K which CeLu DO. allowed c a l c u l a t i o n of the pKa of two k l n e t i c a l l y important func-t i o n a l i t i e s : values of 6.05 and 9*05 were derived with dihydroxy-acetone phosphate (DHAP) as substrate and 6.0 and 9.2 when glyc-eraldehyde 3-phosphate (G3P) was used. The apparent pKa values i n k C a t / K m may r e l a t e to i o n i z a t i o n s i n the free enzyme or i n the-free substrate. Under the experimental conditions, the pKa v a l -ues of substrate alone are 6.0 for DHAP and 6.3 for G 3 P . There-fore, there i s a p o s s i b i l i t y that the lower observed pKa*s of the k ,/K p r o f i l e s have arisen from i o n i z a t i o n of substrates, cat m The upper pKa value of about 9 cannot be r e a d i l y assigned but p o s s i b i l i t i e s are lys i n e or arglnine residues. On the other hand, there i s the p o s s i b i l i t y that the i o n i z a t i o n at pH 9 governs a large-scale conformation change r e s u l t i n g In loss of enzyme ac-t i v i t y owing to a loss of the s t r u c t u r a l i n t e g r i t y or the active s i t e ( 3 9 ) . 12 1 . 3 S t r u c t u r a l Aspects A. The Subunit Monomer When studying the r e l a t i o n s h i p between the quaternary struc-ture of a multi-subunit enzyme and i t s a c t i v i t y , i t i s often of i n t e r e s t to know the enzymatic a c t i v i t y of the monomer, i f i n -deed association of the subunlts i s not necessary f o r c a t a l y t i c a b i l i t y . A useful approach i s to attach the enzyme to a s o l i d Support v i a a single subunit, and to remove the other(s) so that the properties of the i s o l a t e d subunit may be studied under con-di t i o n s where reassoclation i s not p o s s i b l e . Such a procedure was performed recently by F e l l and White ( 4 3 ) . They found that the k i n e t i c properties of chicken TIM were alte r e d by immobilization on Sepharose. The a c t i v i t y , which rep-resented that of the bound monomer gave a K m of 1.7mM while that of the soluble enzyme was reported to be 0.35^M. Upon denatur-ing with guanidine hydrochloride and subsequent reh y b r i d i z a t i o n with rabbit muscle enzyme, an active hybrid of chicken and rabbit muscle enzyme could be formed. It i s therefore possible to dem-onstrate that the 3 2 differences i n amino amino ac i d sequence be-tween these two enzymes (86$ homology) has not s i g n i f i c a n t l y a l -tered the subunit Interface region (44). I t i s Important to exercise caution when evaluating evidence obtained i n such experiments; For example, a contrasting report of the p o s s i b i l i t y of d i r e c t l y l i n k i n g TIM to an agarose support ( l e . no spacer arm between enzyme and agarose was inserted) v i a one subunit was reported by Sawyer and Gracy (45) when they ob-13. served that both subunits of human TIM were linked to the matrix and that the double linkage was preventing d i s s o c i a t i o n . In con-t r a s t , the protein bound to the agarose v i a an acetamidoethyl linkage had k i n e t i c as well as s t a b i l i t y properties closer to the native enzyme. This data challenges the v a l i d i t y of the ex-periments of P e l l and White who used d i r e c t linkage of the protein to the support. The p o s s i b i l i t y that the monomer of TIM i s inactive has been suggested by Waley (46) who performed experiments i n which rabbit or chicken muscle TIM was denatured by guanidiniium hydrochloride and then followed the k i n e t i c s of the renaturation. His scheme involved f i r s t of a l l the r e f o l d i n g of monomer and then the as-soci a t i o n of the two folded monomers to form a dimer. At low concentrations dimerization was a rate-determining step ( k i n e t i c s found to be second order at low enzyme concentrations) and since the reappearance of dlmers was followed by the increase i n act-i v i t y , t h i s was taken to indicate that the monomers showed l i t t l e or no a c t i v i t y . I f t h i s indeed i s true, i t would mean that the active s i t e enzyme conformation induced by the subunit association i s e s s e n t i a l f o r the c a t a l y t i c a c t i v i t y of TIM. It i s clear therefore that t h i s issue i s s t i l l unresolved and any probe of the inter-subunit Interface when rela t e d to enzymatic a c t i v i t y w i l l be of considerable value. B. Protein Modifications There exists very l i t t l e information about the p a r t i c u l a r amino ac i d residues, other than the c a t a l y t i c s i t e hexapeptide, 14. which are most Intimately Involved i n the TIM a c t i v e s i t e and therefore e s s e n t i a l to enzymatic c a t a l y s i s . However, recent studies have investigated the r o l e of the sulfhydryl groups and there are c o n f l i c t i n g reports i n the l i t e r a t u r e concerning the e f f e c t of t h i o l - s p e c i f i c reagent on enzymatic a c t i v i t y ( 2 3 , 2 9 ) . One of the more comprehensive surveys of t h i o l r e a c t i v i t y was presented by Davis et a l (47). Investigation Oof SH r e a c t i v i t y by reaction with malelmldes resulted i n a threefold greater Mich-a e l i s constant f o r the modified rabbit enzyme. Three t h i o l s / mole enzyme reacted. However, mercurials were found to cause a greater change l n K m and reacted with 6 SH's/mole rabbit en-zyme. I t was reported that the rabbit muscle and l i v e r enzyme ap-pear to have s i m i l a r properties but that the chicken muscle enzyme i s l e s s reactive (47). The yeast enzyme does not become in a c t -ivated upon sul f h y d r y l modification. The enzyme from chicken muscle has been found (48) to contain one t h i o l group per subunit that i s more reactive than the others to the reagent 5 » 5 " - d l t h l o -b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB). I t was observed that the ac-cessible t h i o l group was unnecessary to the c a t a l y t i c a c t i v i t y of the enzyme (48) which i s i n d i r e c t contrast to r e s u l t s report-ed i n t h i s t h e s i s . However, i t has been suggested that f o r the r a b b i t muscle protein, one t h i o l per dimer can be modified by DTNB with a 5 0 $ loss of a c t i v i t y and that a second e s s e n t i a l SH group which corresponds to the other active s i t e i s s t i l l f r a c -t i o n a l l y i n t a c t but i s inaccessible through the association of 15. the two subunits (16), However, there i s a r e a l deficiency of knowledge regarding the r e a c t i v i t y of the t h i o l s of TIM and i t i s i n t h i s area that the r e s u l t s of t h i s thesis attempt to expand "the knowledge of the modified p r o t e i n . C. Amino Acid Sequence and X-Bay Cryst a l Structure of TIM The amino ac i d sequence of the rabbit muscle enzyme was f i r s t reported by Corran and Waley (4-9) who found the polypeptide chain had 248 amino ac i d residues and that the molecular weight of the dlmer was 5 3 s 2 5 7 . The t r y p t i c peptides of the rabbit mus-cle TIM was compared with that of chicken ( 5 1 ) . Each chain of the chicken has 24-7 amino a c i d residues and there i s one d e l e t i o n i n each chain. Apart from these gaps, there are 32 differences {86% homology). 22 of the 32 interchanges are consistent with a change of one nucleotide i n the DNA codon. In addition, the amino ac i d sequence of the 15 residue t r y p t i c peptide that con-tains the a c t i v e - s i t e glutamyl residue as determined by Corran and Waley ( 5 3 ) was found to be d i f f e r e n t than the analogous rab-b i t peptide reported by Hartman ( 5 2 ) i n that valine i n chicken was substituted f o r tryptophan i n r a b b i t . The amino a c i d sequence data f o r the chicken breast muscle was published i n 1975 ( 5 4 ) . The sequence data f a c i l i t a t e d the determination of the 2.5 A r e s o l u t i o n c r y s t a l structure which was reported i n the same paper. It was found that each subunit of the chicken muscle TIM i s composed of alternate segments of poly-peptide chain i n the <*.- and ^-conformations that are arranged to form an inner cylinder of p a r a l l e l - p l e a t e d sheet and a l a r g e l y 16 h e l i c a l outer s h e l l . They were also able to indicate the r e s i -dues p a r t i c i p a t i n g i n the subunit Interface as well as those making up the active s i t e . The two subunits were r e l a t e d by a two f o l d a x i s . Interaction between the subunits involves, i n part, the loops 70-80 which form hydrophobic pockets around the Met 14 residue from adjacent subunits. The pockets l i e on the edges of the Interface. The active s i t e s of the protein consists of residues from both subunits as has been found f o r another g l y c o l y t i c enzymejG3PD (89)« This f i n d i n g i s p a r t i c u l a r l y i n -teresting i n l i g h t of the previously mentioned observation that the monomer of TIM i s i n a c t i v e . The x-ray structure as reported by Banner et a l i s p a r t i c u -l a r l y i n t e r e s t i n g l n l i g h t of the s t r i k i n g resemblance which i t has to other g l y c o l y t i c enzymes which are composed l a r g e l y of alternating segments of d- and ^ - s tructure which are folded sim-l l a r l l y . There i s the suggestion by Rao and Rossman that s i m i l a r super-secondary structures may be found l n many protein molecules with widely d i f f e r e n t amino acid sequence as a r e s u l t of converg-ent evolution -(55)• The gap between crystallography and k i n e t i c s can often, i n part at l e a s t , be bridged by spectroscopic studies which make use of techniques such as NMR. NMR has the p a r t i c u l a r advantage of being able to observe i n d i v i d u a l residues; also changes i n con-formation can often be observed and characterized i n d e t a i l . A paper published, by Browne et a l i n 1976 (56) has made con-siderable progress towards assigning the h i s t i d i n e resonances i n rabbit and chicken TIM to i n d i v i d u a l residues and towards char-1 7 . a c t e r i s i n g the change i n conformation when ligands bind. This work, which was made possible through knowledge of the x-ray structure of Banner et a l (75) > weus able to observe the proton resonances of f i v e h i s t i d i n e s i n the chicken muscle enzyme and one h l s t l d i n e i n the rabbit enzyme which were observed to t i t r a t e i n the pH 5«4 to 9 range. The extreme s e n s i t i v i t y of NMR techniques as observed by 19 Browne et a l ( 5 6 ) i s supported by the preliminary P NMR exper-iments presented i n t h i s t h e s i s . We have been able to collabor-ate the a b i l i t y of magnetic resonance to detect changes i n the 19 environment of the observed nucleus 'F when the two most reac-t i v e cysteines of chicken TIM were modified by a t r i f l u r o N-ethyl-maleimlde l a b e l (FEM) and compared to a model compound consisting of the addition product of FEM and N-acetyl c y s t e i n . Clues to the p o s i t i o n of the s i t e of modification of the chicken TIM by FEM are given by examination of the c r y s t a l structure of Banner et a l ( 5 5 ) . The x-ray structure places the two most reactive cysteines, to 2-chlormercuri-4 nltrophenol, at residue 217 which would there-fore be a l i k e l y s i t e f o r the FEM modification of TIM. Cys 217 i s situated on the outer surface of the enzyme as part of the mainly h e l i c a l segments of polypeptide chain which form the c y l -i n d r i c a l outer surface of each subunit. I t i s 5 amino ac i d res-idues away from the active s i t e Val 212 and thus i s about 1.4 complete turns of the d-hellx away from the active s i t e . There-fore Cys 217 i s not i n the intersubunlt area of contact nor i n 18. the immediately adjacent active s i t e area. However, Cys 217 i s close enough to the active s i t e that i t i s possible to envision the p o s s i b i l i t y of i t s chemical modification a f f e c t i n g the cat-a l y t i c a b i l i t y of the active s i t e i f a conformational change oc-curs upon reaction of the t h i o l . However, since Banner et a l also observed modification of a second l e s s r e a d i l y accessible p a i r of t h i o l s at Cys 41 when using the smaller and more hydro-phobic ethyl mercury phosphate, i t cannot be assumed with cer-tainty that FEM modification of chicken TIM proceeds at Cys 217. Cys 41 i s located on the surface of the Inner c y l i n d r i c a l p-pleated sheet structure which i s not adjacent to either a c t i v e s i t e or intersubunlt contact areas. I t i s f a i r l y c l e a r that studies on the actual FEM labeled protein must be c a r r i e d out before any concrete assumptions may be made concerning the actual s i t e of FEM modification. 19. CHAPTER II ISOLATION AND PURIFICATION 2.1 Materials The extracting buffer made use of ethylenediaminetetra-acetate, dlsodlum s a l t (EDTA-2Na+) from Fisher S c i e n t i f i c Com-pany, c e r t i f i e d ACS grade, and 2-mercaptoethanol from Matheson, Coleman and B e l l . Buffers contained Trisma Base from Sigma, Reagent Grade, and sodium chloride, BDH Analar grade. Special Enzyme Grade ( u l t r a pure) ammonium sulfate from Schwarz/Mann was used i n the p r e c i p i t a t i o n s . DEAE Sephadex A50 from Pharmacia Fine Chemicals and DE52 and Biogel A DEAE from Bio-Rad were the resins used i n the chrom-atography experiments. The assay made use of the d i e t h y l a c e t a l barium s a l t of DL-Glyceraldehyde 3-Phosphate, g l y c e r o l - 3 phosphate dehydrogenase, Dowex-50 Hydrogen Form Resin, and nicatinamldeadeninedlnucleo-t i d e , reduced form (NADH) from the Sigma Chemical Company. 20. 2.2 Methods A. Extraction and Isolation A typical protein preparation used 500 gms of muscle dissect ed from the breasts of four chickens. The fresh muscle was ob-tained either from a local butcher shop or from freshly k i l l e d chickens (Department of Poultry Science, UBC); A l l procedures o were carried out at 4 C or on ice (unless otherwise noted) us-ing a modified procedure of McVittie*s (11). The defatted breast muscle was minced in a Waring blender for 1 minute with'an extraction buffer which consisted of 0.2$ mercaptoethanol and 1.5mM ethylenedlamlnetetraacetlc acid (to pH 7.0 at 22°C with NaOH) using 1 ml of cold buffer per gram of muscle. The homogenate was centrifuged in the GSA rotor of a Sorvall BC-2B Centrifuge at 1200Xg for 4 5 minutes. The pellet was resuspended ln 0.5 ml of extraction buffer per gram of mu-scle used, stirred for 30 minutes and recentrlfuged as above. The combined supernatants were f i l t e r e d through cheese cloth three times, and brought to 6 5 $ saturation with solid (NH4)2S0^. 430.4 grams of (NEj^SO^ per l i t e r of supernatant ( 6 5 $ ) were added slowly with gentle s t i r r i n g , a l l the while the extraction buffer being packed in ice. After standing in the cold for at least 18 hours, the 65$ (NHi^SO^ saturated solution was centrl-fuged at 5500Xg for 75 minutes; The pellet was discarded and the supernatant brought to 9 0 $ (NH4) 2S0^ (182;5 gm/liter) sat-uration. Upon standing for about 24 hours, the protein precip-itate was spun down and then dissolved in a minimum amount of 20mM TrisjHCl buffer, pH 7.2; (All buffers were adjusted to pH 2 1 . at 22°C and then stored at 4°C.) The s o l u t i o n was dialyzed a-gainst the T r i s buffer with at l e a s t 8 changes over 3 days.and then applied to a DEAE Sephadex A 5 0 column ( 5 X 7 0 cm) p r e - e q u l l -ibrated with the d i a l y s i s b u f f e r . The column was eluted with a k l i t e r l i n e a r gradient ( i e . 2 1 of low s a l t and 2 1 of high s a l t buffer) of 2 0 to HOmM T r i s pH7.2 at a flow rate of not more than 2 0 ml per hour. Fractions were c o l l e c t e d on a LKB-Produkter f r a c t i o n c o l l e c t o r and the absorbance at 2 8 0 nm deter-mined on a Zeiss PMQ I I UV-Visible spectrophotometer. I n i t i a l l y * a c t i v i t y assays were performed on center protein f r a c t i o n s from each eluted peaki A c t i v i t y assays were then performed at 2 or 3 f r a c t i o n i n t e r v a l s across those protein peaks found to have t r i o s e phosphate isom'erase a c t i v i t y . B. Rechromatography The f i r s t rechromatography experiments were performed with DEAE Sephadex A 5 0 i I n i t i a l l y , the f i r s t TIM peak off the column (Peak A) was rechromatographed using a 3 l i t e r 6 0 to 9 0 mM T r i s pH 7-2 gradient on a 1 1 0 X 2 . 5 cm column with a flow rate of l e s s than 2 0 ml/hour; The eluted protein was assayed, pooled and pre-c i p i t a t e d for storage as before. The •B* TIM peak was rechrom-atographed using a 3 l i t e r 5 0 to 70 mM pH 7 . 2 T r i s gradient on a 2 . 5 X 9 2 cm column, flow rate l e s s than 2 0 ml/hour. Treatment of eluted protein was as before:'. The Isolated A and B peaks from a d i f f e r e n t protein prep-aration were pooled together and rechromatographed, t h i s time by means of a gradient of 0 to 35mM NaCl i n 5mM T r i s pH 7 . 2 over 22 k l i t e r s on a 2:5X70 cm column, flow rate l e s s than 20 ml/hour. The considerable shrinkage of the gel over t h i s s a l t concentra-t i o n range necessitated the Interruption of the gradient period-i c a l l y , i n order to allow the buffer to go down to the top of the bed, followed by resumption of the gradient; The rechromatography of s t i l l another protein preparation u t i l i z e d DE52 c e l l u l o s e r e s i n i The pooled A and B peaks were reseparated with a 0-30mM NaCl gradient i n 5niM T r i s pH 7.8 over 3»5 l i t e r s , on a 2;5X60 cm column with a flow rate of l e s s than 18 ml/hour; D E 5 2 c e l l u l o s e had the advantage of not shrinking with an increase i n the s a l t concentration of the b u f f e r . How-ever, i t s protein binding capacity i s lower than the Sephadex anion exchanger; A f a i r l y high (7«8) pH was required to make the t r i o s e phosphate isomerase adhere to the column. Plaut and Knowles (39) have demonstrated a decrease i n TIM's s t a b i l i t y i n buffers of pH greater than pH 7*5 with the trend increasing e-ven more sharply a f t e r pH 8,0-i With these factors i n mind, a t h i r d r e s i n , Biogel A DEAE, was u t i l i z e d i n the rechromatography experiments. This gel has the advantage of the high capacity of the Sephadex r e s i n but does not shrink or expand with s a l t concentration changes. The pH at which the r e s i n could be used f o r the rechromatography (7-5) also makes i t a better choice than the DE52 c e l l u l o s e . The pooled A peak was rechromatographed using a 3 1. 0 to 20mM NaCl gradient l n 5mM T r i s pH 7.5 on a 2.5X30 cm column, and flow rate of about 18 ml/hour. The B peak was s i m l l a r l l y r e -ohromatographed. 23. C. Assay The t r l o s e phosphate Isomerase assay, which was used In the work described i n t h i s t h esis, i s based upon a coupled enzyme system. A substrate of t r i o s e phosphate isomeras6, glyceralde-hyde-3 phosphate (G3P) i s f i r s t converted to dlhy-droxyacetone phosphate (DHAP) by TIM; DHAP i s next enzymatically reduced by d-glycerol phosphate dehydrogenase (GPD) to g l y c e r o l 1-phosphate. The coupled enzyme reaction of GPD proceeds v i a a nicotinamide adenine dinucleotide coenzyme oxidation (NADH-* NAD+) with concomitant decrease i n A - ^ Q . A si m i l a r assay to the G3P/GPD system i s conversion of the substrate dihydroxyacetone phosphate by TIM to G3P, followed by the enzymatic oxidation of G3P by the NAD requiring enzyme glyceraldehyde-3 phosphate dehydrogenase (G3PD). The G3PD oxidation of G3P proceeds v i a the reduction of 4. NAD;:; to NADH. A schematic representation of the central reac-tions i n the two assays i s as follows t Thus, two assay procedures are possible, the f i r s t being the use of G3P as substrate and GPD as the coupling enzyme with the a c t i v i t y of TIM observable by the rate of decrease i n the absorb-ance at 340nm as NADH disappears; the second uses DHAP as sub-st r a t e , G3PD as coupling enzyme and the a c t i v i t y of TIM i s f o l -24. lowed by an Increase i n the absorbance (at 3^0nm) as the coen-zyme NADH i s formed. The spectra shown below demonstrates that at 3^°nm, NADH i s at i t s maximal absorbance while NAD i s non-i n t e r f e r i n g . Therefore, the convenient wavelength 3^0nm, makes i t possible to follow the disappearance or appearance of NADH and hence follow the i n i t i a t o r y t r i o s e phosphate isomerase reac-t i o n . FIGURE l l U l t r a v i o l e t Spectra of NAD* and NADH 1.0 - -Absorbc 0.5+ . • - . N A D " 1 nee / / v . • 7 . \ " . / \ \ • - ^ NADH 240 280 320 360 X ( n m ) In order to accurately determine the a c t i v i t y of TIM, i t i s necessary that the coupling system not be rate determining, plaut and Knowles ( 3 9 ) , i n an excellent s e r i e s of experiments, demonstrate the conditions underwhich the o v e r a l l reaction was lim i t e d by the t r i o s e phosphate Isomerase c a t a l y s i s . In order to check the v a l i d i t y of the assays ( l e . that the. observed rates of production or oxidation of NADH represents the rate of the TIM reaction), i t was necessary to ensure the l i n e a r dependence of 25. the observed i n i t i a l rate on the concentration of.TIM under the highest substrate concentrations used (for G3P the range of 0.2-l.OmM had been Investigated, while for DHAP the range was 0.2-2.0mM). This was accomplished by studying the i n i t i a l rate as a function of lsomerase concentration (at the highest concentration of.substrate to be used). Variation i n the rate of G3P or DHAP isomerization was studied as a function of coupling enzyme concentration. The ex-periments ruled out any p o s s i b i l i t y of complications a r i s i n g from enzyme-enzyme interactions and, with the evidence of the other studies, allowed one to determine * safe* concentrations of a l l species i n s o l u t i o n . I t was therefore possible to e s t a b l i s h the v a l i d i t y of the assay f o r the conditions used i n thi s account. The G3P/GPD system was used exclusively i n the work presented i n th i s t h e s i s . Procedure s The substrate, G3P, was prepared by the hydrolysis of the die t h y l a c e t a l barium s a l t of DL-glyceraldehyde 3-phosphate. Man-ufacturer's preparative i n s t r u c t i o n s were followed* Dowex 50 Hy-drogen Form Resin (1.5 gm) was suspended i n water (6.0 ml); the barium s a l t (100 mg) was added, mixed thoroughly, and then the mixture was placed into a b o i l i n g water bath f o r 3 minutes. The G3P solution was c h i l l e d quickly by t r a n s f e r r i n g i t to an i c e bath and then i t was poured through a funnel with a glass wool plug. The r e s i n was washed with 2 ml aliquots of water u n t i l the supernatant f l u i d s measured 17.5 ml. The resultant stock was 12mM i n substrate (100/umoles of enzymatically active D-isomer) 2 6 . and had a pH of 2.4. 3torage Involved d i v i d i n g the solution i n -to separate 2 ml portions and freezing u n t i l use. The substrate i s very stable when frozen at -20°C and could be kept f o r sev-e r a l months without any l o s s ; The following stock assay solutions were used i n a 3 ml cu-vette t 2.6 ml of lOOmM triethanolamine pH 7.5 buffer with ad-d i t i o n of EDTA to 5mM, 100jxl of 6mM NADH, lOOyul of 12mM G3P and lOOyul of d-glycerophosphate dehydrogenase. The coupling enzyme was usually protein with a s p e c i f i c a c t i v i t y of about 200 units/mg and stock solutions for the assay had concentrations of about 0.5 mg protein per ml. Therefore, f i n a l concentrations i n the 3 ml volume werei lOOmM triethanolamine pH 7.5, 5mM EDTA, 0.2mM NADH, 0.4mM G3P., and 17yug/ml GPDi The reaction was i n i -t i a t e d by addition of about 6—10 of TIM i n a 5 or v o l -ume. The decrease i n absorbance at 3^ -Onm was followed for 1 minute and usually did not exceed 0.1 absorbance u n i t s . The small volume increase did not s i g n i f i c a n t l y change absorbance so a c t i v i t i e s were calculated upon a 3 ml volume; At the pH used, there i s no detectable decay of NADH over one minute so the re-action was usually followed without a blank. 2.3 Results High a c t i v i t y triose. phosphate Isomerase was eluted from the DEAE Sephadex A50 column i n two peaks at approximately 75mM T r i s ( f i g . 2). The r e s u l t s of a t y p i c a l l i v e chicken preparation are summarized i n Table I. The considerable (36 X ,) p u r i f i c a t i o n , as determined by s p e c i f i c a c t i v i t i e s , can be mainly a t t r i b u t e d to the i n i t i a l DEAE chromatography step. 27. FIGURE Zx I n i t i a l DEAE Sephadex A50 Chromatography Absorbance at 280 nm-o-K>-w w v w i FRACTION NUMBERS (-18 ml) TABLE.It Determination of a c t i v i t y units followed throughout a t y p i c a l l i v e chicken prep-aration of chicken breast muscle For each k i l o of tis s u e : Fraction OD's protein Uhits/OD Total units % recovery P u r i f i c a t i o n A. Crude extract 6 3 , 5 8 5 142 .2 9,041 ,591 1 0 0 . OX B. &5% PPt. 19 , 4 3 6 3 1 0 . 1 6 , 0 2 6 , 9 3 3 6 6 . 7 2.2X C. 90% ppt. 10 , 0 6 4 3 4 0 . 8 3 ,429,661 37.9 2.4X D. Peak A+B of i n i t i a l : DEAE Sephi A 5 0 9 7 1 . 7 ( 7 1 4 . 5 m g ) 5 1 5 2 . 9 5 , 0 0 6 , 8 7 9 55 . 4 3 6 . 2 X Peak A 8 9 8 . 3 ( 6 6 0 . 5 m g ) 5 1 4 7 4 , 6 2 3 , 3 2 3 Peak B 7 3 . 4 (540.Omg) 5 2 2 5 3 8 3 , 5 5 7 29. In order to further p u r i f y and separate the two peaks, the protein was rechromatographedI The r e s u l t s are variable but d i d r e s u l t i n a protein up to twice as a c t i v e . The f a c t that no other protein was observed as separating out i n subsequent steps i n d i -cates that the lowering of s p e c i f i c a c t i v i t y i n the one time chromatographed material i s probably due to small amounts of i n -h i b i t o r y protein or other unknown contaminating substances which have a s i m i l a r e f f e c t . The difference i n s p e c i f i c a c t i v i t y between the A and B peaks appears to vary with i n d i v i d u a l preparations. However, the l i v e chicken preparation d i d usually y i e l d B protein higher i n s p e c i f i c a c t i v i t y than that from chickens obtained from l o c a l butcher shops. The B protein from the l a t t e r source often y i e l d -ed enzyme with s p e c i f i c a c t i v i t y considerably lower than that of the A ( f i g . 2 ) . Figure 3 shows the i n i t i a l DEAE chromatography of a l i v e chicken preparationi Since s p e c i f i c a c t i v i t y i s calculated as the rate of conver-sion of substrate per unit of enzyme (usually per OD^QQ o r P Q r mg), I t i s an i n d i c a t i o n of the e f f i c i e n c y of the c a t a l y t i c pro-cess. I f the protein i s completely homogeneous throughout an eluted peak, i t i s expected that a constant s p e c i f i c a c t i v i t y would be observedi The chromatographic studies on TIM showed changes i n s p e c i f i c a c t i v i t y which indicated that the protein i s not completely homogeneo\is. Contamination of the TIM peaks by protein (which can be e-luted within the same s a l t concentration range as t r i o s e phosphate isomerase) could be a factor i n the observed s p e c i f i c a c t i v i t y 30. -Absorbance at - 280 nm FIGURE 3: I n i t i a l DEAE Sephadex A50 Chromatography of , a Live Chicken Preparation Insert! S p e c i f i c A c t i v i t y and Protein P r o f i l e s of TIM f r a c t i o n s 0 20 Specific Activity units/mc ) -7000 -6000 -5000 -4000 -3000 -2000 -1000 120 130 40 60 80 100 120 140 FRACTION NUMBERS (-18ml) 160 i 31. p r o f i l e . If the contaminating protein(s) i s i n h i b i t o r y (or ac-t i v a t i n g ) , minute amounts could produce substantial changes i n TIM's a b i l i t y to catalyze the lsomerizatlon of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (and hence i n the s p e c i f i c a c t i v i t y p r o f i l e ) . Contaminating proteln(s) which has no e f f e c t upon the en-zymatic c a p a b i l i t i e s of TIM would have to be present i n rather large amounts to produce the p r o f i l e changes observed. Evidence to be presented i n the next section indicates that the protein i s homogeneous as to molecular weight. This would tend to rule out the p o s s i b i l i t y of s u b s t a n t i a l l y contaminated protein being e-luted from the column. The only other p o s s i b i l i t y i s that there i s an inherent molecular or conformational difference i n the t r i -ose phosphate isomerase at various points i n the eluted TIM,peaks. The p o s s i b i l i t y of Isozymes (proteins of s l i g h t l y d i f f e r e n t am-ino a c i d sequence and/or conformation which catalyze the same r e -action) with d i f f e r i n g t r i o s e phosphate isomerase a c t i v i t y capa-b i l i t i e s i s a p o s s i b i l i t y to be discussed i n the protein charac-t e r i z a t i o n section to follow. The v a r i a t i o n found i n multlen-zyme forms of TIM ( l e . number of charges, shape, protein aggre-gation, etc.) could be the basis f o r a chromatographic separation of the various Isozymes. The presence of Isozymes In the chicken breast muscle f i t s well with the observed protein and s p e c i f i c a c t i v i t y p r o f i l e s of the DEAE chromatography experiments. The c h a r a c t e r i s t i c s p e c i f i c a c t i v i t y p r o f i l e was observed throughout the various chromatographies: The double peak observed for the A protein and sing l e one f o r the B might seem to indicate 32. two enzyme forms i n the A with only one l n the B. However, as w i l l be shown l n the characterization work to follow, t h i s does not seem to be the case, l n f a c t , there i s strong evidence to the contrary. The physical basis for the double a c t i v i t y peak of the A protein i s obscure at t h i s point since i n the character-i z a t i o n methods used, no si m i l a r separation occurred. The frequently incomplete separation of the A and B TIM peaks, as well as the observed s p e c i f i c a c t i v i t y p r o f i l e , i n d i -cated that further p u r i f i c a t i o n and i s o l a t i o n of the possible molecular forms of the enzyme should be attempted. To t h i s end, separate rechromatography experiments were performed, The f i r s t experiment u t i l i z i n g DEAE Sephadex A 5 ° r e s i n re-sulted i n a single peak being eluted for the A protein ( f i g . 4A) The peak came off very early l n the gradient (but not with the void volume) and showed the c h a r a c t e r i s t i c s p e c i f i c a c t i v i t y pro-f i l e for the A protein. The early e l u t i o n of the A protein Indi-cated that the rechromatography of the B protein on the DEAE Sephadex A50 should be attempted using a lower s a l t concentration and shallower gradient ( i n order to allow the protein to adhere more strongly to the column). However, an e l u t i o n of B protein early i n the gradient, was observed ( f i g . 4 B ) . The s p e c i f i c ac-t i v i t y p r o f i l e of the rechromatographad B material was similar to that obtained i n the i n i t i a l chromatography. The attempts to rechromatograph t r i o s e phosphate isomerase at s a l t concentrations l n the same range (about 75mM) as that of the i n i t i a l Sephadex-DEAE chromatography seemed to be inadequate. The adherence of the p u r i f i e d enzyme to the anion exchange c o l -Figure 4* Rechromatography of TIM Fractions on DEAE Sephadex A50 (A) Protein from Peak A (B) Protein from Peak B FRACTION NUMBER (-18ML) 34. umn a p p e a r e d t o be d i f f e r e n t f r o m t h a t o f t h e Impure p r o t e i n . The i n t e r a c t i o n s , w h i c h T I M has w i t h t h e o t h e r p r o t e i n s I n t h e 90% ( N H^) 2S0^ s a t u r a t i o n c u t , c o u l d w e l l a f f e c t t h e manner a n d s t r e n g t h w i t h w h i c h TIM i s a b l e t o a d h e r e t o t h e c o l u m n . T h e r e f o r e , w i t h t h e c o n s i d e r a b l e g r a d i e n t changes ( f r o m 60-90 a n d 50»70mM T r i s t o 5mM T r i s w i t h 0-35mM N a C l ) o f t h e r e c o m -b i n e d p e a k A a n d B r e c h r o m a t o g r a p h y ( f i g ; 5)» i t was f o u n d t h a t t h e p r o t e i n was a d h e r i n g much more s t r o n g l y t o t h e c o l u m n . How-e v e r , because, o f t h e i n t e r r u p t i o n s i n t h e g r a d i e n t , t h e f i r s t peak d i d n o t come o f f i n a s m o o t h l y s y m m e t r i c a l p e a k b u t r a t h e r i n a * s t e p - l i k e * m a n n e r . The p r o t e i n p r o f i l e o b t a i n e d ( f i g . 5) i l l u s t r a t e s t h e p r o b l e m s a t t a c h e d t o t h i s k i n d o f r e s i n . The s p e c i f i c a c t i v i t y p r o f i l e o f t h e r e c h r o m a t o g r a p h y o f p o o l e d A a n d B ( d o u b l e p e a k f o r A a n d s i n g l e p e a k f o r B ) a l l o w e d one t o i d e n t i f y w i t h some c e r t a i n t y , t h e A a n d B p r o t e i n s . The c o m b i n -i n g o f f r a c t i o n s b y t h e i n d i v i d u a l a r r o w s o f t h e i n d i v i d u a l A a n d B p e a k s i s shown I n f i g u r e 5* The s e c o n d a c t i v i t y peak o f t h e A p r o t e i n was l o w e r i n s p e c i f i c a c t i v i t y t h a n t h e f i r s t p e a k i n t h e p r o f i l e . T h i s was u n u s u a l i n t e r m s o f t h e p r o f i l e s o b -s e r v e d i n t h e i n i t i a l DEAE c h r o m a t o g r a p h y b u t has b e e n o b s e r v e d i n o t h e r r e c h r o m a t o g r a p h i c e x p e r i m e n t s ( e g ! f i g u r e 7 B ) . The n e x t r e s i n t o be t r i e d , i n a n a t t e m p t t o g e t o v e r t h e Sephadex s h r i n k a g e p r o b l e m , was c e l l u l o s e DE52 ( f i g . 6) w h i c h , h o w e v e r , has a l o w e r c a p a c i t y ; A good s e p a r a t i o n was o b s e r v e d of r e c o m b l n e d A a n d B p e a k s f r o m t h e f i r s t DEAE Sephadex A50 c h r o m a t o g r a p h y a l t h o u g h t h e s p e c i f i c a c t i v i t y p r o f i l e d i d n o t show i t s u s u a l d e f i n i t i o n ( a l t h o u g h i t was g e n e r a l l y t h e s a m e ) . FIGURE 5» Rechromatography of Peak A and Peak B TIM Fractions DEAE Sephadex A50 Absorbance at 280 nm Specific Activity Units/mg AAA 6000 -5000 •WOO r 3000 rEOOO -1000 c n A r T i m i t i i i M D r o I~.AO~.I\ FIGURE 6t Rechromatography of Peak A and Peak B TIM Fractions on DE52 Cellulose 37 Las t l y , an agarose based ion exchange r e s i n , Biogel A DEAE, was employed i n the rechromatography step. The rechromatography of peak A f r a c t i o n s ( f i g . 7A) resulted i n e l u t i o n of a single protein peak (well into the gradient) with the c h a r a c t e r i s t i c double peak s p e c i f i c a c t i v i t y p r o f i l e . The s l i g h t l y assymmetri-ca l nature of the eluted peak i s a feature of the agarose based DEAE r e s i n used. The f a c t that t h i s r e s i n i s capable of as good a separation of the A and B protein peaks as that shown i n the DEAE Sephadex A50 and c e l l u l o s e DE52 chromatographies of recom-bined A and B ( f i g . 5, f i g . 6), i s i l l u s t r a t e d i n fi g u r e 7B. A good separation of r e s i d u a l A protein and the B protein was ob-served. The B peak showed a single s p e c i f i c a c t i v i t y peak while the A protein had two (the second being present as a well defined shoulder of the f i r s t ) ; The second s p e c i f i c a c t i v i t y peak was lower than the f i r s t as was discussed e a r l i e r . In conclusion, i t might be mentioned that the Biogel A DEAE res i n appears to be the best choice for the anion exchanger to be used, i n the rechromatography step. I t combines high capacity, no shrinkage of the r e s i n , and the a b i l i t y to be used at a f a i r -l y low pH, with as good a separation of TIM as has ever been ob-served chromatographically. FIGURE 7* Rechromatography of "TIM Fractions on Biogel A DEAE (A) Peak A (B) Peak B FRACTION NUMBERS H8nril) 39. CHAPTER I I I CHARACTERIZATION OF PEAK A AND PEAK B 3.1 Methods A. SDS Gel Electrophoresis A s l i g h t l y modified procedure of Fairbanks et a l (57) was used. The procedure was carr i e d out i n a v e r t i c a l electrophor-esis apparatus which contained 800 mis of buffer so l u t i o n i n each electrode compartment and no more than 12 g e l tubes ( f i g . 8). The gels contained 1% SDS and 5 a c r y l a m i d e . To f a c i l i t a t e the preparation of buf f e r s , gels, etc., con-centrated stock solutions were f i r s t made up and subsequently used to prepare the solutions which were used i n the electrophor-esis experiment. These are l i s t e d i n Table I I . Gels were made by combining l n a small vaccum f i l t e r f l a s k , 1.4 mis concentrated acrylamide and NN*-Methyleneblsacrylamlde (Ac b i s ) (see Table I I ) , 1.0 ml 10X buffer and 5.6 mis water. The solution was degassed f o r approximately 15 minutes' and then to i t was added 0.5 ml 20$:fSDS, 1 ml of ammonium persulfate (15 mg/ml), and 0.5 ml of 0.5% TEMED (N,N,N*,N*-tetramethylene-diamlne), This mixture was used to f i l l four.0.5X11 cm pyrex tubes (previously cleaned i n a concentrated HCl bath, coated with photoflo solution, and l e f t to dry) to within 1 cm of the top of the tube. The top of each gel was c a r e f u l l y covered with an overlay solution which prevents drying out of the g e l . They were then l e f t to stand f o r at l e a s t 12 hours to ensure complete polymerization. 40. Figure. 8t E l e c t r o p h o r e s i s Apparatus Si C r o s s - s e c t i o n a l side view + 4 -kuffer g e l tube <$ b u f f e r 41. TABLE l i t Stock solutions and buffers f o r SDS gel electrophor-esis A. Stock solutions 1) Concentrated AcBis 40 gms acrylamide 1.5 S m N,N I-methylenebisacrylamlde EgO to 100 mis 2) 10X Buffer 0.4 M T r i s 0.2 M sodium acetate 0.02 M EDTA pH = 7»4 with a c e t i c a c i d 3) 20$ SDS (W/W) B. Electrophoresis buffer (per l i t r e ) 100 mis of 10X buffer 50 mis 20$ SDS H o0 to 1 l i t r e C. Denaturing Solution 2 gms SDS 10 gms sucrose 74.5 mgs EDTA 2 mgs pyronin y 0.242 gm T r i s E.,0 to 100 mis, pH 8 with HCl D. Overlay solution 0.1$ SDS 0.15$ Ammonium persulphate 0.05$ TEMED 42. The proteins were prepared as follox*s. A small amount of the denaturing solution (Table II) was made 80mM i n d i t h i o t h r e i - -t o l , combined with an equal volume of protein solution and heat-ed f o r 15-20 minutes at 37°G to completely denature the p r o t e i n . The molecular weight standards were prepared i n the same way but denatured at 60 G. Upon cooling, up to 100 j*ls of protein sam-ple was c a r e f u l l y applied with a micropipet to the top of the gel (which was now contained i n the electrophoresis apparatus and covered with buffer s o l u t i o n ) . A maximum of lOOy^g of pro-tein was loaded onto each gel which avoided excessively broad bands. Samples were run In dup l i c a t e . Electrophoresis was c a r r i e d out at,a current of 5 mamp/gel and required approximately 3»5 hours under these conditions. In a l l cases, gels were prerun f o r one hour p r i o r to sample a p p l i c a -t i o n which ensured the removal of excess ammonium per s u l f a t e . The molecular weight markers were denatured i n separate test tubes, then combined together i n one test tube and applied on the gel together. The marker proteins were run i n gels separate from the sample proteins. The following proteins were used as standards! bovine serum albumin (68,600), ovalbumin (45,000), t r y p s i n (23,800), and myoglobin (16,900). After removal of the gels from the tubes, the p o s i t i o n of the tracking dye was marked by notching the gels with a needle dipped i n India ink. The gels were then transferred to stoppered glass tubes and agitated sequentially with the following protein staining and destaining s o l u t i o n ! (57) 1) 25$ isopropanol, 10$ a c e t i c a c i d , 0.025$ Coomassie 43 blue (overnight) 2) 10$ isopropanol, 10$ aoetlo a c i d , 0.025$ coomas-sie blue (6-9 hours) 3) 10$ ac e t i c a c i d , 0.0013$ coomassie blue (overnight) 4) 10$ ac e t i c a c i d (overnight) At t h i s point, the proteins were apparent as dark blue bands. The subunit molecular weight of TIM was estimated by f i r s t p l o t -ting log molecular weight against the R^ . values measured r e l a t i v e to the tracking dye. The points for the standard proteins f e l l on a str a i g h t l i n e from which any unknown subunit molecular weight could be obtained by i n t e r p o l a t i o n , ( f i g . 9) B. Disc Gels The apparatus was as f o r SDS gel electrophoresis. A modi-f i e d method of Dietz and Lubrano was used (58). The following stock solutions were made up to f a c i l i t a t e preparation of the gels. Solutions A and B were stored i n the cold, i n the dark for up to one month. Solution C was f r e s h l y made each time. A. 36.3 gms T r i s 0i23 mis TEMED Addition HCl to pH 8.5 H 0 to 100 mis 2 B. 6 gms acrylamide 160 mgs methylene-bls-acrylamide H 20 to 20 mis C. 140 mgs ammonium persulfate H o0 to 100 mis 45 Gel Mixture t Pour 7.5$ gels were made by mixing 1.0 ml A, 2.0 mis B, 1.0 mis water, and 4.0 mis C. The soluti o n was poured into 0.5X10 cm tubes which had been previously coated with Photoflo sol u t i o n . The gels were c a r e f u l l y o v e r l a i d with about 50y-/ls of B^ jO and then l e f t to polymerize at l e a s t 8 hours or overnight. Dialyzed protein was made 20$ i n sucrose and a couple of milligrams of bromophenol blue was added. Electrophoresis buf-f e r f o r the cathode and anode reser v o i r s was made by a 1J9 d i l -ution of a stock 10X buffer which had been previously made: IOX buffer - 6 gms T r i s 28.8 gms glycine to pH 8.3 H 20 to 1 l i t e r The bottom reservoir was f i l l e d with 800 mis of buffer and the gels placed into the apparatus. The various protein solutions to be run were c a r e f u l l y pipetted onto the top of the gels (usu-a l l y about 2 0 j i g protein i n a volume l e s s than lOOyxls were l a i d on each gel) and then the tubes were f i l l e d with b u f f e r . The top reservoir was f i l l e d with buffer, the electrodes connected and the electrophoresis begun. The experiment was performed at 4°C. The protein was concentrated down Into a t h i n layer on top of the gel at 1 mamp/tube and then the voltage was increased to give 2 mamp/tube u n t i l the end of the experiment. Af t e r electrophoresis, the tracking dye was notched with India ink and the protein was f i x e d by a g i t a t i o n of the gels f o r 30 minutes i n stoppered tubes containing 10$ TCA ( t r i c h l o r o a c e t i c 46. a c i d ) . S t a i n i n g o f t h e p r o t e i n was a c c o m p l i s h e d b y a g i t a t i o n I n . 12.5$ TCA w i t h 0 i 0 5 $ C o o m a s s i e B l u e f o r 1 h o u r i F i n a l l y , t h e c o l o r i n t e n s i t y was d e v e l o p e d o v e r a 48 h o u r p e r i o d by a g i t a t i o n i n 10$ T C A . The g e l s were s c a n n e d a t 550nm u s i n g a G i l s o n g e l s c a n n e r . C . I s o e l e c t r i c F o c u s i n g Column I s o e l e c t r i c F o c u s i n g t A d i a g r a m o f t h e 110 m l c a p a c i t y LKB 8101 e l e c t r o f o c u s i n g c o l u m n u s e d i n t h e s e e x p e r i m e n t s i s shown i n f i g u r e 10. D e t a i l e d d e s c r i p t i o n s o f t h e e x p e r i m e n t a l p r o c e d u r e c a n be f o u n d i n s e v -e r a l r e f e r e n c e s (59>60) w i t h t h e p r o c e d u r e u s e d a s f o l l o w s t The v a l v e 12 was o p e n e d a n d c o o l i n g w a t e r p a s s e d t h r o u g h t h e compartments (18 a n d 16) u n t i l t h e a p p a r a t u s was e q u i l i b r a t e d a t 4 ° C . The f i r s t s o l u t i o n t o be a d d e d was t h e c a t h o d e s o l u t i o n ( 0 . 4 m i s e t h y l e n e d i a m i n e , 12:0 gms s u c r o s e a n d 14.0 m i s H 2 0 ) . I t was pumped i n t h r o u g h n i p p l e (1) w i t h a p e r i s t a l t i c pump a n d f i l l e d up t h e b o t t o m o f t h e c o l u m n . A d e n s e s o l u t i o n , c o n t a i n -i n g 3 /4 o f t h e A m p h o l y t e s d i l u t e d t o 42 m i s w i t h w a t e r ( a n d 28 gms s u c r o s e d i s s o l v e d i n i t ) a s w e l l a s a l i g h t s o l u t i o n c o n t a i n i n g ^ o f t h e A m p h o l y t e s a n d t h e d i a l y z e d p r o t e i n d i l u t e d t o 60 m i s were p r e p a r e d . The r a n g e o f A m p h o l y t e s a v a l l a b e f r o m LKB i n c l u d e t pH 3.5-10 p H 5-8 2 i 5-4 6-8 3.5-5 7-10 4-6 8-8.5 5-7 9-11 FIGURE 10i A Sketch of the I s o e l e c t r i c Focussing Column F i g . lOt Electrofocussing column of 110 ml capacity. The outer cooling jacket (18) has an i n l e t at 14, and an outlet at 5» From the outer jacket the water flows through a tube into the c e n t r a l cooling jacket at 4 and leaves the column at 3» Two platinum electrodes are used. One electrode 13» i s l n contact with the plug 7? i s In the upper part of the column. The gas formed at t h i s electrode escapes at 2. The other i s wound on a Teflon bar l l , and gas escapes at 1. Before draining the column the central tube i s closed by l i f t i n g the plug 12 which has a rubber gasket on the upper surface and seals at 15. I s o e l e c t r i c focussing takes place i n compartment 16, which i s f i l l e d through nipple 2. At ' the bottom of the column there i s a plug l b , with an attach-ment for a c a p i l l l a r y tube to enable the column to be -f r a c t i o n a t e d . 48 Ampholytes were used in quantities to yield either a 1$ (2.5 mis of 40$) or a 5$ (12:5 mis of 40$) concentration. In addition to these ampnolytes, a small quantity of pH 3.5-10 range ampholytes was added to the 1$ (0:2 ml of 40$) and the 5$ (1.0 ml of 40$) columns i n order to 'protect' the ends of the pH gradient from the cathode and anode solutions. From 2-20 mgs of protein may be in each protein zone depending upon the range of ampholytes used and the differences in the pi's of the pro-teins. For the experiments descrioed here, about 5 mgs of pro-tein were added; The dense and light solutions were added to the column by means of a LKB density gradient mixer through nipple 2 with the aid of the pe r i s t a l t i c pump. The f i n a l solution to be added was the anode solution which contained 100 j j ls concentrated HgSO^ and 9.9 mis HgO. The experiment was started with a voltage yielding about 2 watts of power. ; The focusing of the carrier ampholytes and proteins was ac-companied by a decrease in the current passing through the sol-ution. The current was checked periodically and the voltage i n -creased (power always kept at or under 2 watts) u n t i l i t had de-creased to a constant value (at constant voltage). At this point, most of the carrier ampholytes should be focused at or near their isoelectric points. To ensure complete focusing of the slower travelling protein, the experiment was continued for a further 12 hours; The entire focusing experiment takes from 24-72 hours depending upon the pH range and concentration used. More concentrated and narrower range ampholyte solutions require ^9. the longer focusing times. Upon completion of the procedure, the power i s turned o f f , and valve 12 i s closed to prevent the central electrode solution from mixing with the e f f l u e n t ; The clamp on the c a p i l l a r y tube (20), i s opened and the column pumped out at a flow rate of a-bout 1 ml/minutei The f r a c t i o n s were c o l l e c t e d on a Gilson f r a c -tionator and t h e i r pH determined at 4°0, the temperature of the experiment. Protein, i f added to the ampholyte procedure i n i -t i a l l y , was then measured by i t s absorbance at 280nm and TIM was assayed f o r using the G3P/c(-glycerolphosphatedehydrogenase pro-cedure . The f i r s t i s o e l e c t r i c focusing experiment performed used pH 7-10 and pH 5-8 range ampholytes (added i n 1 j l r a t i o with a t o t a l range of pH 5-10 obtained) at a f i n a l concentration of 1%, Resolution of the Peak A and of the Peak B protein was i n -adequate so 5% runs of pH 7-10 or pH 5-8 (without protein) were performed and the tubes containing c a r r i e r ampholytes i n the pE 7-8 range were pooled. An appropriate amount of the pooled am-pholytes (about 22 mis usually) was then used In an e l e c t r o -focusing experiment which included p r o t e i n . The amount of su-crose added to the dense s o l u t i o n was decreased to compensate for the sucrose present i n the pooled ampholyte mixture. The best r e s o l u t i o n resulted when no more than 3 absorbance units of Peak A or Peak B was used; Polyacrylamide gel i s o e l e c t r i c focusings The apparatus f o r running t h i s experiment was the same as that used f o r disc or SDS gel electrophoresis. The p o l y a c r y l -50 . amide matrix of the gel served as a substitute for the sucrose gradient of the column. The gels were either photopolymerized or chemically polymerized. Photopolymerlzatlon was the pre-ferred method i n regards to tlmej protein seldom needed to be concentrated before use and the experiment was started 1 hour af-ter the pouring of the g e l s . However, the procedure i s suitable only when the average pH of the ampholyte range i s no greater than 7.0-7.5 (61). Therefore the pH 5-8 or 3»5-10 ranges were suitable for use but not the pH 7-10 range. The main disadvan-tage of the chemically polymerized gels i s the production of a r t -i f a c t s due to the presence of pe r s u l f a t e . This has been comment-ed on by several authors .(63,6.2,63) Electrofocusing i n gels i s f a s t , requires r e l a t i v e l y small amounts of protein and uses much l e s s ampholytes than the column technique., The disadvantage i s that i t i s l i m i t e d i n determin-ing an accurate p i for a protein since the gel i s short. Stan-dards ( i e . duplicate gels without protein) are co-run with the gels containing protein. The pH p r o f i l e of the standard gels Is determined by s l i c i n g the gel rod, soaking the pieces i n water i n order to elute the ampholytes, and determination of the pH of the eluent. The protein i n the other gels i s stained, th e i r p o s i t i o n measured, and the pH at that point determined by com-parison to the pl o t (of pH vs. distance) of the standard g e l . Therefore, the accuracy of the p i depends upon an i n d i r e c t deter-mination of the pH at that point where the protein i s focused and upon the accuracy of s l i c i n g . The method used for the preparation of the photopolymerized 51. gels i s given l n Table I I I . Upon polymerization, the gels were placed i n the electrophoresis apparatus with the anode (bottom) reservoir containing 800 mis of 0.2$ s u l f u r i c a c i d and the cath-ode (top) rese r v o i r containing 800 mis 0:4$ diethanolamlne. The experiment was run at 4°C and an i n i t i a l current of 1 mamp/tube. This was maintained by increasing the voltage u n t i l 350 v o l t s had been reached! The protein and ampholytes were considered f o -cused when a constant amperage (usually 0.5-1 mamp) at 350 v o l t s was observed f o r 1-2 hours (the IEF experimental running time took on average 5 to 7 hours). At t h i s point, the reference gels were s l i c e d at 5 mm i n t e r v a l s and placed l n i n d i v i d u a l test tubes with 1 ml of degassed water and then they were capped. The ampholytes were allowed to elute from the gel at 4°C, the temp-erature of the experiment. The pH of the gel s l i c e s could be determined a f t e r approximately one houri I f the e l u t i o n was a l -lowed to proceed overnight, nitrogen gas was bubbled into the tubes and then the tube was f i r m l y sealed with Parafilm. The gels to be stained (Vesterburg*s quick s t a i n i n g method) (64) were marked with India ink at the anode end and placed l n stoppered tubes; The s t a i n i n g procedure included t a) incubation of the gels i n a 60°C water bath ( i n a fume hood) l n a s t a i n i n g s o l u t i o n (of methanol 75 mis, d i s -t i l l e d water 186 mis, t r i c h l o r o a c e t i c a c i d 30 gms, s u l f o s a l i c y l i c a c i d 9 gms, and Coomassie Blue, 0.1$) for 15 minutes b) replacing of the s t a i n i n g solution with destalnlng solution (of ethanol 250 mis, water 650 mis, g l a c i a l a c e t i c a c i d 80 mis) and a g i t a t i o n of the tubes TABLE I I I : I s o e l e c t r i c focusing solutions f o r gels Photopolymerization— Stock solutions were kept at 4°C i n the dark f o r about one month. A* Catalyst 1.0 ml TEMED 14 mgs r i b o f l a v i n Ho0 to 100 mis B. Acrylamide 30 gms acrylamide 0.8 gm methylene-bis-acrylamide H20 to 100 mis Gel Mixture Mix 3i0 mis of B and 0.3 ml ampholytes (40$). For each set of duplicate gels, take 1.1 ml of the above'and add to I t 2.75 mis H2O (containing d i a l -lzed sample i f the gels are to include p r o t e i n ) . The mixture i s poured into the 0.5X10 cm tubes (to approximately 8 cms height) and exposed to bright l i g h t for approximately 1 hour to complete polymer-i z a t i o n . The tubes had been previously coated with Photoflo s o l u t i o n . Chemical polymerization— Stock solutions were kept at 4°C i n the dark f o r about one month. The c a t a l y s t solution was made fre s h each time; A; Acrylamide 3.05 gms acrylamide H 2 0 to 10 mis B. B i s - a c r y l 100 mgs methylene-bis-acrylamide E o0 to 10 mis C; Catalyst 150 mgs ammonium persulfate HO to 10 mis Gel Mixture Mix 1125 mis A, 1.25 mis B, 3i30 mis H 20, and 2.0 mis pooled ampholyte mlxe. Add to t h i s s o l -ution 25juCLs TSMED and 200 /Us of C. The four gels (of approximately 8 cm height) are poured Immediately into the 0:5X10 cm tubes which had been previously coated with Photoflo s o l u t i o n , and allowed to polymerize for at l e a s t 8 hours before use; 5*. The destaining solution was usually replaced every 30 minutes for the f i r s t 2 hours and then l e f t In a fresh s o l u t i o n over-night to complete the destaining process. The gels were scanned at 550nm when destaining was complete. The pH range 5-8 was found to be Inadequate f o r resolu-tion of the protein bands, since the pl»s of the proteins l n Peak A and B appear to be very s i m i l a r . Therefore, a 5% column i s o e l e c t r i c focusing experiment was performed i n the usual way but with a pooling of those ampholytes i n the pH 7.3-7•9 f r a c -t i o n s . The concentration of the pooled ampholytes was such that 0.5 ml was required f o r each 1$ g e l . Polymerization l n t h i s range of ampholytes was found to occur with l e s s ease than the pH 7-10 range ampholytes (and not at a l l by photopolymeriz-ation) so that 50$ more persulfate than the usual 1$ solution ( i e . a 1.5$ c a t a l y s t used) was required to complete polymeriz-a t i o n . The presence of sucrose from the pooled ampholytes did not i n t e r f e r e adversely with the experiment. I t has been ob-served ( 66) that sucrose s t a b i l i z e s the pH gradient and enables the proteins to be suc c e s s f u l l y focused i n a lower percent a c r y l -amide g e l , with a lower voltage and longer focusing time. Be-cause of the polymerization problems experienced, a lower per-cent acrylamide gel was not used but the gels were prepared as described i n Table I I I . Since TIM i s not excessively large or assymmetrically shaped, i t experiences no problems i n passing through the pores of the higher percent acrylamide gels. One of the main advantages of the 3«5$ gels described by Doerr and Chramback (66) would be that large or assymmetrically shaped pro-55 teins such as immunoglobulins could be focused! Upon polymerization, the gels were placed In the e l e c t r o -phoresis apparatus with the anode (bottom) buffer i n place. The presence of sucrose i n the gels makes them le s s r i d g i d and less able to adhere to the sides of the wall of the glass tubes when placed i n a v e r t i c a l p o s i t i o n : Therefore, small pieces of d i a l -y s i s tubing, held i n place by rubber bands around the bottom of the tube, prevented the gels from s l i p p i n g out into the lower r e s e r v o i r . Protein ( i n a 25$ sucrose sol u t i o n i n a volume under lOOyiils) was c a r e f u l l y l a i d on the top of the g e l with a micro-pipet. Next, 100yAls of 20% sucrose was l a i d on top of the pro-t e i n , followed by lOOyuls of 10$ sucrose: The cathode buffer was added to f i l l the r e s t of the tube and then the top reservoir was c a r e f u l l y f i l l e d with b u f f e r . The buffers, experimental run-ning conditions, temperature, e l u t i o n of ampholytes, and s t a i n -ing of protein were as described f o r the photo-polymerization method. The gels were allowed to focus f o r 13 hours rather than the 5-7 hours used for the photopolymerized g e l s . The narrower pH range of the ampholytes used i n the chemically polymerized gels take longer to t r a v e l to t h e i r i s o e l e c t r i c point. The gels were scanned at 550nm upon completion of destaining. D. Amino Acid Analysis The amino acid data was obtained from duplicate 24 hour hy-drolyses of approximately 0.05yUmoles 2X chromatographed Peak A. Tryptophan was assayed for using Wltkop's procedure (66) of re-action of N-bromosuccinimide (NBS) with the indole r i n g of trypto-56.. phane to y i e l d oxindole. The assay procedure was carr i e d out i n 8M urea (adjusted to pH 4 at 22°C with a c e t i c acid) to ensure complete t i t r a t i o n of a l l tryptophan residues. The t i t r a t i o n proceeds "by stepwise ad-di t i o n s of 5 o f 10mM NBS to 2.0 mis of protein solution (OD 2 8 o = 1»5-2) contained i n a 3«0 ml cuvette. The t i t r a t i o n pro-ceeded with a decrease i n the absorbance at 280nm. 5 y i l addi-tions continued u n t i l a minimum absorbtlon was reached with cor-rections f o r volume increase being made. The absorbance decrease at 280nm may be re l a t e d to the OD of tryptophan i n the protein sample by the empirical factor of 1.31 which allows for the ox-idation product of oxlndole. The E_ Q f o r tryptophan i s 5500M * 3.2 Results A. Subunit Molecular Weight An SDS gel electrophoresis molecular weight determination was performed using protein from the two active TIM peaks of the I n i t i a l DEAE Sephadex A50 chromatograph. The following gels were run ( l n addition to the molecular weight markers)i A. 20 jxes of peak A B. 20y*gs of Peak B C. 20^Ugs of peak A plus 20yjigs of Peak B Protein was taken from the center f r a c t i o n s of the two active TIM. peaks. In a l l cases, single bands were observed upon com-pl e t i o n of the electrophoresis experiment (with exception of gels containing marker p r o t e i n s ) . A subunit molecular weight of 24,500 was observed for Peak A, 24,700 fo r Peak B, and 24,500 when center f r a c t i o n s of peak A and B were co-run i n the same g e l . The presence of single bands was an i n d i c a t i o n of protein homo-geneity. I t would appear that the TIM a c t i v e f r a c t i o n s from both peaks have the same, or nearly the same molecular weight. The values of 49,000-49,400 obtained f o r the dlmerlc molecular weight are within a reasonable deviation from the molecular weight of 48,500 which McVittle calculated from p a r t i a l s p e c i f i c volume and sedimentation equilibrium measurements (11). SDS gel mol-ecular weight determinations are considered accurate to within 5-10$ of the true molecular weight. SDS g e l electrophoresis performed on twice chromatographed material gave r e s u l t s which were within 5$ of the subunit molecu-l a r weight determined for the once chromatographed protein. 58. These second values were 23,300 for Peak A, 23,100 for Peak B, and 23»000 for when Peak A and Peak B were co-run i n the same g e l . Once again, single bands were obtained. The protein was found to be homogeneous by SDS gel electrophoresis. The conclusion which may be drawn from the SDS gel e l e c t r o -phoresis r e s u l t s i s that the heterogeneity of the TIM active pro t e i n (as observed i n the chromatographic.protein and a c t i v i t y p r o f i l e s as well as the i s o e l e c t r i c focusing data which follows) i s not based on molecular weight d i f f e r e n c e s . Even the IX chrom atographed material (with the exception of the FEM modified pro-tein) was homogeneous by SDS gel electrophoresis. B. Disc Gel Electrophoresis The r e s u l t s of the pH 8.5 d i s c gel electrophoresis experi-ment i s shown i n figure 11. The R^, values, reported for the pro-te i n s , were obtained from measurements taken from the g e l s . There i s l i t t l e s i g n i f i c a n t difference i n the electrophoretic mobility of the various samples (4$ greatest d i f f e r e n c e ) . Scope (20) reported only one band for di s c gel electrophoresis of chicken muscle TIM at pH 8.5» hut he also indicated that starch gel electrophoresis had demonstrated the presence of a minor component. The minor component could correspond to the peak B protein which has been shown i n t h i s thesis to be separable chro atographically. The r e s u l t s shown here, support Scope's observation of one band by d i s c gel electrophoresis. The mixing of the protein from the two chromatographic (TIM active) peaks s t i l l r e s u l t s i n one protein zone. Even the peak B protein which was one year FIGURE lit Disc Gel Electrophoresis Using Fresh Protein from Peak A (A) and. peak B (B>, and Old Protein from Peak B (B v) 60 old was homogeneous (by t h i s method). The homogeneity of TIM by t h i s method and the very minor contaminent observed i n the starch g e l electrophoresis has encouraged the b e l i e f i n the l i t -erature that chicken muscle TIM i s r e l a t i v e l y free from Isozymes. However, the i s o e l e c t r i c focusing data to be described i n the following section as well as the chromatographic separa-tion observed, indicates the presence of isozymes. The protein i n peak B comprises almost 8% of the protein found to possess tr i o s e phosphate isomerase a c t i v i t y . The f a i r l y large degree of isozymlc contamination could af-fe c t the v a l i d i t y of crystallographic and amino ac i d sequence data which has been recently published (18). I t appears ijrob-able that both the x-ray c r y s t a l structure at 2.5A resolution and the amino ac i d sequence were performed on protein which was not pure. The implication of t h i s ( i n terms of the correctness of the published r e s u l t s ) could be Important i f the isozymic s t r u c t u r a l differences are s i g n i f i c a n t . In the Interpretation of the electron density map, there were 23 side chains which were a poor f i t with the map i n regions where the electron den-s i t y was r e l a t i v e l y strong (including 6 pa i r s of equivalent res-idues from the two subunits) and there was one short section of polypeptide chain, residues 168-176 i n one subunit, which could not be followed e a s i l y i n the electron density map. These prob-lems were p a r t i a l l y resolved by the authors of the c r y s t a l structure by considering that they were looking at two independ-ent Images of e s s e n t i a l l y i d e n t i c a l structure ( i e . 2 i d e n t i c a l subunits). However, i f there was substantial Isozymic Impurity 61. present i n the protein c r y s t a l s used, the assumption would he no longer v a l i d and. the p o s s i b i l i t y of. incorrect assignments of amino acids e x i s t s . C. I s o e l e c t r i c Focusing; I s o e l e c t r i c focusing (IEF) i s a se n s i t i v e method of sep-arating ampholytesj e s p e c i a l l y proteins, according to t h e i r i s o -e l e c t r i c point. Therefore, the IEF technique, which i s char-acterized by very high a n a l y t i c a l r e s o l u t i o n and by s i m p l i c i t y of apparatus and method, can be used for both preparative sep-arations of proteins as well as i s o e l e c t r i c point (pi) char-a c t e r i z a t i o n of proteins. As l i t t l e as a 0.02 p i difference-i n proteins may be observed. As w i l l be demonstrated i n t h i s section, i t i s possible to observe separations which cannot be seen by disc gel electrophoresis.. Probably the IEF method which has the most p o t e n t i a l f o r s e n s i t i v i t y and accuracy i s the column method, as discussed i n the methods section. I t has allowed, because of i t s high re-solution and r e p r o d u c i b i l i t y of p i value, a characterization of the chromatographically separate Peaks A and B of TIM. The f i r s t experiment to be reported here i s the column Iso-e l e c t r i c focusing of 6 absorbance units of rechromatographed peak A using the narrow range ampholytes (pH 7-8) which had been prepared from a 5$ ampholyte column run. The r e s u l t s shown In figure 12 indicate the presence of a major protein and. a major s p e c i f i c a c t i v i t y peak with a p i of 7*64. The A 2 y 0 plot showed a shoulder with pl=7»57 as well as a minor peak et 7*^7 which ml e f f l u e n t 63. might have some significance when compared to other runs, which contain Peak B protein, which w i l l "be described shortly. There was also a protein with pl=7«73 which has some TIM activity and one with a pl=5»87 which has none. The latter was probably en-zyme which had been aggregated by the pH conditions of the ex-periment. In the electrofocusing of 4 .3 absorbance units of rechrom-atographed Peak. B(see figure 13) the minor peak at pH 7*72 re-appeared as did the nonactive 280nm absorbing peak at pH 5 » 9 2 . However the major features were a protein peak at 7*57 and a well defined shoulder at 7.44. Resolution of the 7 . 5 7 and 7.44 pi peaks could probably be improved by a narrower gradient or less protein. The specific activity profile showed two peaks which appear to correspond to the proteins with isoelectric points of 7'57 and 7.44. The presence of the two small peaks at pH 7 . 5 7 and 7.47 in the protein profile of the isoelectric focusing of Peak A (fig.12) can now be ascribed (with some de-gree of certainty) to the presence of Peak B contaminant. Better resolution of the two proteins in Peak B i s visible in figure 14 which illustrates a column IEF experiment of re-chromatographed Peak A and Peak B. Three major protein peaks were eluted from the IEF column with pi's of 7.66, 7.55 and 7.46. Once again there is a minor amount of protein possessing some TIM activity at about pH 7 * 7 . In addition, there was a small peak of some activity at pH 7 * 3 8 . Less Peak A protein than Peak B was used in this experiment. The specific activity profile showed three peaks but they only roughly corresponded ml e f f l u e n t 65. 66 to the three major peaks v i s i b l e by determining the absorbance of f r a c t i o n s at 280nm. The r e s u l t s of the column IEF indicated the presence- of three proteins with t r i o s e phosphate Isomerase a c t i v i t y ! the f i r s t with a p i of about 7«65 (separable chromatographlcally Into Peak A) and the second and t h i r d with pi's of 7.56 and 7.45 respectively i s o l a t e d chromatographlcally together i n Peak B. There i s approximately 0.1 pH units difference between protein/I (pl=7.65) and protein II (pl=7»56) and between protein II and protein III (pl=7.48). The separation of the three proteins was further demon-strated i n the gel IEF experiment which was performed using the narrow range ampholytes, s p e c i a l l y prepared by $% column IEF runs. The r e s u l t s i l l u s t r a t e d In the g e l scans of figure 15 indicates the focusing of twice chromatographed Peak A into a / single component with pi-7.64, twice chromatographed peak B into three components of p i ' s equal to 7.66, 7.56 and 7.49, and f i n -a l l y the focusing of Peak A and peakB into three components with p i ' s of 7.64, 7.56,7.^-6: The photograph of the gels ( f i g . 16) demonstrates the c l a r i t y of the separation of the three proteins. Minute traces of bands other than the major three proteins are s l i g h t l y v i s i b l e i n the photograph but are not v i s i b l e at a l l l n the gel scans which indicates that contamination i s not present to any s i g n i f i c a n t extent. The presence of the three t r i o s e phosphate isomerase ac-t i v e proteins as observed by the IEF experiments, suggests very strongly three TIM isozymes. 68 FIGURE 161 Sample Gels of Gel I s o e l e c t r i c Focussing of Peak A and Peak B A ( - ) (Hh) B A + B r pi =7.64 pi =756 pi =747 6.9 The presence of more than one enzyme form for chicken breast muscle TIM has an evolutionary b a s i s . Gracy et a l has </ has published r e s u l t s giving the electrophoretic molulity from starch gel electrophoresis of t r l o s e phosphate isomerases from various t i s s u e s . He also indicated t h e number of isozymes pres-ent. A l l vertebrates reported possess three isozymes. It was necessary to go to the evolutionary l e v e l of a crab to observe two isozymes and of a beetle to observe one. I t would there-fore be inconsistent If only one molecular form of TIM was found i n chicken breast muscle. On the basis of the isozymic forms of other vertebrate t r i o s e phosphate isomerase (eg: human and r a b b i t ) , as well as the p i r e s u l t s , i t i s possible to t e n t a t i v e l y suggest t h e basis of the chicken isozymes to be a r e s u l t of two protein .chains ai andp . The presence of 2 d i s t i n c t chains^ ands would give the p o s s i b i l i t y of 3 Isozymes: rip, and p Heterogeneity of subunits has been observed i n other t r l o s e phosphate Isomerases ( 6»24). The r e l a t i v e s t a b i l i t i e s of the three forms as well as the actual r e l a t i v e quantities of and ^ chains would de-termine the r a t i o s of cK^ enzyme to and to f^* with the data available at th i s point a r a t i o of about 80:6:1 for dni^pi^ may be calculated i f the major enzyme form present i n Peak A (pl= ?.64) i s assigned the designation and the two miner isozymes i n the peak B are assigned. <k $ (pl=?.57) andy6'2 (pl=?.49) designa-tions r e s p e c t i v e l y . The two protein chains could have some genetic o r i g i n 70 ( i e . two separate genes) or I t i s possible that they a r i s e from some epigenetic process ( l e . post t r a n s l a t i o n a l changes i n the actual protein I t s e l f which may occur i n the ' i n vivo* s i t u a t i o n ) . The f i r s t p o s s i b i l i t y would r e s u l t from the presence of two d i s -t i n c t genes (possibly a r i s i n g from mutation at one or more s i t e s ) which code for protein chains of d i f f e r i n g amino acid sequence. The second p o s s i b i l i t y of epigenetic processes would involve some sort of chemical modification of the protein occurring l n the c e l l i t s e l f . A well known example of t h i s i s the formation of p r o t e o l y t i c enzymes from pro-enzymes. The presence of a fourth enzyme form which has no ' i n vivo' basis was observed when another gel i s o e l e c t r i c focusing exper-iment was performed which Involved the focusing of fresh twice chromatographed Peak A with older (about 1 year) twice chromat-ographed Peak B ( f i g . 17)« The B protein used, s t i l l possessed at l east 50$ of i t s i n i t i a l s p e c i f i c a c t i v i t y . The focusing of the A protein was si m i l a r to that obtained i n other IEF experi-ments ( f i g . 15) with a pl=7«66 found for the major protein zone. However the B protein was now found to contain 4 proteins of d i f f e r e n t p i * 7»66, 7*62 (not observed previously with fresh B protein), 7»58 and 7«50. Three peaks were accounted for by com-parison to the gel IEF runs of f r e s h A and fresh B proteint protein I (pl=7.66), protein II (pI=7-58) and protein III (pl= 7.50)• The presence of a peak with pl=7.66 l n the B protein probably indicates contamination with A pr o t e i n . The protein with pl=7»62 (from Peak B protein) was a new occurrence. There was no trace of i t when fresh B protein was FIGURE 1?s Gel I s o e l a c t r l c Focussing of Fresh Protein from Peak A and Old Protein from peak B " pH| £.t. T.C. 7.6. 1 7.4, ! 1 .i •7J ^Onm 7.6C A550nm V D I S T A N C E (cm) 72 used ( f i g . 15). While the pI--7«62 protein has taken over as the dominant protein i n the B peak, the protein with pl=7»58 has diminished i n quantity r e l a t i v e to the other B peak pro-t e i n s . I t would seem l i k e l y that, with age, there i s a change i n the pIn.-7.58 protein to give an al t e r e d protein with pl=7»62. The appearance of the pl=7»62 protein a f t e r a considerable length of time and not i n the o r i g i n a l f r e s h preparation of B, makes i t clear that the 7-62 form has no genetic o r i g i n but rather a r i s e s from a modification of e x i s t i n g p rotein. Mod-i f i c a t i o n of reactive carboxyl, amino, or hydroxyl groups i n the ' i n v i t r o ' as well as ' i n vivo' s i t u a t i o n may occur with a concomittant change i n p i . Some of the most l i k e l y changes would be loss of NH3 from asparaglne or glutamine as well as SH oxidation (of cysteine) to -SOH, -S02H, or -SO^E. Replace-ment of even a single carboxyl group l n hemoglobin has been known to cause d i s t i n c t changes i n i t s mobility (67). There i s also the p o s s i b i l i t y that with time, the conformation of the pro-t e i n has changed, with a resultant a l t e r a t i o n i n the exposure of charged amino acid side chains and hence a change i n i t s move-ment i n an e l e c t r i c f i e l d and i n i t s p i . D. Amino Acid Analysis The amino acid data for 1 subunit i s given below, along with the published values (17)• TABLE IV: Amino Acid Analysis of peak A TIM Amino acid 2X chromatographed Li t e r a t u r e value TIM-Peak A cys 4.01 4.0 arg 7.40 7.5 meth 1.98 2.0 tyr 3.80 3.9 his 7-32 7.6 asp 16;52 20.0 v a l 17.29 24.4 i l e u 14.42 16.6 lev 16.00 17.0 phe 7.00 7.8 pro 7.95 8.9 ly s 21.28 23.1 a l a 25.18 28.2 gly 23.90 27.0 glu 24.90 25.8 ser 10.94 13.5 thr 10:26 10.2 trp 5.02 5.0 TOTAL (nearest whole integer) 225 253 74. A molecular weight of 4 8 , 0 6 4 was calculated for the Peak A, (^ 2 isozyme) t r i o s e phosphate isomerase. This i s s i g n i f i c a n t l y lower than the published l i t e r a t u r e value of 5 ^ * ^ 0 0 obtained from the above ( l i t e r a t u r e ) amino acid data. There are s i g n i f -icant differences i n aspartic acid and valine amino acid num-bers with much smaller deviations apparant i n isoleuclne, leucine, phenylalanine, p r o l i n e , l y s i n e , alanine, glycine, glutamic and serine residues. The leucine, phenylalanine, p r o l i n e , and glu-tamic a c i d residues deviate by only 1 residue which could be within the error of the experiments. Amino ac i d data from the other two isozymes w i l l be necess-ary before any assumptions can be made concerning the amino acid differences between the three protein forms. 75-CHAPTER IV PROTEIN MODIFICATIONS 4.1 Introduction Protein modification i s a strategy used by the b i o l o g i c a l protein chemist to probe the structure of a protein. In the case of enzymes, the e f f e c t of modification upon the active s i t e , and hence the c a t a l y t i c c a p a b i l i t i e s of the system, can give valuable information concerning the enzymatic mechanism. For example, i n the case of t r i o s e phosphate isomerase, considerable Interest has been focused on the modification of an enzymatical-l y e s s e n t i a l glutamic a c i d residue. This i s covered i n some de-t a i l i n the introduction to t h i s t h e s i s . Possible s i t e s of modification i n any protein include the su l f h y d r y l group of cysteine, the imidazole group of h i s t i d i n e , hydroxyl group of serine, the f-amlno group of l y s i n e , the u»-carboxyl group of aspartlc and glutamic acids and the phenolic group of tyrosine. The s u l f h y d r y l group of cysteine has attracted attention due to i t s high n u c l e o p h l l i c and redox r e a c t i v i t y and i t s a b i l -i t y to enter into c h a r a c t e r i s t i c and s e l e c t i v e reactions. The s p e c i f i c i t y of the modification i s Important for both the anal-y t i c a l determination of numbers of s u l f h y d r y l residues present as well as a s t r u c t u r a l probe of the p r o t e i n . Therefore i t i s fortunate that the protein s u l f h y d r y l group of cysteine has been found to be very reactive to many reagents. The high n u c l e o p h i l l c l t y of mercaptide ions i s given by the 76. p a r t i c u l a r electron structure of the su l f u r atom with i t s high p o l a r l z a b l l l t y . The t h i o l a t e anion i s considered to he one of the strongest b i o l o g i c a l nucleophlles; i n addition to the po-._ l a r i z a b l l i t y of the su l f u r electrons, there are empty d-orbit-a l s , permitting d - o r b i t a l overlap and thereby increasing nucleo-p h l l i c l t y . For these reasons, cysteine i s a good choice for* modification of proteins. The SH group of cysteine takes part i n most reactions i n the form of the mercaptide anion (RS~). I t was calculated by Eenesch and Benesch ( 68 ) that at pH 7«^> (physiological pH) 6% of the SH's of free cysteine were io n i z e d . In the active s i t e s of enzymes, i t has been found that the pK of cysteine may vary from 7 to 9» Examples Include the active s i t e SH of phosphoenolpyruvate carboxykinase with a pH of 7»3 (69 ) and f i -c in which has been found to include a cysteine residue with pK of 8.55 ( 70 ) i n i t s active s i t e . Microscopic environments which cause t h i s v a r i a t i o n include proximity to p o s i t i v e charges (pK 0TT decreases) and negative charges (pK„ T T increases). Ioniz-ation of sulfhydr.yls are p a r t i c u l a r l l y depressed when the c y s -teine i s i n a hydrophobic microenvironment, buried within the protein. The pK fs i n t h i s case are commonly found to be above 9« The protein chemist"s i n t e r e s t i n cysteine i s a r e s u l t of the s i g n i f i c a n c e of the SH group for s p e c i f i c functions of a number of enzymes, hormones and other b i o l o g i c a l l y active pro-teins which play a central r o l e i n the normal course of many physi o l o g i c a l processes^as well as th e i r exceptional r e a c t i v i t y . 77 As a b i o l o g i c a l l y active functional group, the su l f h y d r y l has been known to be responsible for noncovalent binding of sub-strates and cofactors, d i r e c t covalent p a r t i c i p a t i o n i n the cat-a l y t i c act and maintenance of the native c a t a l y t i c a l l y active conformation of an enzyme. With the b i o l o g i c a l Importance of cysteine i n mind as well as the po t e n t i a l role of chemical s u l f h y d r y l modifications i n elucidating protein structure and function, i t i s well to note the high r e a c t i v i t y and d i v e r s i t y of chemicals reactions which distinguishes the SH group* a l k y l a t l o n , a c y l a t i o n , oxidation, t h i o l - d i s u l f i d e exchange, reactions with s u l f e n y l halides, and the formation of mercaptides, hemimercaptols and mercaptols. The following i s a short l i s t i n g of some of the reaction classes along with examples of reagents. Reaction Class Reagent Comment 1 . Transition. Metals C o l l , N i l l , C u l l , Hgll mercury complexes among the most stable 2. Oxidation H2°2 i n absence of metals, f a i r l y s p e c i f i c for cysteine and me-thionine 3- Nucleophilic Addition N-ethyl malelmide vary from r e v e r s i b l e conden-sations with aldehydes and ketones to f a i r l y i r r e v e r s -i b l e reactions with malelmide 4. Displacement a) haloacetol phosiD hates b) DTNB highly s p e c i f i c , r e s u l t i n g i n stable products 78« The a l k y l a t i n g l a b e l s include some of the more reactive r e -agents although the problem does exi s t of the r e s u l t i n g t h i o l product being quite unstable i n the aqueous environment and hy-drolyzlng. However, one class of labels, maleimides, usually have considerable success i n forming a stable covalent linkage with cysteine. The reaction i t s e l f involves addition of the s u l f -hydryl to an activated double bond: This Michael-type conjugate addition i s I r r e v e r s i b l e and goes rapidl y i n a l k a l i n e media. There i s an enhancement of rate as pH increases since the mercaptide anion of cysteine i s the most reactive species. An important competing reaction could be the addition of the £-amino groups of l y s i n e or the imidazole group of h i s t i d i n e to a malelmlde v i a an analogous mechanism. The s p e c i f i c i t y of the reagent for cysteine i s maintained by keeping the pH at or be-low pH 7«0 at which point the reaction of £-amino groups (or imi-dazole) i s i n s i g n i f i c a n t i n the time period required for t i t r a -t i o n of the SH groups which i s usually under an hour. At pH 7«0, the rate of reaction for simple t h i o l s i s on the order of 1000 times f a s t e r than for simple amines ( 71 ). Therefore, maleimides can be highly s p e c i f i c reagents although there i s always the pos-s i b i l i t y that the malelmlde may react with some residue other than cysteine which possesses increased r e a c t i v i t y as a r e s u l t of i t s p a r t i c u l a r environment l n the p r o t e i n . E-S + 79 • A widely used malelmide i s N-ethyl malelmide (NEM) which i s employed as a s u l f h y d r y l reagent. NEM absorbs strongly at - 1 - 1 around 300nm (£=620M cm ) which allows one to follow the re-action of the l a b e l by observing the decrease i n absorbance at 300nm as the reaction proceeds. This reagent was found to be suitable for chemical modification of t r i o s e phosphate isomer-ase, the r e s u l t s of which are reported i n t h i s section. A f l u o r i n e containing analogue of NEM was synthesized by D.G. Clark for t h i s work: | [ ^ 0 + HgN-CHg-CF^ —»ir^N-CE^CF^ + H 20 0 The r e s u l t i n g t r l f l u r o - N - e t h y l malelmide (FEM) has a broad max-imal absorbance centered at about 280nm (£=390). This 280nm ^ makes d i r e c t spectrophotometric observation of SH modifica-t l o n by FEM d i f f i c u l t since there i s a strong interference with protein absorbtion (and hence problems with blanking). The values obtainable by the d i r e c t spectrophotometric observation have a p o s s i b i l i t y of about 10$ error. Figure 18 i l l u s t r a t e s the u l t r a v i o l e t spectra of both NEM and FEM. The electron withdrawing e f f e c t of the f l u o r i n e atoms appears to activate the mallelmlde double bond even more strong-l y r e s u l t i n g i n a more f a c i l e addition to cysteine than the hy-drogen analogue, NEM. The FEM modifications reported, here have a s p e c i a l i n t e r e s t i n l i g h t of the reagent's p o t e n t i a l as a NMR l a b e l . FIGURE IVt U l t r a v i o l e t Spectra of NEM and FEM -i ———i ~ r ~ 250 300 350 \ ( \M) 81. Modification of t r l o s e phosphate isoraerase was also car-r i e d out using SH-disulphide interchange methods based on r e -actions with d i s u l f i d e s which are considered to be among the most s p e c i f i c reagents f o r protein SH groups, A p a r t i c u l a r i l y e f f e c t i v e reagent which has been used i n t h i s work i s Ellman's ( 7 2 ) reagent, 5 , 5 f - d i t h i o b i s (2-nitrobenzroic acid) which reacts with t h i o l s as follows : E-S~ + N 0 2 - ^ ^ S _ S - ^ ~ J J - N 0 2 — E - S » S - ^ ~ ^ - N 0 2 + ~S-<^^-NG2 ""00C COO" COO" COO" (DTNB) (E-TNB) (TNB") The strongly colored thionitrobenzoate anion which i s l i b e r a t e d 4 —1 —1 may be quantated by i t s absorbance at 4l2nm (£=1.36X10 M cm at pH 8.0). However, I t must be r e a l i z e d that with some proteins ( 73 , 74, 75 )» two reactions are possible a f t e r some SH*s have reacted: (1) the normal intermolecular reaction of the f i r s t SH*s (as above) (2) an intramolecular reaction of SH with the mixed d i s u l f i d e product which r e s u l t s from the reac-t i o n of protein -SH with DTNB. P S _ ^ •NO, 82. Whether or not the Intra molecular reaction succeeds i n compet-ing successfully with the I n i t i a l lntermolecular reaction, the end r e s u l t i s the same* release of one equivalent of n i t r o t h l -opholate anion (TNB") f o r each SH group that reacts. The absorbance of the reaction products at 4l2nm i s highly dependent upon pH. Consequently, there i s a l i m i t to the range of pH's which may be used with DTNB (usually pH 7.5-8 only ) . In addition, there i s a problem with autoxidatlon of the n i t r o -phenolate anion as well as d i f f i c u l t i e s l n using the reagent with colored proteins (eg. heme containing Proteins) which ab-sorb strongly at 412nm. Some of the d i f f i c u l t i e s may be gotten around by following Butterworth*s procedural changes ( 7 6 j * the protein i s f i r s t modified with DTNB, then Isolated and f i n a l l y reacted with d l -t h i o t h r e i t o l (DTT). The DTT very r a p i d l y l i b e r a t e s TNB~ from the protein and allows determination of the number of SH's by observing the Increase i n absorbance at 4l2nm. This method may not be used for those proteins which are able to undergo the i n tramolecular, d i s u l f i d e formation and resultant elimination of TNB". However, when t h i s method Is used, i t i s possible to ob-tai n values as correct as by the method I n i t i a l l y described a l -though i t i s not possible to observe the k i n e t i c s of the mod-i f i c a t i o n of the protein with DTNB. Another method of l i b e r a t i n g TNB~ i s by displacement with cyanide j (CN") E-S-(TNB) ^ E-S-CN + TNB" 83* This reaction also has the advantage of allowing the protein to become l a b e l l e d with C 1 3 v i a C 1 3N~ or C 1^ v i a C^N". The 13 - l 4 C N protein could be used In CMR studies while the C N pro-t e i n would make the modified protein r a d i o a c t i v e . A second aromatic d i s u l f i d e was used i n the modification studies to be described. There are two analogues of the d i t h i -odipyridine d i s u l f i d e described by Gra s s e t t i and Murray ( 7 7 ) possible i (1) (2) The 4,4»dithiopyridlne(2), i s the most sensitive reagent since upon reaction with a SH group i t releases a 4-thiopyridone which has as extinction c o e f f i c i e n t of 19,800 at 324nm which i s consider-ably larger than the maximum molor extinct i o n c o e f f l c l e n t , o f 7060 at 3^3 f o r the 2-thiopyridone. The 4,4* reagent was used ex c l u s i v e l y : (4-PD3) (4-TP) The pyridine d i s u l f i d e s have the advantage of being able to be used over a much wider pH range than DTNB. I t i s p a r t i c u l a r i l y useful f o r the lower pH's, where DTNB cannot be employed, since the diminishing l e v e l of E-S~ i s compensated fo r by the increas-ing r e a c t i v i t y of the reagenti The electron withdrawing proper-t i e s of the pyridine r i n g (and hence the r e a c t i v i t y of the d i -s u l f i d e ) becomes stronger with protonation of the nitrogen. •84* 4.2 Methods A. Modification with Malelmld.es (a) N-ethylmaleimlde (NEM)1 Approximately 20 mgs of twice chromatographed t r i o s e phos-phate i s o m 3 r a s e from Peak A i n a lOOmM pH 6.5 phosphate buffer was gently s t i r r e d overnight.in the cold with 25mM d i t h i o t h r e i -t o l (DTT) to ensure complete reduction of cysteine residues. The protein was desalted by passing i t down a Sephadex G25 c o l -umn (1 .5X20cm) e q u i l i b r a t e d and eluted with the lOOmM pH 6 . 5 phosphate b u f f e r i Approximately 2 ml f r a c t i o n s were c o l l e c t e d on a Gllson microfractionator and the absorbance at 280nm for each f r a c t i o n was determined; A f r a c t i o n with A 2 3 0 equal to 8.02 was selected f o r the modification and k i n e t i c study. A Zeiss PMQ II vislble-UV spectrophotometer was set up f o r constant temperature runs at 20°C. The reaction was i n i t i a t e d by placing 1 ml of protein into a 3 ml cuvette containing 2 mis of a N-ethyl malelmide s o l u t i o n i n the pH 6 . 5 b u f f e r . The mal-elmide: solu t i o n was of such a concentration as to give 20X molar excess of malelmide over protein; f i n a l concentrations were 4l.8yA.M In protein and 850y^M i n NEM. A reaction blank consist-ed of 2 mis of the NEM soluti o n and 1 ml cf bu f f e r . The modifi-cation of the protein was monitored by following the change i n absorbance at 300nm. as a function of time. ( f i g . 21) A decrease was observed u n t i l a constant value was obtained i n l e s s than 1 hour. (b) T r l f l u o r o - N - e t h y l malelmide (FEM) Peak A TIM was prepared f o r chemical modification as was 85 • the protein In the NEM experiments. However, either pH 6.0 or pH 6.5 lOOmM phosphate buffer was used fo r the modification re-action conditions. When pH 6.0 buffer was to be used i n the modification, the protein was s t i r r e d overnight i n pH 7.0 buf-fer so as to ensure the s t a b i l i t y of the p r o t e i n . In a l l other cases, the protein was reduced at the same pH as the modifica-t i o n was c a r r i e d out. In the f i r s t series of experiments (pH 6.0), 70 mgs enzyme was allowed to react In the cold with 20X excess FEM. The mod-i f i e d protein was desalted on a Sephadex G25 column, eluted with pH 6.0 lOOmM phosphate buffer and those f r a c t i o n s with an absorb-ance at 280nm over 1.0 were kept for s u l f h y d r y l determination J with DTNB. The DTNB reaction was i n i t i a t e d by adding 1 ml of enzyme containing about 1 absorbance unit protein to a 3 ml cu-vette containing 2.0 ml of the reagent stock s o l u t i o n containing lOOmM phosphate pH 8.0, lOmM EDTA and 5.25mM DTNB. The blank consisted of 2.0 mis of stock reagent and 1.0 ml of pH 8.0 lOOmM phosphate buffer. The reaction was followed by observing the increase i n absorbance at 4l2nm u n t i l a constant value was observed a f t e r 2 hours. In the same experimental s e r i e s , an a l i q u o t of the FEM modified protein was s t i r r e d In DTT overnight (25mM) and then reacted with DTNB. A s i m i l a r series of experiments was performed but with the FEM modification at pH 6.5• Both the reduction of the unmod-i f i e d protein and i t s desalting on the Sephadex G25 column were performed at pH 6.5. The protein was modified under 20X molar 86 excess reagent conditions and then the modified protein was de-salted on a pH 8.0 lOOmM phosphate conlumn. The DTNB determin-ation containing 1% sodium dodecyl3ulfate (SDS) i n addition to the lOOmM phosphate pH 8.0, lOmM EDTA and 5'25mK DTNB. An attempt was made to follow the k i n e t i c s of the FEM mod-i f i c a t i o n by obsarving the decrease i n the absorbance at 280nm as the protein reacts with trifluoromaleimide at pH 6.5. Thus, complete reaction taking place i n under 30 sec. Thus the modi-f i c a t i o n was too rapid (and would necessitate use of a stop-flow spectrophotometer) to follow using normal spectrophotometry techniques. However, i t d i d allow a d i r e c t determination of the number of SH*s which had reacted with FEM. The s p e c i f i c a c t i v i t y of the NEM and FEM modified protein was determined using the TIM assay procedure described e a r l i e r . Protein which had been modified by NEM as above at pH 6.5 was desalted on a Sephadex G25 column which was eluted with lOOmM pH 7.5 triethanolamine buffer, the appropriate d i l u t i o n was made, and the a c t i v i t y was determined. The same procedure followed i the FEM modification experiment. A c t i v i t y measurements were also performed on a control of unmodified Peak A protein a f t e r DTT reduction. ation proceeded as before but made use of a reagent stock s o l -was allowed to react with 87 B. Modifications with D i s u l f i d e s (a) 5,5*-dithiobis ( 2-nltrobenzoic acid) (DTNB) Twice chromatographed Peak A protein was reduced overnight i n the cold with 25mM d i t h l o e r y t h r e t o l (DTE) or DTT i n pH 7.0 lOOmM phosphate, and then desalted on a Sephadex G25 column e l u t -ed with lOOmM phosphate pH 8 . 0 buffer. Eluted f r a c t i o n s (~2mls) of A28O greater than 0 . 8 were used f o r the SH determination. The reaction was i n i t i a t e d by addition of 1 . 0 ml enzyme solut i o n to 2 . 0 mis of stock reagent (lOOmM phosphate pH 8 . 0 , lOmM EDTA, 5.25mM DTNB) i n a 3 ml cuvette. The reaction c e l l was blanked against a cuvette containing 1 . 0 ml buffer and 2 . 0 mis reagent. The reaction was followed v i a an increase i n the absorbance at 4l2nm u n t i l a constant value was obtained a f t e r approximately 2 hours. Upon completion of t i t r a t i o n of the 'exposed* SH's, a small quantity (^10 mgs) of s o l i d SDS was added to the reaction c e l l . Within one minute, the protein was completely denatured, thus f a c i l i t a t i n g the reaction of the *burled» SH*s. The f i n a l absorbance at 412 was determined giving a value f o r t o t a l pro-t e i n s u l f h y d r y l . The above procedure was repeated f o r twice chromatographed Peak B pr o t e i n . Experiments involving the displacement of TNB were performed on twice chromatographed Peak A protein which had been modified with DTNB as before except that reaction was ca r r i e d out i n a 37°C water bath Instead of room temperature i n order to decrease the reaction time: The modified protein was desalted on a Seph-adex G25 column ( 1 . 5 X 2 0 cm) eluted with a pH 7*5 triethanolamine bu f f e r . Approximately 4 mis of A 2 8 o = 0 , 1 8 1 Protein sample was 88. obtained. One half of the protein ( i e . 2 mis) was placed i n a cuvette and 1 mg of d l t h i o e r y t h r e l t o l was added. Liberat i o n of TNB~ (as observed by an Increase i n absorbance at 4l2nm) was almost instantaneous. The second half of the protein sample ( i e . 2mls) was also placed i n a cuvette and a c r y s t a l of KCN (1 mg) was added. The reaction c e l l holder i n the spectrophoto-meter was thermostated at 20°C. Liberat i o n of TNB~ was again observed by following the increase i n absorbance at 4l2nm. The dispacement by CN*" was slower than that by DTE and data f o r a k i n e t i c analysis was c o l l e c t e d , (b) 4,4* di t h i o p y r i d i n e The modifications of TIM with the second d i s u l f i d e , 4,4*-dithi o p y r i d i n e were performed i n a s i m i l a r manner to the DTNB reaction except that pH 6.5 was chosen f o r the reaction condi-t i o n . As before, the protein was reduced overnight ( i n the cold) i n 25mM DTE or DTT i n preparation for the chemical modifications. Desalting of the reduced, unmodified protein was performed on a Sephadex G25 column (1.5X20 cm) eluted with pH 6.5 lOOmM phos-phate bu f f e r . Fractions containing protein with &2Q0~^*® were employed i n the experiment. The reaction was i n i t i a t e d by plac-ing 50juls of stock reagent (26 mgs of 4,4"dithiopyridine per ml) delivered v i a a Lang-Levy pipet into a 3 ml cuvette containing 1.95 mis of pH 6i5 lOOmM phosphate buffer and 1.0 ml enzyme. The modification was observed by following the increase i n ab-sorbance at 324nm u n t i l a constant value was obtained, usually ..less that 2 hours. A determination of t o t a l s ulfhydrals was 8 9 . accomplished by adding a small amount of s o l i d SDS at t h i s point and observing the increase i n A ,. 324 A 4,4'dithiopyridine t i t r a t i o n at pH 6.5 was also performed on protein which had been modified by DTNB at pH 8.0. The un-modified Peak A protein was reduced i n the usual way, desalted at pH 8.0, reacted with DTNB ( i n the cold f o r several hours), desalted at pH 6 i 5 and f i n a l l y allowed to react with the p y r i -dine d i s u l f i d e . Upon completion of the t i t r a t i o n of reactive groups, SDS was added and an a d d i t i o n a l increase i n the absorb-ance at 32^nm was observed. A series of s p e c i f i c a c t i v i t y measurements of the various modified proteins was performed. In a l l cases, the proteins were modified under the conditions described previously. The pro-t e i n samples includedj (1) peak A TIM modified with DTNB (2) DTNB modified protein which had been allowed to react with CN" (3) DTNB modified p r o t e i n which had been allowed to react with DTE (4) 'control' sample of unmodified protein 90 . 4.3 Results A. Maleimides (a) N-ethylmaleimida Figure 19 i l l u s t r a t e s the pseudo f i r s t order modification of t r i o s e phosphate isomerase with N-ethylmalelmide. Data points were plotted to the t h i r d h a l f l i f e and a l e a s t squares technique was employed to fi:.t the best s t r a i g h t l i n e to the data. A pseudo _*a -1 f i r s t order rate constant of 2.39X10 J sec was calculated (20 C). A value of 2.05 was calculated f o r the number of sulfhy-dryls modified. At the pH of the experiment (6.5) i t i s improb-able that any residues other than cysteine was being modified. Since TIM i s a dimer, the near i n t e g r a l value of 2 suggests that one cysteine per monomer i s exposed to attack by NEM when the protein i s i n a native s t a t e i However, i t should be r e a l i z e d that there i s a p o s s i b i l i t y that the conformation of the protein i s such that 2 cysteine residues are exposed on one monomer but none on the other. In view of the f a c t that recent x-ray work on chicken muscle TIM demonstrated a symmetrical molecule (18) t h i s appears u n l i k e l y . The k i n e t i c analysis demonstrates that both SH's appear to be reacting with NEM at the same r a t e . This i s i n contrast to r e s u l t s which w i l l be reported i n the l a s t chapter with k i n e t i c s of d i s u l f i d e modification. With the data a v a i l a b l e from these experiments, i t cannot be decided with any certainty which cys-teines are being modified by NEM. 91. FIGURE 19: Pseudo F i r s t Order Analysis of the Mod i f i c a t i o n of TIM with NEM In s e r t : Increase i n the Absorbance as the Reaction Proceeds - I 1 r-4 8 12 TIME (MIN) 92* (b) T r i f l u o r o N-ethylmaleimide (FEM) Peak A t r i o s e phosphate isomerase, when modified at pK 6.0 or 6.5 by FEM, was found'to also be able to react with DTNB. I t w i l l be shown i n l a t e r segments of t h i s section that both FEM and DTNB are able to modify 2 cysteine residues of TIM. There-fore, i f one assumes that the same residues are being modified by a FEM modification or a DTNB modification, i t i s necessary to account for the type of reaction occurring If DTNB i s shown to react with protein which i s already modified by FEM. In t h i s case protein which had been modified by FEM at pH 6.0, and hence had 2 residues react was observed to have 1.10 residues exposed to reaction with DTNB under nondenaturing conditions and a t o t a l of 7.28 exposed to reaction imder denaturing conditions. The difference, 6.28, probably denotes the number of residues which are buried and not exposed to reaction with either DTNB or FEM. This value i s sim i l a r to the value of 6 'hidden' SH's usually obtained by DTNB t i t r a t i o n s alone (see d i s u l f i d e modification segment of t h i s s e c t i o n ) . When the FEM modified protein was allowed to s t i r overnight In DTT and was then assayed with DTNB, s i m i l a r r e s u l t s were ob-tained t 1.01 residues reacted under nondenaturing conditions. The difference i n t h i s case i s 5«67 which i s s t i l l quite close to the i n t e g r a l value of 6 usually obtained f o r a t i t r a t i o n of hidden SH's by DTNB. I f the FEM modified protein i s denatured : by SDS, 5*77 residues were found to react with DTNB, vihich again denotes a value close to 6 f o r the number of hidden residues. I f i t i s again assumed, that FEM and DTNB attack the same residue, a mechanism f o r displacement of the FEM by DTNB must be given. Under normal circumstances, maleimide modifications are considered to be i r r e v e r s i b l e . However, a mechanism such as the following could explain the r e s u l t s obtained i n t h i s p a r t i c u l a r modification of TIM* The general base (Bs) could well be a basic amino ac i d side chain i n the v i c i n i t y of the FEM modified s i t e which i s uniquely set up to accept a proton from the malelmide. The basic pH (8.0) of the DTNB modification makes the pos-s i b i l i t y of basic residues being i n the unprotonated form more l i k e l y . When the protein which had been modified by FEM was allowed to react with DTNB i n nondenaturing solution f i r s t and then i n denaturing s o l u t i o n , the t o t a l number of equivalents of DTNB averaged out to about 7 (one residue reacts i n solution without SDS, the remaining s i x react when SDS i s added). However, when the modified (FEM) protein was denatured before addition of DTNB, . only 5-77 residues were found to react. This gives support to a maleimide displacement which i s as s i s t e d by a s t r a t e g i c a l l y placed basic amino acid side chain. The displacement cf malei-mide appears to not be able to occure i f the protein i s denatured. 94'. The f a c t that only one equivalent of maleimide i s l i b e r a t e d suggests that there i s a nonequivalence of the two modified s i t e s which i s due to some conformational d i f f e r e n c e . I f the displace-ment i s indeed dependent upon some basic amino ac i d side chain, even small differences could a f f e c t i t s a b i l i t y to catalyze the reaction. The FEM modification of TIM has -a p r a c t i c a l a p p l i c a t i o n i n fl u o r i n e NMR of the p r o t e i n . Figure 20 gives the spectra ob-tained from 200 mgs of modified TIM along with a reference and a model compound ( a l l at pH 6.5). The reference i n t h i s case i s t r l f l u r o a c e t l c a c i d (TFA) which has been a r b i t r a r i l y set at zero. The model compound, which was formed by combining equimolar portions of N-acetyl cys-teine and FEM (pH 6.5), was found to absorb 1192 hertz downfield from TFA. However the protein sample gave a s i g n a l at 375 hertz u p f i e l d from TFA which i s a t o t a l of 1567 hertz from the model compound. A l l spectra were performed, at 100MHz on the XL100. I t was necessary to accumulate 100 transients for the model compound. (.02 hours) and 100,000 (5.57 hours) for the protein sample. The large chemical s h i f t difference between the model com-pound and the FEM l a b e l l e d protein i s i n d i c a t i v e of both the sen-s i t i v i t y of the f l u o r i n e nucleus to changes i n environment and also the considerable a l t e r a t i o n of the chemical environment of the FEM l a b e l . The single preliminary experiment i l l u s t r a t e d i n figure 22 can only give l i m i t e d Information but i t does indicate the f e a s i b i l i t y of further i n v e s t i g a t i o n with t h i s system. " FIGURE 20» F NMR of. FEM Modified Trlose Phosphate Isomerase 0 HERTZ ^ 96. F u r t h e r s t u d i e s a t v a r i o u s pH"s and b o t h l n the presence and i n the absence of s u b s t r a t e should g i v e v a l u a b l e i n f o r m a t i o n con-c e r n i n g the i o n i z a t i o n of groups i n the v i c i n i t y of the f l u o r i n e nucleus as w e l l as changes l n conformation of p r o t e i n upon the b i n d i n g of s u b s t r a t e . 97 . (c) S p e c i f i c A c t i v i t y Measurements on Modified TIM A s i m i l a r k i n e t i c analysis as that done f o r the NEM modi-f i c a t i o n of TIM could not be performed f o r FEM (without stop flow techniques) because of the r a p i d i t y of the modification r e -actio n . As discussed i n the introduction to t h i s section, t h i s Is probably due to a c t i v a t i o n of the double bond due to the e l -ectron withdrawing properties of the-CF^ group. In addition, there i s the p o s s i b i l i t y that the fluoroanalogue i s able to more r e a d i l y 'reach' the reac t i o n s i t e s i n the protein than the NEM i s , due to the changed e l e c t r o n i c character of FEM. However, the experiment which was performed allowed a d i r -ect determination that I.96 SH's had been modified. Thus, the extent of modification i s the same as that occurring with NEM. The two exposed SH's found i n the FEM t i t r a t i o n are es p e c i a l l y i n t e r e s t i n g i n l i g h t of the a b i l i t y of DTNB to t i t r a t e one SE equivalent per mole of FEM modified p r o t e i n . There appears to be a nonequivalence of the two FEM modified groups to modifica-tion by DTNB. As w i l l be demonstrated i n the k i n e t i c s section to follow, there i s also a nonequivalence of the unmodified protein to DTNB modification. The nonequivalence of the two cysteine residues could be pre-existing i n the native unmodified protein but also might be induced by the modification of either one or both of the SH's. S i m i l a r i t i e s between the NEM and FEM modified proteins are demonstrated by the data on the s p e c i f i c a c t i v i t y of the modi-f i e d species, as summarized belowt 98" Protein S p e c i f i c A c t i v i t y (units) % A c t i v i t y #SH Modified 1. Control 5233 100.0 0 2. NEM modified 1204 23.0 2.0 3. FEM modified 1307 25.0 2.0 The s i m i l a r i t y of the s p e c i f i c a c t i v i t i e s of the modified proteins suggests that the e f f e c t of the respective maleimides on the t r l o s e phosphate isomerase molecule i s also s i m i l a r . Even though the rates of modification of the two maleimides were quite d i f f e r e n t , i t i s l i k e l y that the a c t u a l modification ( i e . the s i t e ) i s the same. At t h i s point, i t would be premat-ure to assume that since the s p e c i f i c a c t i v i t y i s decreased by the modificationj that the s i t e of modification i s at or near the active s i t e . However, the recent 2.5A r e s o l u t i o n x-ray structure places the cysteine residue 126 In the c a t a l y t i c active s i t e of the enzyme. At the same time, i t i s possible that modification remote from the active s i t e could induce a conformational change that would a l t e r TIM cs c a t a l y t i c a b i l i t y . Thus, i f a change i n conformation did r e s u l t i n actual changes i n the structure of the a c t i v e s i t e which made c a t a l y s i s more d i f f i c u l t , a decrease i n the s p e c i f i c a c t i v i t y would be observed. This p o s s i b i l i t y has a s p e c i a l s i g n i f i c a n c e upon examination of the t r l o s e phosphate Isomerases prepared f o r the 2.5A r e s o l -ution x-ray structure which indicated that the most reactive cysteine was at a s i t e remote from the c a t a l y t i c center. I t was 99 • found that the SH of residue 21? was the most reactive to mercury compounds such as 2-chloroBiercuri-4-nItrophenol. However, a smaller and more hydrophobic mercurial, ethyl-mercury phosphate, reacted with a l e s s r e a d i l y accessible p a i r of sulphydryl groups at residue 41. B. D i s u l f i d e s In addition to the aforementioned maleiisldes, i t was found that TIM reacted r e a d i l y with d i s u l f i d e s . This i s i n contrast to reaction with 3-hromo-l,l,l-trifluropropanone or brompacetic acid which did not occur to any noticeable extent (Andrew M. Goetze, BSc t h e s i s , UBC, 1973). However, when DTNB i s allowed to react with Peak A TIM, the r e s u l t s Indicate that there are 2 SH groups which are exposed for ready modification by the d i s u l f i d e : upon denaturation 6 more are able to react to give a t o t a l of 8 cysteines present. This agrees well with the 8 cysteine residues per dimer found l n the amino ac i d a n a l y s i s . Repeated experiments usually gave r e s u l t s which varied only one or two percent from the Integer values of 2.0 and 8.0 for the respective 'exposed' and t o t a l SH t i t r a t i o n . The r e s u l t s f o r Peak B protein were si m i l a r with 1.7 SH's being t i t r a t e d i n i t i a l l y by DTNB, and an average of 8.0 being obtained upon addition of SDS. The cyanide displacement of TNB" from the DTNB modified protein occurred quite r e a d i l y . The pseudo f i r s t order k i n e t i c analysis with data given to the t h i r d half l i f e i s i l l u s t r a t e d i n f i g u r e 21.. A l e a s t squares treatment yielded the best straight FIGURE 21» Pseudo F i r s t Order Analysis of the Cyanld Displacement of TNB~ from the DTNB Modified peak A Protein Insert i Increase i n the Absorbance as the Reaction Proceeds I , . , r — 6 12 T (MIN) ~i f : d : r 1 2 3 4 TIME (MIN) IOI. l i n e for the data and the slope thus calculated gave a value of —3 —1 1 1 . 1 6 x 1 0 " - ' sec f o r the pseudo f i r s t order rate constant f o r the displacement of TMB by CN" at pH 7 * 5 and 20°C. I t was observed that CN" i s ablo to displace 0 . 8 5 equivalents of TNB" (average of 3 runs). Thus, once again there appears to be a nonequivalence In the a b i l i t y of modified cysteine residues to react with reagents. The same explanations of nonequivalence due to conformational difference may be invoked here a l s o . A schematic summary of the reactions includess DTNB C N " E > E(TNB) ? —-» E(TNB)CN pH 8 pE 7 . 5 The nonequivalence of the DTNB modified protein was not ob-served upon ac t i o n of d i t h l o e r y t h r e i t o l on the E(TNB) 2 form of the enzyme. Upon addition of DTE 2 . 0 8 equivalents of TNB~ per mole of TIM were l i b e r a t e d . The difference between the r e s u l t s of the CN" and DTE experiments might be explained by d i f f e r e n t a b i l i t i e s of the reagents to enter the s i t e of DTNB modification , on the enzyme. Results from the s p e c i f i c a c t i v i t y measurements performed upon the various modified proteins are summarized belows S p e c i f i c A c t i v i t y S p e c i f i c A c t i v i t y Protein (units) {%) ' ( 1 ) Control 8 3 6 8 1 0 0 ( 2 ) E(TNB) 2 2059 2 4 . 6 ( 3 ) E(TNB)CN 2 4 1 0 2 8 , 8 . ( 4 ) E(TNB) 2 + DTE. 8 4 7 7 1 0 1 . 3 102 The t o t a l a c t i v i t y of the enzyme was regained upon addition of DTE. This along with the observation of 2.08 equivalents of TNB" l i b e r a t e d indicates that the TIM molecule has resumed a na-ti v e unmodified state. I t i s i n t e r e s t i n g to note that the s p e c i f i c a c t i v i t y of the E(TNB) 2 form of the enzyme (24.6$) i s e s s e n t i a l l y i d e n t i c a l to that of the malelmlde modified species (25.0$). This lends sup-port to our e a r l i e r assumption that DTNB and maleimides ( l e . NEM and FEM) modify the same s i t e s on TIM. The s p e c i f i c a c t i v i t y of the E(TNB)CN form of TIM (28.8$) i s also very s i m i l a r . The reaction of TIM with the second d i s u l f i d e 4,4-dithlopyr-idine (4-PDS) followed along the same l i n e s as DTNB to y i e l d v a l -ues of 2.0? exposed SH t i t r a t e d under nondenaturing conditions and 8.02 t o t a l SH's t i t r a t e d under denaturing conditions to y i e l d a t o t a l of 5.95 SH's 'hidden*. The s p e c i f i c i t y of DTNB appears to apply also to 4-PDS when t i t r a t i n g TIM. However, the greater working range of pH could make It more u s e f u l f o r further modifi-cation studies of TIM (or indeed any other cysteine containing p r o t e i n ) . A rather Interesting example of the nonequivalence of the DTNB modified s i t e s of TIM was observed when 4-PD3 was allowed to react with the E(TNB) 2 enzyme form. The experiment i l l u s t r a t e s that 1.01 equivalents of 4-PDS were able to react with the enzyme under non-denaturing c o n t i t l o n s . When SDS was added (after t i t -r a t i o n of the 1.01 residues) 5.53 a d d i t i o n a l groups reacted to y i e l d a t o t a l of 6.61 reactive groups. Again i t appears that there i s one of the two DTNB modified s i t e s which i s able to r e -i03 act further. The 6 'hidden* cysteines are only able to react when the protein Is unfolded. In T o r c h l n s k i i * s excellent text dealing with s u l f h y d r y l and d i s u l f i d e groups of proteins, he gives a possible account of the reactions occurring i n a d i s u l f i d e exchange reaction ( ?8). R'-S-S-R' + R»-S-S-R* s 2R'-S-S-R* Three mechanisms for the exchange are given: one fo r neutral and basic solutions, one fo r a c i d i c and f i n a l l y a free r a d i c a l mechanism. At the pH of the experiment (6.5) there should be a p o s s i b i l i t y of more than one occurring. They are given below: (a) Basic or neutral s o l u t i o n The reaction i s catalyzed by t h i o l s ( c a t a l y t i c quantities can aris e by hydr o l y t i c cleavage of d i s u l f i d e s ) which carry out nucle-o p h l l l c attack on a sulfur atom of the d i s u l f i d e as i n a t h i o l -d i s u l f i d e exchange. (1) R\S~ + RWS-SR* » R'S-SR« + R WS" (2) R^S" + R«S-SR f ===== R*S-SR' + R'S~ (b) Acid Media The exchange takes place through a sulfenlum cation which i s formed through attack of a proton on the S-S bond. (1) R'S-S-R' + H + ==? R-S-S-R ===== R'SH + R'S + H. (2) R'S + + R*S-SRW ===== R»S-SR* + R WS + (3) R*S + + R'S-SR* ===== R"S-SR' + R'S + The reactlon(s) would be i n h i b i t e d by t h i o l s : BSH.+ R'S + ==* RS-SR' + H + 104. o) Free radical The reaction occurs with participation of free radicals which can arise either from high temperatures (>100°C) or ult r a -violet radiation. Since the reaction i s followed at 324nm, there i s the possibility of some photolysis occurring. Radicals arising from thermal cleavage can be ruled out. (1) R'S-SR1 2R»S« (2) H ' S ' + . R«-S-SRW R*S-SR' + RMS* (3) R*S« + R'S-SR" R®S-3R' • B ' S -Assuming that one, two or a l l mechanisms are operative to some extent in the case of E(TNB) 2 reacting with 4-PDS, the f o l -lowing scheme can be writtent DTNB 4-PDS E E (TNB) g pH 6.5 E(TNB) (4-TP) PH 8 105, CHAPTER V • KINETICS OF DISULFIDE MODIFICATION OF TIM 5.1 Methods Twice chromatographed Peak A protein was prepared for k i n e t i c runs by gentle s t i r r i n g overnight at 4°C i n 25mM d i t h i o t h r e i t o l or d i t h l o e r t h y r e l t o l . The protein was desalted on a 1 .5X20 cm Sephadex G25 column eluted with 200mM phosphate buffer, pH 6.5 i n the case of ^ j ^ ' d l t h i o p y r i d l n e runs and pH 8.0 f o r d i t h i o -bis-(2-nitrobenzoate) k i n e t i c s . Approximately 2 ml f r a c t i o n s were c o l l e c t e d by a Gllson Microfractionator, and t h e i r absorb-ance at 280nm determined on a Zeiss PMQII UV-Visible spectro-photometer. Fractions with Aggg^O.S were kept i n ice u n t i l ready for use. The most extensive series of k i n e t i c s involved modification of t r i o s e phosphate isomerase with ^ j V d l t h l o p y r i d i n e . K i n e t i c s of the modification of TIM were performed at 37°,31°,2?°,25°,22°, and 15°C. The k i n e t i c s were followed both l n the presence and i n the absence of substrate (G3P) f o r each temperature c i t e d . The progress of the reaction was followed by observing the l i b -eration of the thiopyrldone v i a an increase i n the absorbance at 324nm. The runs were done i n either duplicate or t r i p l i c a t e ; the calculated rate constants being an average of the i n d i v i d u a l values obtained from each i n d i v i d u a l experiment. A l l c o n s t i t -uents of the reaction mixture as well as the blank were e q u i l -ibrated at the temperature of the experiment. The temperature 106. was held constant throughout the experiment by means of a c i r c u -l a t i n g water bath which passed water of a constant temperature through the c e l l holder of the spectrophotometer. Individual k i n e t i c runs were i n i t i a t e d by rapid delivery of SOjxls of 4, V d l t h l o p y r l d i n e , 26 mgs/ml, v i a a Lang-Levy pipet into a 3 ml cuvette which contained l ml of protein sample (A 2g 0= 1) and 1 . 9 5 mis of 200mM pH 6 . 5 phosphate b u f f e r . The re-action mixture was blanked against a 3 ml cuvette containing 50 ^utls of reagent and 2 . 9 5 mis bu f f e r . For those runs which were to include substrate, lOOy^ls of glyceraldehyde 3-phosphate (0.57mM) was added to both reaction c e l l and blank i n place of 100 jO .B of b u f f e r . At the completion of each experiment, a small amount (~10 mgs) of sodium dodecyl s u l f a t e (SDS) was added to the reaction c e l l and the f i n a l A-^ gZ* r e s u l t i n g from t i t r a t i o n of a l l cysteines, both hidden and exposed was determined. The k i n e t i c experiments involving modification of TIM by DTNB were performed s i m i l a r i l y . Runs at 3 5 - 5 ° and 22°C were done, both i n the presence and i n the absence of G3P. Preparation of the protein was at pH 8.0 as described previously; reagents, buffer, and protein were pre-equllibrated at the temperature of the experiment. The k i n e t i c runs were performed at constant temperature. The experiments were i n i t i a t e d by the p i p e t t i n g of 1.0 ml of protein ( A 2 5 Q = 1»0) Into a 3 ml cuvette containing 2.0 mis reagent stock solu t i o n consisting of lOOmM phosphate pH 8.0, lOmM EDTA, and 5»25mM DTNB. For those k i n e t i c runs which were 107 to contain substrate, lOOyil of G3P («57mM) was added along with 1.90 mis reagent, and 1.0 ml protein s o l u t i o n . The modification of TIM by DTNB was followed by observing the l i b e r a t i o n of TNB~ as shown by the increase i n absorbance at 4l2nm. The absorbance at 4l2nm was determined at various time i n t e r v a l s u n t i l a con-stant value was obtained. The t o t a l t h i o l content of the protein was then determined upon addition of s o l i d SDS; with a further Increase i n A ^ l 2 r e s u l t i n g from denaturatlon of the protein and exposure of hidden cysteine residues to modification by DTNB. Data Treatment The pseudo f i r s t order rate equation may be written as f o l -lows with "a 1 equal to i n i t i a l concetratlon of reactant ( l e . r e -active t h i o l s ) , (a-x) meaning concentration of reactant at time • t* and k being the pseudo f i r s t order rate constant. a In . - kt a-XL Since a i s proportional to the f i n a l absorbance at 4l2nm (for DTNB ki n e t i c s ) or 324nm (for 4-PDS k i n e t i c s ) the substitution may be made whereby In . - kt Therefore a p l o t of In versus time t h e o r e t i c a l l y gives a Ao~ At straight l i n e going through the o r i g i n with slope k. A l l data was plotted to the t h i r d h a l f - l i f e . However, the pseudo f i r s t order p l o t s were not always ob-served to be l i n e a r since there were two (or more) reactive t h i -ols and each SH not necessarily reacting at the same ra t e . When 3.0S. the k i n e t i c p l o t was bi-phasic, i t was necessary to f i r s t deter-mine the slope of that l i n e a r portion of the curve, at l a t e r time (t) where reac t i o n of the more rea c t i v e t h l o l ( s ) was complete and the slope represents the rate of reaction for the slower reacting t h i o l . This slope was then subtracted from the e a r l i e r non-l i n e a r portion and a r e p l o t of the new values against time was performed. In the case of bi-phasic k i n e t i c s , the r e p l o t should now be l i n e a r and the slope would represent the rate constant for the f a s t e r reacting t h i o l . A t y p i c a l example of mono-phasic k i n e t i c s i s i l l u s t r a t e d i n f i g u r e 22 which shows the pseudo f i r s t order plo t f o r 4 , 4 " - d i -thiopyridine (4-PDS) modification of TIM at 22°C In the absence of substrate (G3P). A t y p i c a l bi-phasic p l o t , along with i t s r e p l o t , i s shown i n figure 23 which demonstrates the 4-PDS mod-i f i c a t i o n of TIM at 25°C i n the absence of substrate (G3P). The r e s u l t s of these plus the other temperature runs are given i n the section following. In a l l cases, a l e a s t squares analysis was performed on the l i n e a r p l o t s . A u s e f u l quantity to know i s the 1 h a l f - l i f e * or * half-period' of a reaction which i s the time i t takes f o r half the o r i g i n a l substance to disappear ( i e . r e a c t ) . There i s a simple r e l a t i o n -ship between the h a l f - l i f e of the reaction and the f i r s t order rate constant • k't 0.693 TJL = — f o r T i n minutes when k i s i n min . * k The h a l f - l i f e , as a useful concept for semi-quantitative discus-sions, was calculated for a l l rates determined. FIGURE 22 : A Typical Example of Monophaslc K i n e t l c s -4-PDS Modification of TIM at 22°G i n the Absence of Glyceraldehyde 3-Phosphate 5 10 15 20 25 T I M E (MIN ) n o . "FIGURE 23J A T y p i c a l Example of Ei-phasic K i n e t l c s -4-PDS Modification of TIM at 25°G i n the Absence of Glyceraldehyde 3-Phosphate Insert: Replot of Bi-phaslc Area .2(h 5 10 20 40 • 60 T I M E ( M I N ) i l l . An important r e l a t i o n s h i p i n k i n e t i c s which provides much information concerning mechanism,, i s one that connects the rate constant of a reaction with the temperature! the Arrhenius Law. The law may be expressed as follows $ k = : A e - E a / R T with 'k* the rate constant, 'A* the frequency factor of the re-action, s E a f the a c t i v a t i o n energy, *R" the universal gas con-stant and *T* as temperature (°Kelvin). Therfore a p l o t of log kCsec""1) versus 1/T K y i e l d s a slope of -Ea/2.303R or -Ea/4.57 with intercept A. This energy of a c t i v a t i o n represents the energy that the reactants must acquire i n order to undergo reaction and as such i s one measure of the ease of reac t i o n ; 112. 5.2 Results A. Modification of TIM with 4-PDS The r e s u l t s obtained from the pseudo f i r s t order k i n e t i c plots of the modification of TIM with 4-PDS are shown belows TABLE V i Modification with 4-PDS (1) In presence of Glyceraldehyde 3-phosphate , , •. T°C S p e c i f i c Rate Constant (mln-1) T.L (min) #SH"s' modified * 37 k l = .3950 1.75 2.0 31 k 2 .3259 .1681 2.13 4.12 2.0 27 k l = .2804 2.47 2.0 25 k2 = .2413 .0607 2.87 11.42 2.5 22 k 2 = .1297 .0637 5.34 10.88 2.4 15 k l = .0149 46.51 2.6 In a l l cases, 8.0 SH's were modified under denaturing conditio] (2) In absence of Glyceraldehyde 3-phosphate 37 .3421 2.03 2.0 31 = .2880 2.41 2.0 27 K2 .2760 .1432 2.51 4.84 2.0 25 k l k2 = • 2731 .0372 2.54 18.63 2.0 22 k l = .0788 8.79 2.0 15 .0289 23.98 2.0 113 From r e s u l t s presented i n Table V -1 ,2 , there does not seem to be any s p e c i a l dependence upon temperature or presence or ab-sence of substrate when observing mono- or bi-phaslc behaviour i n the k i n e t i c p l o t s . However, the presence of bi-phaslc behav-i o r does indicate that under certain conditions (of temperature or a v a i l a b i l i t y of substrate) there was a non-equivalence i n the p o t e n t i a l s i t e s of modification such that the reaction rates were able to d i f f e r by a f a c t o r of as much as eight (eg: 25°C> absence of substrate). As previously discussed i n the chapter concerning protein modifications, there i s the p o s s i b i l i t y of reaction of a single t h i o l Inducing a conformational change In the protein such that the second t h i o l i s now i n a less favor-able p o s i t i o n to react. I t i s evident from the l i s t i n g of the number of SH's which were modified i n the various experiments that the binding of sub-strate at temperatures at or below 25°C Is able to influence the t i t r a t i o n of a portion of a t h i r d t h i o l . I t Is known that the substrate G 3 P or DHAP i s able to change the conformation of TIM to a s i g n i f i c a n t degree ( 3 8 ) which might account, i n part, for the exposure of the a d d i t i o n a l t h i o l . However, an a d d i t i o n a l temperature dependent mechanism appears to be at work since mod-i f i c a t i o n of more than 2.0 sulfhydryls does not occur at temp-eratures above 25°C. At the same time, the complete absence of a t i t r a t i o n of a t h i r d t h i o l i n those experiments which were per-formed i n the absence of substrate, seems to indicate some de-pendence upon the conformational changes which the binding of i i 4 . substrate can Induce. The observation that only a portion of the t h i r d t h i o l (0.4 to 0.6) was t l t r a t a b l e could indicate the existence of an equilibrium between some •reactive* and "non-reactive* state of the t h i r d t h i o l and hence i n i t s p a r t i a l mod-i f i c a t i o n by 4 , 4 ' d i t h l o p y r i d i n e . The rate constants of the most ra p i d l y reacting t h i o l ( k 1) were employed i n an Arrhenius p l o t of log k versus 1/T K. The re s u l t s for those experiments which contained G3P i s shown i n figure 24 and fo r those which lacked G3P i s i l l u s t r a t e d i n f i g u r s 2 5 . A very prominent bi-phasic c h a r a c t e r i s t i c i s apparent i n both p l o t s . For those experiments performed with substrate,, the break comes at 24.6°C with the a c t i v a t i o n energy of the 4-PDS modification being 7 » 2 kcal/mole above t h i s temperature and 50»0 kcal/mole below i t . However, the second plot (without G3P) ex-hi b i t e d a break at 2 5 . 7 ° C with the upper limb (T> 2 5 . 7°C) giving an a c t i v a t i o n energy of 4 . 4 kcal/mole and the lower limb (T< 2 5 . 7°C) 3 9 . 9 kcal/mole. The presence of t h i s type of Arrhenius p l o t i s not unknown i n the l i t e r a t u r e . Copious examples are av a i l a b l e which i l l u s -trate temperature dependent a c t i v a t i o n energies of the c a t a l y t i c process of an enzyme, which i s analogous to the temperature de-pendent r e a c t i v i t y of a s p e c i f i c group, that i s the t h i o l of cysteine, which has been i l l u s t r a t e d l n the Arrhenius plots of figure 2 4 and 2 5 . Some ins i g h t into the temperature dependent mechanism i n TIM U 5 . 126, ^FIGURE 25J Arrhenlus P l o t for the 4-PDS Modification of TIM i n tha Absence of Glyceraldehyde-3-Phosphate 117 • might be gained by looking more closely at some of the examples i n the l i t e r a t u r e of temperature dependent processes occurlng i n enzymes. Glutathione reductase from P. chrysogenum has been shown to o have a c a t a l y t i c a c t i v a t i o n energy of 14.3 kcal/mole between 20 and 30°C and 11.8 kcal/mole between 30°and 40°C (79). The argu-ment of conformational change was Invoked to explain the r e s u l t s . Massey et a l (80) also obtained nonlinear Arrhenius plots for amino acid oxidase which was found to undergo a temperature de-pendent conformational change as observed by changes i n the sed-imentation constant,difference Bpectra, and fluorescence. The c r i t i c a l temperature f o r these phenomena coincided with the break i n the Arrhenius p l o t . An even more r a d i c a l example of a b l -phasic Arrhenius plo t a r i s i n g from temperature dependent conform-a t i o n a l change was observed i n the CMII isozyme of chorismate mutase (81). The plo t I l l u s t r a t e d an a c t i v a t i o n energy of 9«i80 kcal/mole above 25°C and 99.000 kcal/mole below 25°C. No further temperature, pH, or substrate influenced cooperative i n t e r a c t i o n was observed. However, the large (11X) a c t i v a t i o n energy d i f f e r -ence displayed by the mutase i s an i n d i c a t i o n of the s i g n i f i c a n t changes l n conformation which may take place as a function of temperature. The r e a c t i v i t y of the most e a s i l y modified t h i o l of TIM (by 4-PDS) changes by a factor of about 7 l n the a c t i v a -t i o n energy i n the presence of substrate and approximately 9 when substrate i s not present. The modification r e s u l t s i n the preceding chapter do suggest that Peak A TIM i s sens i t i v e to conformational change. However, an i n t e r e s t i n g continuation of 118 the discovery of bi-phasic Arrhenius p l o t s f o r the Peak A protein, would be inv e s t i g a t i o n Into whether the phenomenon occurs i n the other two isozymes. This could be an i n d i c a t i o n of e s s e n t i a l differences between the d i f f e r e n t enzyme forms of chicken muscle TIM. An i n t e r e s t i n g example of a break i n an Arrhenius plot was found i n muscle phosphorylase kinase (82) i n which the phenom-enon was e n t i r e l y absent at 7^gs/ml protein but present at 28 jlgs/ml. The marked d i s c o n t i n u i t y was centered at l 5 ° C with an a c t i v a t i o n energy of 18.200 kcal/mole at higher temperatures and 2.600 kcal/mole at lower temperatures. This unusual example of an enzyme with a higher a c t i v a t i o n energy at the higher temp-erature was explained by i t s c a t a l y t i c a c t i v i t y being controlled by a combination of substrate binding and a s s o c i a t i o n - d i s s o c i a -t i o n of the enzyme. This example has some i n t e r e s t since i t has been reported that TIM's c a t a l y t i c a b i l i t y i s affected by i t s concentration (83). The k i n e t i c t i t r a t i o n s of TIM SH's i n t h i s thesis were carried out at protein concentrations of about 0.3 mg/ml. In the study of Poole et a l (83) c r y s t a l l i n e TIM i s o l a t e d from rabbit muscle was d i l u t e d to concentrations between 1 mg/ml and 5 mgs/ml. Pro-gressive increase of enzymatic a c t i v i t y with time was observed with rate of c a t a l y s i s by TIM increasing up to twice the i n i t i a l value observed immediately a f t e r d i l u t i o n . In addition, i t was found that the concentration of mercurial i n h i b i t o r s required to reduce TIM a c t i v i t y by h a l f , as well as the rate of reaction of t h i o l s to iodoacetate, was dependent upon concentration of protein^ 119 I t has been suggested that the observed e f f e c t s are due to con-formational differences of the enzyme at d i f f e r e n t protein con-centrations which could be due to the proteins protein interactions present i n aggregation. The concentration of protein used i n the k i n e t i c experiments reported In t h i s thesis (0;3 mg/ml) are high enough to show these conformation and aggregation e f f e c t s . The p o s s i b i l i t y of temperature dependent mechanisms other than conformational change was further expanded by Hipps and Nel-son^s reports on four esterases (84). They found that the ester-ases, as p u r i f i e d from the American cockroach were characterized by double sloped Arrhenius plots with a c t i v a t i o n energies be-tween 6.3 and 8 . 5 kcal/mole at higher temperatures and between 11.6 and 14.5 kcal/mole at lower temperatures. The data was l i n k e d to association d i s s o c i a t i o n phenomenon ( l e . formation of enzyme aggregates) which indicated that the cockroach gut ester-ases probably e x i s t as thermally dependent molecular aggregates with d i f f e r e n t rates of h y d r o l y t i c a c t i v i t y i n the associated and dissociated forms. Thus, lowering the temperature dissociates the aggregates into l e s s active subunits (84). As mentioned e a r l i e r TIM i s known to form concentration dependent aggregates. In addition, i t has been well substantiated that there i s some association of g l y c o l y t i c proteins (including TIM) Into large units In the 'In vivo' s i t u a t i o n (85,86,87,83): Therefore, the p o s s i b i l i t y of some sort of a s s o c i a t i o n - d i s s o c i a t i o n mechanism fo r the blphasic Arrhenius p l o t behavior of TIM cannot be ruled out. In conclusion, I t i s d i f f i c u l t to suggest with any certainty, 120 the operative temperature dependent mechanism i n TIM with the data a v a i l a b l e at t h i s point; However, the mechanism i s not obviously s e n s i t i z e d by substrate binding although the (-) sub-strate case does have somewhat lower a c t i v a t i o n energies. The break i n the plo t s i s about i°C apart (24.6*0 versus 25*7*0) which i s within the experimental error. There i s a great de-gree of d i f f i c u l t y i n drawing accurate Arrhenius p l o t s with the few temperatures which were investigated. B. Modification of TIM with DTNB The k i n e t i c s of the DTNB modifications of Peak A protein was investigated at two temperatures, 22°C and 35»5 < >C. The re-su l t s are shown belowt (1) (+) substrate (2) (-) S p e c i f i c Rate Constant #SH modified 35-5 k-, = .3625 kg = .1885 2.0 22 kj = .0425 2.0 substrate T° S p e c i f i c Rate Constant #SH modified 35-5 K = .3555 kg = .2311 2.0 22 k 1 = .0355 2.0 121 The phenomenon of t i t r a t i o n of more than 2.0 t h i o l s i s ab-sent i n the DTNB modification of TIM. However, biphasic behavior was s t i l l observed i n the case of those runs performed at 35«5°C which indicates some s i m i l a r i t y i n enzyme changes occuring. However, the biphasic behavior was not nearly as marked as that obtained with 4-PDS and so, with the time l i m i t a t i o n s which were present f o r t h i s segment of the t h e s i s , the modifications with DTNB were not pursued beyond two temperatures. However, the close chemical r e a c t i v i t y of DTNB and ^ j V d i t h i o p y r i d l n e indicates the p o t e n t i a l i n t e r e s t of more extended temperature v a r i a t i o n . With the l i m i t e d data a v a i l a b l e , i t would seem inappropriate to report a c t i v a t i o n energies of the (+) substrate and (-) sub-strate systems. Only two points would be a v a i l a b l e f o r each Arrhenius p l o t and thus a low degree of confidence would be placed i n the values obtained. In addition, caution must be exercised i n deriving any i n t e r p r e t a t i o n from r e s u l t s obtained from such Arrhenius plots since the temperature i n t e r v a l (22-35.5°C) cuts across the breaking point (about 25°C) observed i n the Arrhenius plots of 4-PDS modification. 122 . CONCLUSIONS In conclusion, i t might he advantageous to b r i e f l y discuss the s i g n i f i c a n c e of the r e s u l t s reported i n t h i s thesis i n re-l a t i o n to the e x i s t i n g knowledge of t#riose phosphate isomerase. An i n d i c a t i o n of the puri t y of the i s o l a t e d chicken muscle enzyme i s that I t was found to possess s p e c i f i c a c t i v i t y up to 12,000 units/mg which i s at l e a s t as high or higher than the optimum of 10,000 units/mg which has been reported. Also, the chromatographic separation of TIM c l e a r l y demonstrated the f i r s t separation of chicken muscle isozymes. The f i r s t eluted peak, designated as "A" was shown to consist of one electrophoretic moiejby while the second *B* peak was observed to contain two. The presence of the double a c t i v i t y peak i n the s p e c i f i c a c t i v -i t y p r o f i l e of the DEAE-Sephadex chromatography of Peak A i s d i f f i c u l t to account f o r . The p o s s i b i l i t y of protein aggrega-t i o n has been discussed In t h i s thesis and might provide some explanation f o r the observed a c t i v i t y p r o f i l e ; I t has been well established that TIM forms part of an i n vivo g l y c o l y t i c aggreg-ate which consists of glyceraldehyde 3-phosphate dehydrogenase, aldolase, pyruvate kinase, and l a c t a t e dehydrogenase., (65,86,87) Therefore i t would not be too su r p r i s i n g i f the i s o l a t e d protein aggregated as w e l l . This p a r t i c u l a r type of protein:protein In-teraction could have some e f f e c t upon the enzyme's c a t a l y t i c a b i l i t y and hence on i t s s p e c i f i c a c t i v i t y . U l t r a c e n t r i f u g a t i o n sedimentation measurements could lead to Information concerning the p o s s i b i l i t y that these aggregates do, i n f a c t form. 123 • There i s very l i t t l e p o s s i b i l i t y that the observed, three Isozymes are pseudo-isozymes which or i g i n a t e from various ex-tents of s u l f h y d r y l oxidation as was found for ra b b i t muscle phosphoglucose isomerase (90) : The c a r e f u l reduction of a l l protein with d i t h i o l t h r e l t o l before any experimental manipula-tions, have ensured that a l l t h i o l s are i n a reduced state as shown by DTNB assay; However, t h i s does suggest a p o s s i b i l i t y f o r the double s p e c i f i c a c t i v i t y p r o f i l e observed f o r the Peak A isozyme. The chromatographies were not c a r r i e d out i n the presence of a reducing agent and i f the t h i o l s of the Peak A isozyme are p a r t l c u l a r l l y l a b i l e , i t could r e s u l t l n pseudo-isozyme-formation: These pseudo-isozymes would not be detected i n the electrofocusing of the protein since the enzyme i s re-duced Immediately before-hand; The type of f a c i l e oxidation which t h i o l s are p a r t i c u l a r i l y prone to i s usually r e v e r s i b l e . The Implications of the Isozymic separation on the struc-t u r a l determination already performed has already been discussed i n section B of the characterization chapter. Extreme caution w i l l have to be exercised i n i n t e r p r e t i n g the published amino ac-i d sequence and x-ray structure, expeclally i n l i g h t of the fac t that there were s i g n i f i c a n t differences i n many amino acid res-idues i n the amino a c i d analyses reported i n t h i s t h esis, for the Peak A p r o t e i n : In addition, the calculated molecular weight was d i f f e r e n t from the l i t e r a t u r e value by at l e a s t 5»000 gms/mole: The chemical modifications reported were i n d i c a t i v e of s i g -n i f i c a n t temperature induced conformational changes. Information 124. concerning t h i o l modification has, to date, been scanty. The foundations l a i d 'with these observations w i l l be important not only f o r general understanding of the protein, but also f o r such spectroscopic techniques,,as-NMR. .There i s a promising beginning with the F NMR spectra observed f o r the PEM modified TIM. The large chemical s h i f t difference of the labeled protein complex j and the labeled model compound gives some i n d i c a t i o n of the sen-s i t i v i t y with which t h i s technique w i l l be able to detect changes i n environment and hence the substrate-induced protein conforma-t i o n a l changes; The k i n e t i c s chapter was able to expand upon the modifications performed and l e d to some e x c i t i n g discoveries of temperature de-pendent mechanisms; L i t t l e information i n t h i s area i s a v a i l a b l e but the lower a c t i v a t i o n energy of the substrate enzyme complex correlates with some information a v a i l a b l e i n the l i t e r a t u r e (16) concerning reaction of rabbit muscle TIM with DTNB. In one paper, Krletsch et a l reported a second order rate constant for the DTNB modification i n presence of substrate DHAP which was only a t h i r d of that observed i n the absence of substrate. 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