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Modification of histidine in K-casein by 2-phenyl-1, 4-dibromoacetoin Styles, William George 1973

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MODIFICATION OF HISTTDIKE IN K-CASEIN BY 2-PHENYL-l,4-DIBR0M0ACST0IN by WILLIAM GEORGE STYLES B.Sc,(Hons) University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Food Science We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH. COLUMBIA . A p r i l , 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of p&-tre£. S<UJL>^-C*~ The University of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT The main object of this thesis was to specifically modify histidine resi-dues in K-casein and KA1 ani KA2 with the reagent 2-phenyl-l ,'4-dibromoacetoin (PDA) for the overall purpose of testing the hypothesis that histidine plays an important role in the stabilizing a b i l i t y of /c-casein. Experiments de-signed to test the hypothesis that PDA causes K-casein to aggregate by cross-linking were also carried out and these included the preparation of 2-phenyl-4-bromcacetoin (PKA) and reacting this with /c-casein and KM and KA2, as well as reacting PDA with histidine. Preliminary objectives of this thesis were to purify large amounts of rf-casein by scaling up the method of Z i t t l s (66), to improve the electrophoresis technique described by Perrin (^l) and Pever-idge (2) so that K-caseir; subfractions could be seen, and to purify the sub-fractions by DSAE cellulose chromatography using the method of Mercier (28). The lowest yield of /f-casein during four attempts of scaling up Z i t t l e ' s method was 6 . 0 ; ? and the highest yield was ?.5^ < Two preparations of K-casein resembled those published by KcKenzie (23) both in amino acid composition and electroohoretic heterogeneity. Modifications in the electrophoresis technique which gave promising re-sults included steps to increase the voltage through the gel equilibrating the gels by carrying out preliminary runs without samples, lowering the ionic strength of the system, changing the bridges, and increasing the concentra-tion of urea in the gel. Suggestions by Mercier (2^) which proved to be important in the senara-tion of the subfractions of V-casein by DSAB cellulose chromatography i n -cluded s i f t i n g the cellulose before use, recrystallizing the urea, and using concentrations of NaOH and HCl below 1.0 molar during regeneration cf the cellulose. Reactions of PDA and PMA with <-casein, KA.1 and. KA2 did not support the hypothesis that histidine plays an important role in the stabilizing a b i l i t y of K-casein because l ) PDA K-casein was prepared, in low ionic s t r e n g t h buf-fer, which dissolved, normally and had the same stabilizing a b i l i t y as un-treated '< -casein, even though 0,f> histidine residue was modified; and ?.) PMA KA1 and PMA KA2 were prepared which, although having 1.05 h i s t i d i n e and 1,60 lysine residues less than untreated KA1 and KA2, nevertheless had the same stab i l i z i n g a b i l i t y as the untreated protein. Rather, these results sutvDort the contentions that l ) the low stabilizing a b i l i t y of PDA-*.'-casein results from aggregation caused, by cross-linking of histidine residues on separate w-easein molecules; and 2) the cross-linking may be dependent upon the ionic strength of the reaction. Removing one bromine from PDA to make PMA seemed to result in a change of selectivity from histidine to lysine, PMA did. not react on K-casein in the same way as i t did on KA1 and KA2 and this supports the statements that future work on the modification of amino acids in *-casei.n and. studies on the interaction of this protein with * i-casein should be carried out on the subfraction rather than on ^-casein. Reactions involving PDA and histidine did not lead to an explanation of the mechanism of action of PDA because the yields of the products were too low to allow chemical analysis. Products having Rf's of 0.62, 0,53, and O.kn were produced and purified by cellex and paper chromatography in Jones' solvent (33) for the thin layer chromatographic separation of amino acids. These products reacted positively to Pauley's reagent, indicating that they were histidine addition products. i i i TABLE OF CONTENTS , p g cr g ABSTRACT 1 LIST OF FIGURES v LIST OF TABLES v i i INTRODUCTION 1 CHAPTER I LITERATURE REVIEW A. D e f i n i t i o n s ^ 3 . The Discovery and P u r i f i c a t i o n c f ^-casein 5 C. The E l e c t r o p h o r e t i c A n a l y s i s of Caseins 6 D. The P u r i f i c a t i o n of ^-casein S u b t r a c t i o n s 10 E. The M o d i f i c a t i o n of H i s t i d i n e Residues i n - c a s e i n 13 CHAPTER TI PRACTICAL EXPERIENCE IN LARGE SCALE PRODUCTION OF ZTTTLE'S CASEIN M a t e r i a l s 1? Methods 18 Results and Dis c u s s i o n 18 P r o p e r t i e s of the P u r i f i e d ic'-casein 27 CHAPTER I I I ATTEMPTS TO IMPROVE THE ELECTROPHORESIS OF x-CASEIN M a t e r i a l s 30 Methods 31 Results and Dis c u s s i o n 33 Sunmaiy 3? iv CHAPTER IV SEPARATION OF 3UBFRACTI0NS OF x'-CASEIN BY DEAE CELLULOSE CHROMATOGRAPHY Pa.~e Experimental P l a n 3~: M a t e r i a l s 39 Methods 0^ Results and Di s c u s s i o n Summa.ry 51 CHAPTER V MODIFICATION .OF HISTIDINE RESIDUES WITH PDA M a t e r i a l s 5^  Methods 55 R e s u l t s and D i s c u s s i o n 56 Summary 90 CHAPTER VI STUDIES TO DETERMINE THE MECHANISM OF ACTION OF PDA WITH HISTIDINE I n t r o d u c t i o n 71 Theory 7 6 Experimental P l a n 7^ M a t e r i a l s 73 Methods 73 Results and Discussi o n 81 Summary 95 CHAPTER VII SUMMARY AMD CONCLUSIONS 97 BIBLIOGRAPHY l no V LIST OP FIGURES Figure ¥a.F.e 1 The p r e p a r a t i o n of case i n f r a c t i o n s according to Waugh 6 • and von H i p p e l . 2 E l e c t r o p h o r e t i c a n a l y s i s of the pH 1.3, 2.2 M p r e c i p i t a t e 23 i n Z i t t l e ' s method. 3 E l e c t r o p h o r e t i c a n a l y s i s of the ammonium s u l f a t e p r e c i p i - 25 t a t e i n Z i t t l e ' s method. k E l e c t r o p h o r e t i c a n a l y s i s of the f i r s t p r e p a r a t i o n of 28 /r-casein. 5 E l e c t r o p h o r e s i s of «-casein showing good r e s o l u t i o n of 36 s u b t r a c t i o n s , 6 P a r t of a s u c c e s s f u l chromatographic fro.ctiona.tion of vrhcle c a s e i n on DEAE c e l l u l o s e u s i n g the method, described. by M e r c i e r , 5^ 7 E l u t i o n p r o f i l e of one of the f i r s t attempts to f r a c t i o n -ate /r-ca.sein on DEAE c e l l u l o s e u s i n g the r . o d i f i c a t i o n s of M e r c i e r et a l . k6 8 S u c c e s s f u l f r a c t i o n a t i o n of K-casein on DEAE c e l l u l o s e . 4 7 9 F r a c t i o n a t i o n of K - c a s e i n d u r i n g which gra d i e n t was a p p l i e d by g r a v i t y . kQ 10 Rechromatography of shaded a.rea.s of Figure 7. k9 11 Rechromatogra.phy of KA1 and KA2, 50 12 E L e c t r o p h o r e s i s of /c-casein and PDA- K - c a s e i n . 59 13 Change i n h i s t i d i n e r e s i d u e s i n KA1 w i t h time o f r e a c t i o n w i t h 2-phenyl-^-bromoacetoxn (PMA). 61 lk E l e c t r o p h o r e s i s of KA1 and PMA-KA1, ' 62 15 S t a b i l i z i n g a b i l i t y of KA1, KA2, PMA-KA1, *-ca.sein and SSS- K-casein. 63 16 S t a b i l i z i n g a b i l i t y of untreated /c-casein and PDA- K-casein reacted i n low i o n i c s t r e n g t h b u f f e r . 68 1.7 F r a c t i o n a t i o n on c e l l e x of 0.5 ml of r e a c t i o n mixture of PDA and h i s t i d i n e . 82 Rechromatography on c e l l e x of s e v e r a l p r e p a r a t i o n s of F r a c t i o n Gj shown i n Figure 17. F r a c t i o n a t i o n of 1.5 ml of s o l v e n t c o n t a i n i n g 80 mg of r e a c t i o n mixture of PDA and h i s t i d i n e on a l a r g e c e l l e x column. F r a c t i o n a t i o n of J.O n l of s o l v e n t c o n t a i n i n g 80 mg of r e a c t i o n mixture and h i s t i d i n e cn a l a r g e c e l l e x column. Thin l a y e r chromatography of the tubes spanning F r a c t i o n I of Figure 2QA. Rechromatography of 1 ml of s o l v e n t c o n t a i n i n g m a t e r i a l r i c h i n Rf 0.62 substance. F r a c t i o n a t i o n cn c e l l e x of PDA and h i s t i d i n e r e a c t i o n mixture c o n t a i n i n g a 10 molar excess of PDA. Thin l a y e r chromatograms of r e a c t i o n mixtures of PDA and h i s t i d i n e c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of r e a c t i v e s . Thin l a y e r chromatography of r e a c t i o n mixtures of PDA and h i s t i d i n e r e f l u x e d i n the presence of Aluminium Amalgum. Thin l a y e r chromatography of r e a c t i o n mixtures of PDA and h i s t i d i n e r e f l u x e d i n 6N HCl f o r 1 and 2k hours. LIST OF TABLES Amino a c i d composition of two pr e p a r a t i o n s of k-casein p u r i f i e d by the method of Z i t t l e . The e f f e c t s of b u f f e r and bridge systems on voltage measurements taken at v a r i o u s l o c a t i o n s on the e l e c t r o p h o r e s i s apparatus, Amino a c i d a n a l y s i s of K-casein and PDA- K - c a s e i n Amino a c i d a n a l y s i s of K-casein and PMA- K - c a s e i n Amino a c i d a n a l y s i s o f PDA- K-casein reacted i n low i o n i c s t r e n g t h b u f f e r , The e f f e c t of c o n c e n t r a t i o n of r e a c t a n t s on the pH of the r e a c t i o n mixture (PDA + h i s t i d i n e ) a f t e r 2h and 72 hours. v i i i ACKNOWLEDGEMENTS I would l i k e to thank Dr. S. Nakai f o r s u p e r v i s i n g t h i s work, Miss Arlen e Gelder f o r conducting the amino acid, analyses, Miss Dianne Sher-wood f o r her s e c r e t a r i a l help, Mrs. Gerry S t y l e s and Mrs. Pat Chan f o r t y p i n g the f i r s t d r a f t , a.nd Mrs. P h y l l i s Spencer f o r t y p i n g the f i n a l copy. INTRODUCTION It has been known for some time now that k -casein has the a b i l i t y to interact with <^s - and 3 -caseins and form a stable micelle in the presence of calcium. Thus k-casein i s important in the over-all s t a b i l i t y of the milk system. This i s manifested when milk i s processed in various ways. For instance, when milk i s heated at 85°C for 5 minutes A"-casein interacts with o i . -lactoglobulin to form a complex. When steps are taken to interfere with this interaction, for example, by stopping the disulfide interchange reaction, then the heat s t a b i l i t y of the milk i s increased because the ^•-casein i s available to stabilize the system ( 2 9 , ^ 5 ) « Rennin acts on zf-casein at least 1 0 0 0 times more rapidly than i t does on the other caseins ( 2 3 ) . The enzyme s p l i t s a glycomacropeptide from the k-casein and thus renders the protein unable to protect % - and £-caseins. Heat processing and enzymatic action are preliminary steps in the formation of many dairy products. Thus, i t i s important to know the factors which govern the a b i l i t y of AT-casein to stabilize the other caseins. It has been known since the early 1 9 6 0's that there are two genetic variants of k-casein, These have been cla s s i f i e d as /(r-casein types A or B according to their electrophoretic mobility on starch and polyacrylamide gels containing 2-mercaptoethanol, It i s possible to obtain a single genetic variant from the milk of cows which are homozygous for the desired type. Early experiments involving the electrophoresis of K-casein indicated that this protein consists of at least six distinct subfractions joined together by disulfide bonds. Each subfraction has been partially purified and characterized. Schmidt in 1 9 6 6 found that N-acetyl neuraminic acid increased as the fraction mobility increased and that the cystine and phosphorous content did not d i f f e r between the subfractions. Further, i t vras found t h a t the a b i l i t y to s t a b i l i z e «** -ca s e i n a g a i n s t p r e c i p i t a t i o n by calcium d i d not vary between the s u b f r a c t i o n s . The e l e c t r o n h o r e t i c p a t t e r n s of K-casein as pu b l i s h e d i n the l i t e r a t u r e have not been con-s i s t e n t i n t h a t the s u b f r a c t i o n s have not appeared i n a very d i s t i n c t way. Tn many cases the s u b f r a c t i o n s cannot be seen, What appears i s a l a r g e s t a i n e d area which shows the heterogeneity of the ^.-casein molecule. Attempts to p u r i f y the i n d i v i d u a l s u b f r a c t i o n s have been p a r t i a l l y success-f u l and probably f o r t h i s reason most of the work that has been done to ex p l a i n the mechanism o f i n t e r a c t i o n of K-casein with d$ - c a s e i n has been carried, out on whole K-casein, I t wa.s s t a t e d above that each sub-fraction: of K-casein can s t a b i l i z e cly-casein e q u a l l y as w e l l as whole /C-casein. This i n d i c a t e s that the carbohydrate moiety on the molecule i s not r e s p o n s i b l e f o r the s t a b i l i z i n g a b i l i t y . This hypothesis vras supported, by experiments which involved, the removal o f neuraminic a c i d from K - c a s e i n by neuraminidase p r i o r to c a r r y i n g out the i n t e r a c t i o n w i t h ^ / - c a s e i n (^O), I t was found t h a t the k - c a s e i n so t r e a t e d d i d not l o s e i t s s t a b i l i z i n g a b i l i t y , P h c t c o x i d a t i o n s t u d i e s cn K - c a s e i n i n d i c a t e d t h a t h i s t i d i n e might be an important amino acid, i n the s t a b i l i z i n g a b i l i t y of K - c a s e i n ( 6 ? ) , However, Nakai's work ( 3 2 ) did. not support t h i s hypothesis. No attempt was made by the above workers to check the s p e c i f i c i t y of t h e i r m o d i f i c a t i o n r e a c t i o n s . Therefore, they could draw no d e f i n i t e c o n c l u s i o n s as to the r o l e of h i s t i d i n e i n the a b i l i t y of rc-casein t o s t a b i l i z e -J^ - c a s e i n . The o b j e c t of t h i s t h e s i s was to s p e c i f i c a l l y modify h i s t i d i n e residues i n K-casein with the reagent 2-phenyl-l ,^4-dibromoacetoin (PDA), and to i d e n t i f y the products of the r e a c t i o n of PDA with h i s t i d i n e f o r the o v e r - a l l purpose of t e s t i n g the hypothesis that h i s t i d i n e p l a y s an important r o l e i n the s t a b i l i z i n g a b i l i t y of *--casein. Since K -casein i s a heterogeneous 3 protein, i t was also decided to react subfractions one and two with this reagent. Preliminary objectives, therefore, were to purify ^casein, to improve the electrophoresis technique to the point where the subfractions of /f-casein could be readily seen, and also to separate the subfractions of /C-casein by DSAE cellulose chromatography. Early in these studies i t was observed that PDA caused l f - c a s e l n to aggregate and for this reason tests were carried out that involved changing the reaction conditions to see i f a workable derivative of PDA- K -casein could be obtained. In order to test the mechanism of aggregation the rnono-bromo derivative 2-phenyl-^-bromoacetoin (PMA) was prepared and reacted with whole ^-casein and subfractions one and two; also, PDA was reacted with the amino acid histidine and attempts were made to purify and identify the reaction products. LITERATURE REVIEW D e f i n i t i o n s In t h i s t h e s i s the main components of casei n are c a l l e d f r a c t i o n s and are r e f e r r e d to by t h e i r proper names, e.g. - and ft - c a s e i n , The genetic v a r i a n t s are designated as "types", f o r example, K - c a s e i n type A, This i s the f a s t e s t moving major /C-casein band t h a t appears f o l l o w i n g polyacryiamide o r s t a r c h g e l e l e c t r o p h o r e s i s i n the presence of urea and 2-mercaptoethanol of whole casein prepared from pooled milk . The term s u b t r a c t i o n a p p l i e s only to k-casein. S u b f r a c t i o n s are the macrornolecules which r e s u l t a f t e r r e d u c t i o n of whole A"-casein and chromatography, on DEAE c e l l u l o s e columns i n the presence of urea. According to K i l l and Wake ( l ? ) " s u b f r a c t i o n s are i n t a c t /<-easein molecules which d i f f e r from each other l a r g e l y i n t h e i r carbohydrate content". The o p e r a t i o n a l d e f i n i t i o n of casein used i n t h i s t h e s i s i s t hat quoted from Nakahori ( 3 0 ) : "Casein i s a heterogeneous group o of phosphoproteins p r e c i p i t a t e d from mi l k a t pH h.c and 20 C". In 1939 Melander f i r s t observed the r e s o l u t i o n of a c i d c a s e i n by moving boundary e l e c t r o p h o r e s i s at pH 8 . 6 i n t o three peaks which he named -, and £ - i n order of decreasing m o b i l i t y ( 2 7 ) . I t was o r i g i n a l l y thought t h a t <i-casein was a s i n g l e p r o t e i n and. t h a t i t was the f r a c t i o n upon which the enzyme rennin acted. However, i t was found i n 1956 that p a r t of the ^-complex was calcium i n s o l u b l e and p a r t of i t was c a l c i u m - s o l u b l e . The enzyme rennin acted at l e a s t one thousand times more r a p i d l y on the l a t t e r than i t d i d on the other c a s e i n s . At t h i s time the term ^ - c a s e i n was a p p l i e d to the c a l c i u m - s e n s i t i v e f r a c t i o n and the term K-casein was a p p l i e d to the r e n n i n - s e n s i t i v e , c a l c i u m - s o l u b l e p o r t i o n . The di s c o v e r y and. p u r i f i c a t i o n of the l a t t e r w i l l now be b r i e f l y d i s c u s s e d . 5 B. The Discovery and Purification of K-casein The idea that one of the caseins acts as a protective colloid to stabilize the milk system dates back to the work of Hammerstein in 1 8 7 2 - 7 7 ( 1 3 ) . This view was supported by Linderstrc/ni-Lang in 1 9 2 3 ( 2 1 ) . He stated that casein consisted of at least two components, one insol-uble and the other soluble, in the presence of calcium ( l l ) . This l a t t e r component or protective colloid interacted with the calcium-insoluble component to prevent i t s precipitation. This protective co l l o i d was demonstrated by Waugh and von Hippel in 1956 ( 6 0 ) . These workers centrifuged ( 4 5 , 0 0 0 x g) skim milk after addition of 0 . 0 6 M calcium chloride and obtained calcium caseinate gel ( f i r s t cycle casein fraction). After removal of calcium ions from f i r s t cycle casein and dissolution in water, the calcium concentration was brought to 0 . 2 5 M with calciun chloride. A precipitate was obtained, designated second cycle casein - fraction P. The supernatant of second, cycle casein was then centrifuged ( 9 0 , 0 0 0 x g at 2°C) and the sediment discarded, Cal-cium was removed from the supernatant and this fraction was called second cycle casein - fraction S. The procedure i s shown in Figure 1 . It was found that the addition of calcium to the second cycle casein fraction P at concentrations markedly lower than those found in milk, led invariably to the formation of a coarse, heavy precipitate. Addition of calcium to the f i r s t cycle casein led to the formation of stable micelles of skim milk. It was reasoned, therefore, that a st a b i l i z i n g factor was removed during the separation of the secondary cycle fraction P, Support for this possibility was obtained when i t was demonstrated that stable micelles formed when appropriate amounts of fraction P and the corresponding supernatant, freed of calcium, were combined in the presence of calcium levels similar to those found in .6 Figure 1. The preparation, of casein f r a c t i o n s a c c o r d i n g to V/augh and von Hip p e l . P r e c i p i t a t e ( F i r s t c y c l e ca.sein) Remove G a + + D i s s o l v e i n water 0 . 2 5 H Ca' ++ P r e c i p i t a t e ( F r a c t i o n P) Sediment - d i s c a r d SKIM MILK 0 . 0 6 M Ca'+ Centr i f u g e (45,000 x g) Supernatant (Second Cycle Casein) C e n t r i f u e J (90.000 x c at 2°C) Supemate Remove Ca I (Second Cycle Casein F r a c t i o n s ) 7 skim milk. Thus i t was at this time that the -casein fraction as named by Mellander ( 2 7 ) was shown to consist of a calcium-sensitive fraction called - and a calcium-insensitive fraction called /('-casein. Since then many methods have been suggested for the purification of /:-casein. By 1952 several workers had succeeded in fractionating whole casein into the three major electrophoretic components mentioned earlier. Wake ( 2 3 ) succeeded in the isolation of highly pure ^-casein from second cycle casein fraction S, KcKenzie and Wake (24) improved upon this method by isolating d. -casein by the alcohol fractionation method of Hipp et al ( 2 3 ) , followed by removal of d.s -casein by calcium pre-cipitation, Swaisgood and and Brunner ( 1 9 6 2 ) ( 4 9 ) dissolved whole acid casein in 4 . 6 M urea and prepared /(f-casein by preliminary removal of the other caseins with 12% trichloroacetic acid. H i l l ( 1 9 6 3 ) ( 1 * 0 com-bined Waugh and von Hippel's method with a DEAE cellulose chromatography step for the purification of /r-casein. These and several other methods ( 2 2 , 55f 56) have been proposed and each one has i t s advantages and disadvantages. The main disadvantage of a l l of these methods i s that they are tedious and time-consuming and do not give high yields. Z i t t l e and Custer ( 1 9 6 3 ) ( 6 6 ) proposed a method that has had the widest use because i t is relatively simple to perform and gives high yields. Its main disadvantage i s the harsh chem-i c a l treatment that the method entails, Whole casein dissolved in 6 , 6 M urea i s brought to pH 1 . 3 - 1 . 5 with sulfuric acid, Reduction of the urea concentration by the addition of two volumes of water results in a precipitate containing oCs - and ft -caseins. The AT-casein in the supernatant i s removed by ammonium sulfate, and this i s followed by two alcohol fractionation steps. Experiences with large scale preparations of /("-casein using this method are given later in this thesis. Since a 8 necessary preliminary aspect of the present work involved the improve-ment of the electrophoretic technique to the point where the sub-fractions of /C-casein could be readily observed, the subject of the electrophoresis of caseins w i l l now be reviewed, G, Electrophoretic Analysis of Caseins For many years following Mellander's work on moving boundary electrophoresis of casein, d i f f i c u l t i e s regarding the interpretation of the electrophoretic patterns existed. It became increasingly appar-ent that the caseins interacted, with themselves and with one another ( 2 3 ) . Many workers demonstrated that considerable interactions took place in the °*--peak (5,6,48,54,59). Moreover, the results of Krecji et al (1941) ( 2 0 ) indicated that interactions between U - and $ - peaks took place. These early studies showed the heterogeneity of casein in that inclusion of calcium in the buffer systems resulted, in a decrease in intensty of the °L peak and an increase in material remaining at the origin. It became more and more apparent that, i f relia.ble information on the heterogeneity of casein was to be obtained, a method of resolu-tion would have to be used in which the caseins were dissociated to their monomelic forms. A careful study of this problem was conducted by Wake and Baldwin in 1961 ( 5 8 ) . They carried out electrophoresis of whole acid casein in starch gels using t r i s - c i t r a t e buffer (pH 8.6) containing 7 M urea. They found over twenty bands, A number of cA. -casein fractions were examined and found to have a major band identifiable with the major bands in whole casein, but they also exhibited a number of minor bands. Preparations of B- casein showed considerable variation in heterogeneity, a l l giving one major band in common with the second most pronounced band in the whole casein, but a number of preparations showed other bands as w e l l . A smeared zone was observed f o r /{'-casein with some sharp bands superimposed on the smeared bands. N e e l i n et a l (1963) (36) added to t h i s work by examining the e f f e c t of urea c o n c e n t r a t i o n on causing d i s s o c i a t i o n and of pH and b u f f e r type on r e s o l u t i o n . M o b i l i t i e s i n creased w i t h i n c r e a s i n g PH, but above pH ?,0 the sep a r a t i o n of major zones diminished. Neelin et al_ p r e f e r r e d pH 7.0-7.2 (cacodylate b u f f e r , 5-5 M urea) f o r comparison of more mobile bands, but pH 8.2-8,4 ( v e r o n a l , 4,8 M urea) f o r slower moving bands. The problem of "smearing" and l a c k of complete r e p r o d u c i b i l i t y of ic - c a s e i n behavior s t i l l remained, Peterson (1963) (^2) obtained good r e s o l u t i o n of - and.# -caseins by u s i n g nolyacrylamide g e l with a 4,5 M urea, Tris-EDTA-boric acid. (pH 9.0) mixture, This has been widely used and Aschaffenburg (1964) ( l ) used i t f o r t y p i n g ds - and/ 5-caseins by e l e c t r o p h o r e s i s of whole milk samples, Thompson (1965) (53) c a r r i e d out a. c o l l a b o r a t i v e survey of the r e p r o d u c i b i l i t y of t y p i n g ^  - Bnd.fi -caseins by the var i o u s g e l e l e c t r o p h o r e t i c procedures used i n a number of l a b o r a t o r i e s . On the whole the agreement between the v a r i o u s workers has been s a t i s f a c t o r y . However, ac c o r d i n g to McKenzie (23), the present procedures are e m p i r i -c a l and fundamental s t u d i e s of r e s o l u t i o n i n zone e l e c t r o p h o r e s i s i s necessary before the heterogeneity of the caseins can be more o b j e c t -i v e l y understood. S e v e r a l workers concentrated on s o l v i n g the smearing problem t h a t was evident when e l e c t r o p h o r e s i s was c a r r i e d out on /c-casein, even i n the presence of 7 M urea. MacKinley and Wake (1964) (25) t r e a t e d /C-casein prepared by the method of McKenzie and Wake (1961) (24) with sodium s u l f i t e to break any S-S bands that might be present. 10 They then examined the product by the urea-starch gel electrophoretic method of Wake and Baldwin (1961) ( 5 8 ) . The S-sulfo- AT-casein showed two major sharp bands and three minor sharp bands. Likewise Neelin ( 1 9 6 4 ) ( 3 6 ) found two major sharp bands in /T-casein which had been prepared from pooled milk by the method of Z i t t l e and Custer ( 1 9 6 3 ) ( 6 6 ) and reacted with 2-mercaptoethanol to reduce S-3 bonds. On the other hand, reduced /C-casein preparations from milk of individual cows showed the two main bands or either one of them. Woychik published almost identical results at about the same time ( 1 9 6 4 ) ( 6 l ) and sug-gested that the presence or absence of these bands was under genetic control. Minor bands were also present, the fastest of which migrated slightly slower than the @-casein band. The notion that these two major bands were under genetic control was supported by Schmidt ( 1 9 6 4 ) (46) and MacKinley and Wake ( 1 9 6 5 ) ( 2 6 ) . The l a t t e r workers ( 2 3 ) also prepared S-carboxymethyl /C-casein (SCM- t< -casein) purified from pooled milk. The K -casein showed 5 bands on urea-starch gel electro-phoresis, This indicated, that /c-casein consists of subfractions joined together by disulfide bonds. The K-casein samples used were a mixture of types A and B and i t remained for other workers to show that each type consists of disulfide-linked subfractions. D, The purification of -casein Subfractions Dumas ( l 9 6 l ) ( 9 ) achieved partial fractionation of whole casein on DEAE cellulose columns using 0 . 0 2 M imidazole-HCl-3.3 M urea buffer, pH 7i0, and a linear sodium chloride gradient from 0 to 0 , 6 M, Rose ( 1 9 6 3 ) ( 4 3 ) unsuccessfully applied this technique to the fractionation of ^-casein. Thompson ( 1 9 6 6 ) ( 2 ) , recalling Neelin's use of 2-me.rcaptoethanol in the electrophoresis of /^"-casein ( 3 6 ) , incorporated this component into the chromatographic buffer system and obtained 11 f a i r l y extensive fractionation of whole casein. In the same year Schmidt et al ( 4 7 ) reported an attempt to fractionate A'-casein on DEAE cellulose using an imidazole-HCl buffer system and stepwise elution with sodium chloride. Five peaks were obtained from the homozygous K-casein samples applied to the column. Only one sub-fraction from each of the K-A and K-E was obtained which was homo-geneous on starch gel electrophoresis. This degree of separation was good enough, however, to show that N-acetyl neuraminic acid, increased as the fraction mobility increased and that the cystine and phosphorous content did not d i f f e r between the subfractions. Further, i t was found that the a b i l i t y to stabilize <?/.-casein against precipitation by calcium did not vary between the subfractions. Amino acid analysis of K-A and K-B revealed that K-B contains one aspartic acid less and one proline, alanine and isoleucine more than K-A. This work supported the theory of Wake and Baldwin ( 2 3 ) that the major difference between /c-casein subfractions l i e s in their s i a l i c acid content while K-A and K-B d i f f e r in peptide backbone and are therefore under genetic control. Woychik et al ( 1 9 6 6 ) ( 6 2 ) chromatographed reduced alkylated (carboxamidomethyl-) and reduced unalkylated ^casein on DEAE cellu-lose columns in the buffer system of Dumas ( 9 ) using a linear gradient of sodium chloride from 0 to 0 . 1 5 M. They obtained a major peak and four or five minor peaks from each of /(-casein types A and B. Amino acid analysis showed that each of the subfractions had approximately the same composition; the carbohydrate content was larger in the minor components and this was shown to be the main reason for their higher charge. The subfractions obtained from the reduced, unalkylated K-casein again aggregated due to intermolecular disulfide bonding. 12 Sedimentation analysis showed that the molecular weights of the major components, designated A-l and 3-1, were around 20,000. These findings supported the hypothesis that ^-casein i s an aggregate that results from intermolecular disulfide bonding and that the basic monomer unit i s obtained by reduction. A l l of the functional a b i l i t y of AT-casein resides in the polypeptide unit since a l l components stabilized <^V -casein equally well. It i s important to add at this time that H i l l and Vfake (I969)(l8) feel that the strength of the interaction of each subtraction with -casein should be tested in order to see i f the hydrophilic carbohydrate portion of /c-casein i s important in i t s st a b i l i z i n g a b i l i t y . Another reason why i t i s necessary to use subfractions of /C-casein to test the mechanism of interaction i s that the subfraction i s the monomer unit (18), Nakai and Clarke (3^) have obtained con-f l i c t i n g results in the reaction ratio of i< - and -J-s -casein as measured by ultracentrifugation and spectrofluorometry, Other workers (12,38,39.60) have reported various reaction ratios ranging from unity to four <Ks molecules to one K -casein molecule. Experiments involving the modification of particular amino acids in the protein have been carried out on whole A"-casein. The results using whole /c-casein are d i f f i c u l t to interpret because of the heterogeneity of the protein, It i s very d i f f i c u l t , for instance, to separate unreacted molecules from reacted ones as has been done with other proteins that have been modified at a single point on the primary-structure (37). These d i f f i c u l t i e s have shown up in the variation between results of different workers. Talbot and Waugh ( 5 0 ) found a decrease in stabilizing a b i l i t y after one lysine was modified, whereas 13 Pepper et al (40) found i t necessary to modify five residues of lysine in /C-casein before there was any loss in stabilizing a b i l i t y . H i l l (1970) (16) reported that arginine residues were easily modified by the reagent glyoxal whereas Clarke could not agree (7). It i s clear that one cause for conflicting results in the same modification experi-ments performed in different laboratories i s the heterogeneity of the ("C-casein molecule. This accentuates the importance of working with the subfractions of k-casein in order to better understand how this protein stabilizes -casein, Woychik's work on this problem (62) was the most successful at the start of this present thesis. The subfractions were not absolutely pure even after rechromatography, Approximately one year after the start of the present work, a paper was published from France (28) which reported much more success at obtaining subfractions of /<-casein. From that point on, attempts were made to repeat their work. Pure sub-fractions were obtained and the f i r s t two of these were used to modify histidine with the reagent 2-phenyl-l, 4-dibromoacetoin (PDA), Hi s t i -dine was chosen because i t had been implicated as playing an important role in the stab i l i z i n g a b i l i t y of /r-casein. The work which has led to this hypothesis will now be mentioned, E. The Modification of Histidine Residues in Ar-casein Z i t t l e (67) photooxidized t<-casein with methylene blue in the solution in illuminated Warburg flasks with an atmosphere of ai r . The respective decreases in tyrosine, tryptophan, and histidine were 35. 80 and 100% when /^-casein was oxidized by an uptake of 15 umoles of oxygen per mole of K-casein. The sta b i l i z i n g a b i l i t y of the oxidized K-casein was greatly reduced and at this level, a product resulted which was unable to stabilize -casein. Rennin did not hydrolyze 14 this product and i t also did not act on a mixture of untreated and oxidized /C-casein, It was reasoned that, whereas the oxidized A"-casein could not react with -casein i t i s possible that i t could react with native /^-casein. Since histidine was the amino acid most affected by photooxidation, i t was postulated that the amino acid plays an important role in the stabilizing a b i l i t y of /f-casein. Nakai added support to this contention when he demonstrated that modifying tryptophan residues with N-bromosuccinimide resulted in no decrease in st a b i l i z i n g a b i l i t y ( 3 1 ) , Later, Nakai ( 3 1 , 3 2 ) found that alkylation of k- casein with 2-phenyl-l,4-dibromoacetoin for 24 hours at pH 7 . 5 resulted in a modification of 1 , 5 to 2 residues of histidine per molecular weight 28,000 and a decrease in sta b i l i z i n g a b i l i t y of 55%, The product(s) of alkylation at pH 6 . 6 eluted with a Kav of 0 and 0.19 on Sephadex G200, whereas that at pH 7 . 5 eluted at only one peak with a Kav of 0 , The optical density ratio of the l a t t e r at 2 8 0 nm, pH 12.0 to 220 nm, pH 7.0 was 0 . 136 as opposed to 0 . 1 0 3 and 0 , 0 9 1 for the Kav 0 and 0 . 19 peaks of the pH 6 . 6 modification, This indicated that more alkylation occurred at pH 7«5< Nakai later showed that the reagent i s less specific at the higher pH- ( 3 3 ) . The higher optical density ratio of the pH 6 , 6 Kav 0 peak as opposed to that of the pH 6 . 6 Kav 0 . 19 peak indicated that more alkylation occurred, on the aggregated peak and that aggregation followed histidine modification, Treatment of K-casein with iodoacetic acid and with diisopropyl fluorophosphate respectively decreased the histidines by 1 , 5 and 0 , 5 residues in 5 per molecular weight 28,000. Aggregation was not ob-served, nor was there any decrease in stabilizing a b i l i t y . The d i f -f i c u l t y with these reagents, however, i s that they are not specific ( 3 3 ) . The s t a b i l i z i n g a b i l i t y of c - c a s e i n a l k y l a t e d w i t h PDA at pH 6 . 6 was r e s t o r e d by treatment with base, whereas that of k-casein a l k y -l a t e d at pH 7.5 w a s not, Nakai has shown that c y s t i n e i s decreased d u r i n g a l k y l a t i o n at the l a t t e r pH ( 3 3 ) . Treatment with 0.2N NaOH at 2 5°G f or 30 minutes r e s t o r e d most of the s t a b i l i z i n g a b i l i t y of s t e r i l i z e d and stored fcr-casein, s i m i l a r to the PDA-alkylated c a s e i n at pH 6 . 6 , Reoxidation of PDA-alkylated *-casein at pH 6 . 6 a f t e r r e d u c t i o n with 0 , 0 3 M mercaptoethanol i n 6 M urea and d i a l y s i s ( 3 2 ) r e s t o r e d s t a b i l i z i n g a b i l i t y ; whereas 0 , 0 6 M mercaptoethanol r e s t o r e d only one-half of the s t a b i l i z i n g a b i l i t y of K-casein stored f o r 90 days. The e l u t i o n p a t t e r n s d i d not change r e g a r d l e s s of how much the s t a b i l i z i n g a b i l i t y was r e s t o r e d . Furthermore, both peaks f o r PDA-alkylated X-casein at pH 6 , 6 showed decreased s t a -b i l i z i n g a b i l i t i e s r e g a r d l e s s of t h e i r d i f f e r e n c e s i n aggregation, Sulfo-compound.s i n s t o r e d ^ - c a s e i n may be i m p l i c a t e d i n decreased s t a b i l i z i n g a b i l i t y . Nevertheless, i t i s f e l t t h a t h i s t i d i n e could be i n v o l v e d i n t h a t aggregation f o l l o w s the m o d i f i c a t i o n of these r e s i d u e s , p o s s i b l y due to a s t r e n g t h e n i n g of the hydrophobic bonds, s i n c e h i s t i d i n e i n i t s n a t i v e form takes part i n hydrogen bending in p r o t e i n s ( 3 2 ) . Nakai found t h a t the t o t a l amount of h i s t i d i n e r e s i d u e s decreased a f t e r d i a l y s i s o r g e l f i l t r a t i o n of s t o r e d f c -casein ar.i concluded that "the sum of f r e e and masked h i s t i d i n e r e sidues were r e s p o n s i b l e f o r decreased s t a b i l i z i n g a b i l i t y and con-c e i v a b l y deeply masked residues may not be a v a i l a b l e for m a i n t a i n i n g s t a b i l i z i n g a b i l i t y " ( 3 ? V L a t e r , Tatto and Nakai (51) i d e n t i f i e d low molecular weight amine compounds d e r i v e d from, s t e r i l i z e d and stored K-ca.sein as NH^ and. cysteic a c i d , They a t t r i b u t e d the presence of NH, to the heat decomposition of amide and h i s t i d i n e 16 residues in the /('-casein and the cysteic acid to the oxidation of cysteine. Again i t was suggested that histidine may play a role in the sta b i l i z i n g a b i l i t y of /r-caseln and the destruction of this amino acid may be one of the causes of gelation of stored milk products, such as ultra high temperature pasteurized milk, condensed milk, and chocolate milk. The major purpose of this thesis was to specifically modify h i s t i -dine residues in AT-casein and two of i t s subfractions. Much prelimi-nary work had to be done in order to get at the major problem, and i t i s f e l t that important observations can be made from experience at each preliminary step, CHAPTER II 17 Practical Experience in Large Scale Production of  Z i t t l e ' s fr-casein The purpose of the present chapter i s to report experiences with at-tempts to scale up the method of Z i t t l e ( 6 6 ) so that approximately 60 grams of K-casein could be purified at one time. MATERIALS • '. The starting material for 60 grams of /C-casein was 600 g of whole casein. This was obtained from approximately 6 gallons of whole milk from a cow known to be homozygous for /C-casein type A, The milk for the f i r s t three batches of Jr-casein was obtained from a cow named "Ideal Model" and that for the fourth batch was obtained from "Tune", Both cows were free of mastitis, • Other materials required were: (1) A container to hold at least 20 l i t r e s of li q u i d . In the present work one 5-gallon, two 10-gallon, and one 17-gallon plastic buckets were used, (2) A cream separator. Alternately, milk may be skimmed by centrifuging (800 rpm) for 20 minutes at room temperature, followed by centrifugation at the same speed and time at 4°C. The skim milk i s simply poured off. This i s a very effective procedure but not practical for large quanti-ties of milk, (3) A detachable electric mixer. (4) An electrophoresis apparatus. This was used to test the purity of the f i n a l product and to test the efficiency of the fractionation steps. The apparatus used were those described by Perrin (41) and by Beveridge (2), ( 5 ) A pH meter or pH Hydrion papers. (6) Chemicals, The following quantities of chemicals were required for the preparation of 4-0-60 grams Zit t l e ' s /c'-casein, 3 gallons of 95% ethanol 2 l i t r e s of concentrated hydrochloric acid 400 ml of concentrated sulfuric acid 5 2 . 3 pounds of urea 5 . 5 pounds of ammonium sulfate 10.0 grams of ammonium acetate METHODS A, Typing of Cows Ten cows were typed, by a modification of the polyacrylamide gel electrophoresis technique of Thompson ( 5 3 ) « Two tenths m i l l i -l i t r e of whole milk were dissolved in approximately 0.1 ml of a solution consisting of 0 . 5 ml 2-mercaptoethanol and 5»0 ml with tris-glycine buffer, pH 9 . 1 . Alternately, sufficient urea was added to 0 ,2 ml of milk to disperse the casein, then 1 drop of 2-mercaptoethanol and 1 drop of N NaOH were added. B, Purification of /^-casein by Z i t t l e ' s Method Each step w i l l be commented on during the Results and Dis-cussion section, RESULTS AND DISCUSSION Observations and comments on each step of the procedure are as follows, A, Skimming the milk It took two f u l l days to collect enough milk to start the separation. During this time the milk was stored at 4°C. It was 19 found that when milk was run through the separator at this temperature i t did not separate at a l l . When i t was run through at room tempera-ture i t had to be recycled four times in order to achieve sufficient separation. When the milk was heated to 45°C excellent separation was obtained by one passage through the separator, B, Preparation of Whole Casein During the f i r s t t r i a l i t was found that a poor yield of casein was obtained. The reason for this i s that the milk was at the low temperature of 15-20°C during the separation of casein, It was found during a l l four t r i a l s that the amount of 0 . 1 N HCl required to bring the pH of the milk to 4 , 5 was exactly half the volume of skim milk. The speed at which the acid i s added to the skim milk i s very important and thorough mixing must be done throughout the addition; otherwise isolated areas of coprecipitation occur which are not readily redissolved. C, Fi l t e r i n g Casein It i s recommended that close knit material such as s i l k be used instead of cheesecloth because too much casein i s lost using the latter. The f i l t r a t i o n and washing processes are very slow using the former but very l i t t l e casein i s lost through the material, D. Washing Casein It i s recommended that the f i l t e r e d casein be placed in a large bucket and approximately 20 volumes of d i s t i l l e d water added. The casein should be washed thoroughly by mixing. The casein should not be l e f t standing in the wash water but a l l three washings should be done as soon as possible. This i s a tedious part of the operation and requires approximately eight hours to complete. Red.issolving Gasgin The casein should be made up to the concentration i t was in the skim milk. For instance, i f 24 l i t r e s of skim milk i s the starting amount then the casein precipitated from this should be redissolved in a total of 24 l i t r e s of water. Upon adding the water to the casein the l a t t e r should, be broken up into very small.pieces and then stirred rapidly. The pH can be main-tained at ?,5 by attaching the electrodes of an automatic t i t r a t o r to the sides of the bucket with masking tape and allowing IN NaOH to drop in through the automatic switching unit. The casein takes at least three hours to dissolve. During the f i r s t three t r i a l s the pH of the solution was measured by pH Hydrion papers and IN NaOH added as needed. The use of the t i t r a t o r therefore speeded up the process and also saved a great deal of labor, Repreclpitating Casein This was done as before and again half the volume of 0.1N HCl was used to precipitate a l l the casein. The HCl again was added slowly, the casein collected as before and then squeezed by winding the cloth around i t . It was then hung up and allowed to drain in the cold room. Before discussing the next step there are several things which should be kept in' mind about the scheme followed up to this point. The practice of collecting the milk over a two-day period, warming i t up to 45°C, allowing i t to cool to room temperature or lower i s not a good practice from a bacteriological standpoint. It meant that by the time the separation of casein was started part of the milk was 72 hours old, and that five days elapsed before commencing purification of the casein. It i s recommended that the casein be separated from several lots of afternoon milk from the desired cow and frozen in blocks 21 u n t i l enough i s c o l l e c t e d . This procedure would be safe from a bacte-r i o l o g i c a l p o i n t of view and a l s o a l l o w skimming and ca s e i n p r e c i p i t a -t i o n w h i le the mil k i s s t i l l warm, which w i l l r e s u l t i n higher y i e l d s of c a s e i n . I f the temperature of the milk decreases markedly d u r i n g the skimming, the milk should be c a r e f u l l y warmed to JQ°C before pre-c i p i t a t i n g the c a s e i n , G. D i s s o l v i n g the o a s e i n i n 6.6H urea Z i t t l e ' s o r i g i n a l procedure ( 6 6 ) c a l l e d f o r the d i s s o l u t i o n o f from 60 to 9 5 gram.s of dry casein i n 1 l i t r e of n.6M urea.. I f the c a s e i n used i s frozen i t i s good, to know the moisture content so tha t the cor-r e c t f i n a l volume of s o l u t i o n i s obtained, The urea (52.3 l b s . ) was a.dded d i r e c t l y to the c a s e i n and mixed thoroughly. At f i r s t a very viscous paste was formed. The c a s e i n was broken up i n t o small pieces by cr u s h i n g i t against the s i d e s of the con-t a i n e r w i t h a l a r g e s t i r r e r . E v e n t u a l l y the ca.sein was completely d i s -s o l v e d and water was added to make the t o t a l volume up to c.O l i t r e s . This u s u a l l y r e q u i r e d approximately 1 . 5 l i t r e s of water. I t took ap-proximately two hours f o r the c a s e i n to completely d i s s o l v e during t h i s step. A f t e r l e a r n i n g how much water i t would take to make up the f i n a l volume, d u r i n g a l l o t h e r batches the water was added i n w i t h the ursa. so a.s t o l i m i t the c o n c e n t r a t i o n o f the l a t t e r to as c l o s e to 6 . 6 M as p o s s i b l e . H. Decreasing the pH to 1.3-1.5 This sten was accomplished by dropwise a d d i t i o n of 1 , 2 0 0 ml of 7N HgSO^. The s o l u t i o n was s t i r r e d very thoroughly with a detachable e l e c t r i c s t i r r e r . The a d d i t i o n of a c i d was made by p l a c i n g a sepa-rator?/ f u n n e l on a r i n g stand and a l l o w i n g the a c i d ' t o drop i n t o the s o l u t i o n . 22 Diluting with water Twelve l i t r e s of water were then added by allowing the water to run in very slowly with thorough s t i r r i n g throughout the addition. It is im-portant to decrease the pH before adding the water, otherwise the casein w i l l precipitate simply by a.cid precipitation. As the addition of the water nears completion a precipitate forms which becomes mo.re a.nd more flocculent. The solution was allowed to stand for another lj-Z hours and the precipitate was separated. Separation of the Precipitate The procedure recommended by Z i t t l e was to f i l t e r the precipitate. This was attempted but found to be unsuccessful because i t was a very slow process and also because the f i l t r a t e was s t i l l very milky after f i l t r a t i o n . Better separation was obtained when centrifugation was done at 4°C at 2 , 5 0 0 rpm for 10 minutes in a Sorvall refrigerated centri-fuge. Figure 2 shows that the precipitate contained no ^-casein. This indicated that this step in the procedure was successful. The precipi-tate was frozen for later use in the purification of </j- and ^-casein. It was found that during the centrifugation period, which lasted 20 hours, that the mixture of the precipitate and supemate should be stored at room temperature as the Us - and /^-caseins are soluble at re-frigeration temperature (3). A d i f f i c u l t question and perhaps a source of error at this point i s the wisdom of holding the caseins in this amount of urea (2.2M) for such a long period. There i s one theory which states that the exposure of K-casein to urea during the purification procedure could give rise to the subfractions through the process of carba.mylation (l?). Ammonium Sulfate Precipitation The volume of the f i l t r a t e was measured, and ammonium sulfate was 2 3 Figure 2. E l e c t r o n h o r e t i c a n a l y s i s of the pH 1.3, 2,2 H p r e c i p i t a t e i n Z i t t l e ' s nethod. 2'+ added to make a fina l concentration of 1 molar. For 16 l i t r e s of liquid this amounted to 2,112 kilograms. : Stirring was not done during addition of the ammo'nium sulfate, the solid material was simply added slowly and allowed to sink to the bott om of the liq u i d . After a l l the ammonium sulfate was added the mixture was stirred slowly with a long knife and the precipitate separated by f i l t r a t i o n . Figure 3 shows the results of electrophoresis analysis of the pre-cipitate obtained. Very l i t t l e ^-casein was present a,nd no ^-casein could be seen. Material at this stage could be used for the chromato-graphic separation of subfraotions. But when i t was desired to use whole K-casein, the impurities were removed by alcohol fractionation. L. Alcohol Fractionation The precipitate obtained by ammonium sulfate fractionation was dissolved in water at a concentration of approximately 1% protein and dialyzed for 72 hours in the cold room against 80 l i t r e s of water. The f i n a l volume of dialyzed solution was four l i t r e s . The concentration of protein wa.s checked by the optical density method of Nakai (33) using the conversion factor of 11,69 as being equivalent to 1% Ar-casein. The solution was diluted with the necessary amount of water to adjust the'final protein concentration to 1%. Two volumes (usually 8-10 litres') of 95% ethanol were slowly added and then ?.M ammonium acetate was added dropwise until a dry precipitate formed. The preci-pitate was mucilaginous and gathered at the bottom of the container, A mistake was made during the preparation of a small batch of A:-casein in that the mixture was stirred during the dropwise addition of ammonium acetate. The precipitate was very gummy - like thick glue. The s t a b i l i z i n g a b i l i t y of this <-casein turned out to be as good as 2 5 Figure J, E l e c t r o p h o r e t i c a n a l y s i s of the ammonium s u l f a t e p r e c i p i t a t e i n Z i t t l e ' s method. '•/hole Ppt. O r i g i n the other batches and the subfractions obtained from this batch of /C-casein were like those from the other batches; but this indicates a d i f f i c u l t y that can occur during alcohol fractionation. Stirring in some way leads to greater aggregation of the protein. The precipitate obtained from this step was dissolved in water by breaking i t up and blending small amounts in a blender jar. The pH was raised to 7 . 5 by the addition of N NaOH. Blending, admittedly, could lead to further aggregation but the precipitate could not be handled in any other way. The solution was dialyzed in the same way as was the ammonium sulfate precipitate. Following dialysis the concentration of protein was again adjusted to 1% and the alcohol fractionation procedure repeated. The sticky pre-cipitate was again dissolved, dialyzed., and this time freeze-dried. The desired yield from this amount of starting material was 60 grams of /c-casein. This would be 109? of the starting material. Z i t t l e ' s o r i g i -nal paper ( 6 6 ) stated yields of 7 to 10% of the total starting material. The highest yield that was obtained during the present studies was 4 5 grams of /c-casein. This was 7.5% of the starting material. The lowest yield was 36 grams or 6.0% during the f i r s t attempt. It i s f e l t that one suggestion that w i l l increase the yields i s that the casein be prepared from several small batches of milk that can be carefully warmed to 30°C unless an automatic system for the continuous preparation of casein i s used at a dairy. 'The pilot plant pasteurizer could be used for large scale production with the only d i f f i c u l t y being that i t would take a long time to collect a l l the milk and a storage problem arises. Two people should, work on this together because i t i s too long a job for one person, especially such stages as the washing of the casein and. the centrifugation of the Jif - and /^-casein precipitate. 27 P r o p e r t i e s of the P u r l f l e d ^-ca.sein Figure 4 shows t h a t the K-casein p u r i f i e d by the above method was f r e e of i m p u r i t i e s . E l e c t r o p h o r e t i c a n a l y s i s agrees with t h a t of MacKinlay and Wake ( 2 5 ) which showed K-casein to be a heterogeneous p r o t e i n . The K-casein from the f i r s t batch had lower s t a b i l i z i n g a b i l i t y than the o t h er p r e p a r a t i o n s . I t was a l s o d i f f i c u l t to d i s s o l v e t h i s K-casein. Treatment of t h i s K-casein w i t h urea and mercaptoethanol a.ccording to Nakai ( 3 2 ) r e s u l t e d i n a complete r e s t o r a t i o n of s t a b i l i z i n g a b i l i t y and s o l u b i l i t y . I t i s f e l t t h a t the reason f o r these observations i s t h a t there was g r e a t e r aggregation of the f i r s t p r e p a r a t i o n of K - c a s e i n , probably due to l a c k of experience w i t h the method and because the a l c o h o l p r e c i p i t a t i o n step was accompanied by too vigorous s t i r r i n g throughout the procedure. Again i t i s s t r o n g l y recommended t h a t the very minimum of s t i r r i n g be c a r r i e d out d u r i n g the a l c o h o l f r a c t i o n a t i o n step. Table I g i v e s the amino a c i d composition of two batches o f K-casein. These r e s u l t s agree very c l o s e l y with those published i n a recent review by McKenzie ( 2 3 ) . I t w i l l be n o t i c e d t h a t the s u b f r a c t i o n s of K-casein cannot be seen i n the e l e c t r o p h o r e t i c a n a l y s i s (Figures J,k), Attempts to modify the method of e l e c t r o p h o r e s i s to the poi n t where the s u b f r a c t i o n s can be r e a d i l y seen w i l l be the subject of the next chapter. 28 Table I . Amino a c i d composition of two pr e p a r a t i o n s of -casein p u r i f i e d by the method of Z i t t l e , k - c a s e i n Amino A c i d F i r s t P r e p a r a t i o n Second P r e p a r a t i o n Residues per 2 0 , 0 0 0 g* A s p a r t i c a c i d 14, 9 1 5 . 0 Threonine 1 6 . 5 1 6 . 5 Serine 14 . 6 14.4 Glutamic a c i d . 3 1 . 0 3 1 . 5 P r o l i n e 2 2 . 6 2 3 . 0 G l y c i n e 2 . 9 2 . 9 Alanine 1 6 . 6 1 6 . 6 2 Cystine 2 . 5 2 . 5 V a l i n e 1 2 , 9 1 2 . 9 Methionine 2 . 0 2 . 0 I s o l e u c i n e 1 3 . 9 1 3 . 9 Leucine 10.4 10.4 Tyrosine 8 . 0 8 . 0 P h e n y l a l a n i n e 4 . 5 4 . 5 Lysine 9 . 6 9 . 6 H i s t i d i n e 3 . 0 3 . 1 A r g i n i n e 5 . 9 5 . 9 * S t a t i s t i c s based on e a r l i e r analyses done by Nakai ( 3 3 ) . CHAPTER III Attempts to Improve the Electrophoresis of ^-casein A large number of attempts have been made by various workers in this and other laboratories to obtain the distinct separation of subfractions of ^-casein by starch and by polyacrylamide gel electrophoresis. The la t t e r was chosen in these studies because i t was f e l t that i t was easier to prepare and handle. Several discouraging i n i t i a l runs showed that results were very inconsistent and therefore i t was decided to thoroughly test at least three factors known to be important in determining the suc-cess of an electrophoretic run. Attempts were then made to control these parameters closely. These variables included ( 1 ) the type of bridges used; ( 2 ) the voltage through the gel; and ( 3 ) the ionic strength of the buffers. MATERIALS The electrophoresis apparatus used in this study was the one described in detail by Perrin ( 4 l ) and by Beveridge ( 2 ) . A paper electrophoresis apparatus was also adapted to test the effect of high voltage (maximum 2 50 volts) through the polyacrylamide gel. This apparatus was equipped with a water jacket for cooling the system. Tris-glycine buffer was prepared by dissolving 2 0 7 . 4 grams of glycine in water and bringing the total volume up to 6 l i t r e s with d i s t i l l e d water. The pH was adjusted to 9 . 1 by the addition of 2 . 4 ml of 3 0 % NaOH. For gels of 10% strength 1 0 grams of acrylamide and 0 . 5 grams of N, N 1 methylene-bis acrylamide as well as 32 grams of urea were dissolved in t r i s glycine buffer diluted 1 : 1 . The fi n a l volume was made up to 1 0 0 ml with this buffer. Then 1 ml each of 1 0 5 c (w/v) ammonium persulfate, JOJi (v/v) tetramethylethelenediane (V/.?,T>) and 0.34 n l of 2-mercaptoethanol were added. The g e l was then poured i n t o a mold made of p l e x i g l a s s and a cover made of the same m a t e r i a l was p l a c e d on top of the gel. A f t e r the g e l had s o l i d i -f i e d the cover of the g e l was replaced by saran wrap. S p e c i a l bridges were c o n s t r u c t e d and tested, d u r i n g the f i r s t phase o f the study. These are l i s t e d below, (a) Cheesecloth and. F i l t e r Paper Bridges L i s t e d i n Table I I are the words Cheesecloth and F i l t e r Paper with various, s u b s c r i p t s . These s u b s c r i p t s represent the t h i c k n e s s o r number of f o l d s of the r e s p e c t i v e m a t e r i a l s . The f i l t e r paper used was Whatman No, J. (b) A.2*ar Bridges These were prepared by p l a c i n g 2% Difco Flake Agar d i s -solved i n cj% K G i n t o g l a s s U-tubes, On two occasions t h i s agar was poured onto three t h i c k n e s s e s of f i l t e r paper and i n Table I I t h i s i s designated as F i l t e r Paper & Agar. (c) Polyacrvlamide Gel Bridges These were co n s t r u c t e d by pr e p a r i n g a 10% gel as de-s c r i b e d before but l e a v i n g out the urea and rnercaptoethanol, (d) Sponge Bridges This type o f bridge c o n s i s t e d simply of s t r i p s of the sponge used to l i n e the drawers i n the l a b o r a t o r y , METHODS A, P r e l i ^ i ^ a w s+ud* a c ; :—'. in v " " Voltage measurements were taken at v a r i o u s p o i n t s on the e l e c t r o -p h o r e s i s apparatus (2,4l) equipped w i t h d i f f e r e n t b ridges and b u f f e r s . B. Changes i n the technique The f o l l o w i n g m o d i f i c a t i o n s i n the technique were a p p l i e d : 32 1. Based on a suggestion by Dr. Yaguchi ( 6 3 ) , 10:.3 g e l s were pre-pared without urea and mercaptoethanol and e q u i l i b r a t e d f o r one day i n d e i o n i z e d water and f o r at l e a s t two days i n 8M urea/0.08?5M T r i s - g l y c i n e b u f f e r , pH O.l/O.OOIM 2-mercaptoethanol. Mercaptoethanol -was added d a i l y . F o l l o w i n g e q u i l i b r a t i o n the gels were d r i e d o f f with paper towels before being used f o r e l e c t r o p h o r e s i s . 2. The high voltage paper e l e c t r o p h o r e s i s apparatus was adapted, as f o l l o w s ; Each t e r m i n a l has three separate b u f f e r conroartments so t h a t paper s t r i p s can be run. Three laj^ers of f i l t e r paper were cut to a.ct as one bridge connecting the three b u f f e r compartments on each s i d e to the g e l , A cover was placed on the g e l and water run through the c o o l i n g j a c k e t d u r i n g the e l e c t r o p h o r e s i s run. The e l e c t r o p h o r e s i s was ran f o r 24 hours with a. voltage o f up to 2 6 5 p a s s i n g through the g e l , 3 . The m o d i f i c a t i o n based on the suggestion of Yaguchi was combined w i t h the use of the adapted high voltage paper e l e c t r o p h o r e s i s apparatus d e s c r i b e d above. The run was f o r only 4 hours, 4. Ten per cent g e l s c o n t a i n i n g 4,_5M urea were prepared by b r i n g -i n g the f i n a l volume up to 100 ml w i t h 0.0875M or 0.0175M T r i s - g l y c i n e b u f f e r a.nd d i s s o l v i n g the i n g r e d i e n t s w h i l e warming to 25°C. One-half m i l l i l i t r e of 2-mercaptoethanol was added, f o l l o w e d by 1.0 ml each o f 10% (w/v) ammonium p e r s u l f a t e and j,0% (v/v) tetramethylethylenediamine (TMBD). The g e l s were poured i n t o the molds and p l e x i g l a s s covers p l a c e d on them. A f t e r the g e l s were set (40 - 6 0 minutes) the p l e x i g l a s s covers were r e -placed by saran wrap and the g e l s run f o r 24 hours without samples. Next day the samples were a p p l i e d and the e l e c t r o p h o r e s i s run f o r 24 hours w i t h a g e l voltage of 100 v o l t s a.nd a current of 20-2 5 mill:iamperes. RESULTS AND DISCUSSION P r e l i m i n a r y s t u d i e s Table I I snows the r e s u l t s of v o l t a g e measurements taken through d i f f e r e n t p o i n t s on the e l e c t r o p h o r e s i s apparatus as a f f e c t e d by d i f f e r -ent combinations of bridge and b u f f e r systems. The f i n a l system t h a t was chosen on the b a s i s of these experiments wa.s the one c o n s i s t i n g of 0.1M NaCl i n the outer chambers, 0.0875M T r i s - g l y c i n e b u f f e r , pH 9.1 i n the i n n e r chamber and g e l , and bridges c o n s i s t i n g of ten t h i c k n e s s e s of cheesecloth, Cheesecloth bridges were chosen over f i l t e r paper bridges because they are l e s s expensive and can be used longer. P o l y a c r y l a m i d e g e l , agar, and sponge bridges were not chosen because they were found to be d i f f i c u l t to handle and o f f e r e d no advantage i n r e -gard to e l e c t r i c a l conduction over the cheesecloth b r i d g e s . M o d i f i c a t i o n i n the technique F i g u r e 5 - s a drawing of e l e c t r o p h o r e s i s runs t h a t show good sepa-r a t i o n of K-casein s u b f r a c t i o n s . This type of s e p a r a t i o n was obtained, when i ) the m o d i f i c a t i o n based on Yaguchi's suggestion, i i ) the com-b i n a t i o n of the adapted, high v o l t a g e apparatus w i t h Yaguchi's suggestion and i i i ) the technique based on suggestions of Nakahori and. Nakai were used. Use of the h i g h voltage apparatus on g e l s which were not pre-v i o u s l y e q u i l i b r a t e d gave good mi g r a t i o n but no s e p a r a t i o n of the sub-f r a c t i o n s . I t i s suggested t h a t t h i s m o d i f i c a t i o n c o u l d be used suc-c e s s f u l l y i f the e q u i l i b r a t i o n step was i n c l u d e d . The one disadvantage of t r e a t i n g the g e l s according to Yaguchi i s t h a t they s w e l l up and become very b r i t t l e . Table II, The effects of buffer and bridge systems on voltage measurements taken at various locations on the electrophoresis apparatus. VOLTAGE Expt. Buffer used Mo, Outer Inner 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 5 16 Gel Outer 0.1 M NaCl T.35gMy T. ?5 gM y Repeated 0.1 M NaCl T r i p l y Tri^gly •i it >• f/0 KC1 Repeated 5 * K d T r i p l y T r i p l y 0.1 M NaCl 0 . 3 5 M/4 0 . 3 5 M Tri-gly Trx-gly E. P.I F. P.2 P.P. 3 F. P.3+Agar Agar F.P.3+Agar F.P.. Agar ChC_* ChC?* ChC3* ChC 1 0* ChC 20' ChC 1 5* rstem er & Gel mA I n i t i a l Voltage Outside Ter'l. Outer Chamber Inner Chamber Gel F.P.I 30 340 340 325 3 2 5 100 F.P.2 30 270 268 258 190 100 F.P,3 30 197 197 188 148 83 tt 30 180 180 169 145 76 M 30 222 220 210 1 6 8 80 If 30 262 260 252 1 6 5 80 30 259 2 5 9 240 162 80 II 30 180 178 169 1*5 75 ll 30 192 190 190 149 78 »l 30 262 261 2 52 172 82 30 2 5 5 252 252 170 90 II 30 180 180 175 150 90 ChC-]_* 30 92 89 82 72 10 ChC2* 30 99 9 5 90 79 18 Chfi^* 30 102 98 90 7 5 22 ChC 1 0* 30 195 1 9 5 185 160 90 ChC2o* 30 170 170 160 132 85 GhCx e* 30 180 180 17^ 140 80 Table II. (Cont'd.) VOLTAGE Expt. No. Buffer Outer used Inner Gel Bridge System Outer Inner & Gel mA I n i t i a l Voltage Outside Ter'l. Outer Chamber Inner Chamber Gel 17 0.1 M NaCl 0 . 3 5 M Tn -g! y 0 . 3 5 MA Tri-gly ChCj 0* chc10* 30 22 5 2 2 5 2 1 5 180 140 18 11 0 . 3 5 M/4 Tri-gly l l ChC 1 0* ChC 1 0* 30 270 2 7 0 2 5 5 2 2 5 168 3 9 I t 0 . 3 5 M Tri-gly 0 . 3 5 M Tri-gly P.P.3 F.P.3 30 190 190 178 169 138 20 5% KC1 I I 11 I I 30 218 218 2 1 0 188 118 21 0.1 M NaCl o.?5 MA Tris-gly 0 . 3 5 MA Tris-gly I I I I 30 190 190 180 170 99 22 5% KC1 0 . 3 5 M Tris-gly 0 . 3 5 MA Tri-gly KC1 Agar I I 30 300 2 9 8 2 9 0 188 110 2 3 f l l l l l l l chc10* 30 260 2 5 9 249 150 110 24 I t I I 0 . 3 5 MA Tris-gly CnC 1 0* I I 30 207 2 0 7 200 190 130 2 5 I I 0 . 3 5 MA Tris-gly 0 . 3 5 MA Tri-gly l l I I 30 159 1 5 9 149 130 100 26 0.1 M NaCl 0 . 3 5 M T-gly l l F.P.3 PAG in 5 A 30 172 171 1 6 5 150 100 2 7 M l l I t chc10* PAG in 0 - ? 5 / A 30 170 1 6 8 1 5 5 140 90 28 I I 11 M 2% KC1 Agar 30 2 1 5 2 1 5 2 0 8 150 99 2 9 I I 0 . 3 5 MA T-gly I I P.P. 3 H 30 1 6 5 1 6 5 1 5 5 140 100 Cheesecloth Figure "5. E l e c t r o p h o r e s i s of of s u b f r a c t i o n s . f - c a s e i n showing good r e s o l u t i o n SUMMARY Several modifications of the e l e c t r o p h o r e s i s technique described by P e r r i n (41) and Beveridge ( 2 ) gave promising r e s u l t s i n the se p a r a t i o n of the subf r a c t i o n s of - c a s e i n . These changes i n c l u d e d steps to increa.se the voltage through the g e l , e q u i l i b r a t i o n of the g e l s with the b u f f e r s , increasing the urea c o n c e n t r a t i o n , decreasing the i o n i c s t r e n g t h o f the b u f f e r system, and changing the b r i d g e s . CHAPTER IV Separation of Subtractions of fr-casein by  DEAE Cellulose Chromatography As mentioned in the Introduction and Literature Survey, several workers have attempted to fractionate >r-casein by various modifications of DEAE cellulose chromatography. The f i r s t attempt in this laboratory was reported by Beveridge in 1968 (2), His thesis stated quite a number of serious d i f f i c u l t i e s involved in achieving good separation. The pur-pose of the present chapter i s to describe in detail several important modifications of the method followed by Beveridge which have resulted in the successful purification of ^-casein subf ractions. Experimental Plan Series one The f i r s t series of experiments was a direct repeat of the method described thoroughly by Beveridge, with the only modification being that a micro pump was used to pump the gradient elution buffer onto the column. Several successful runs were obtained using this method but success was not consistent. Series two The second series of experiments was a slight modification as sug-gested by Mercier in 1968 (28). This method gave consistently good sepa-ration once experience with the method was obtained. After achieving good separation on a small column using J00 mg of fc-casein, a large column was used on which 8,8 grams of k-casein was successfully fractionated into six subfractions. 39 Series three The third phase of this study was to rechromatograph subfractions ob-tained from the f i r s t fractionation. An attempt was made to completely purify subfractions which were not well separated on the f i r s t run as well as those that were very pure from the f i r s t run. MATERIALS Some of the K-casein applied to the column was not carried through a l l of the purification steps mentioned in chapter two but was /c-casein which was carried only to the ammonium sulfate precipitation stage. Thus the K-casein contained some ds - and B -casein. During several of these runs the NaCl gradient was stepped up at the end of the /^-casein subtraction separation so that the buffer would also elute the Uj - and 6 -caseins. This provided a stock of -casein which was used to carry out st a b i l i z i n g a b i l i t y determinations. The DEAE cellulose was obtained from Whatman Chemical Company, Puri-fied grade urea, imidazole and sodium chloride were obtained from Fischer Chemical Company. Reagent grade mercaptoethanol was obtained from Eastman Kodak Company, The chromatographic buffer used in the f i r s t series of experiments was as follows: 0 , 0 1 M imidazole, 4 , 0 M urea, J ml. of 2-mercaptoethanol per l i t r e , pH adjusted to 7 . 0 by the addition of 1 . 0 5 ml, of concentrated HCl per l i t r e of buffer. The buffer was made up fresh each time and f i l t e r e d before being used. In the second and third set of experiments the buffer was 0 . 0 2 M imidazole, 3 . 3 M urea, 3 ml. of 2-mercaptoethanol per l i t r e , pH 7 . 0 . METHODS Se r i e s one The f i r s t set of t r i a l s was done according to the method described by Eeveridge (2) with the only d i f f e r e n c e being t h a t a micropump was used t o ' punp the b u f f e r onto the column. S e r i e s two Several important m o d i f i c a t i o n s suggested by Mercier i n 1968 (28) were i n c o r p o r a t e d i n t o the procedure. The treatment of the DEAE c e l l u l o s e i s the f i r s t major change. The powdered c e l l u l o s e i s f i r s t o f a l l s i f t e d overnight on a shaker and o n l y tha.t between 120 and 2 0 0 mesh s i z e i s used. This i s placed i n ap-proximately 10 volumes of IN NaOH and soaked t h e r e i n f o r 30 minutes. This i s then washed w i t h d e i o n i z e d water on a f i l t e r pad i n a Bucnner funnel u n t i l the wash water reaches approximate n e u t r a l i t y . I f the wa.ter i s s t i l l y e l l o w then a repeat of the NaOH soaking and water wash i s suggested.. The c e l l u l o s e i s then held f o r 30 minutes i n approximately 5 volumes of a s o l u -t i o n of 0.1N HCl/ 2 5 % NaCl f o l l o w e d by washing w i t h 10 volumes of water and suspension i n t o the pH 7,0 b u f f e r described, above. A 20 cm x 7 cm column r e q u i r e s approximately 100 grams o f s i f t e d DEAE c e l l u l o s e . A f t e r each chromatographic run the 20 cm x 7 cm column i s regenerated by the su c c e s s i v e passage of the f o l l o w i n g three s o l u t i o n s : 1 . 5 to 2 l i t r e s of O .25N NaOH (time of passage approximately 3 hours) 2 . 5 to 3 l i t r e s of 0.1K HCl / 2 5 % NaCl (time of passage, approximately 4 to 5 hours) 3 . 5 to 4 l i t r e s o f d e i o n i z e d water 3 . 5 to h l i t r e s of pK 7.0 b u f f e r (the f l o w of b u f f e r i s stopped when the column reaches n e u t r a l T>H). 41 In the present studies i t was found that the last step could be done with a preliminary passage of 3»5 l i t r e s of 0.02M imidazole buffer, pH 7.0 without the urea and mercaptoethanol followed by approximately 1 l i t r e of the complete buffer. This cut down somewhat on the amount of urea required. The use of low molarity KaOH and HCl compared to the procedure used in the f i r s t series of experiments i s believed to be an important step which would greatly lengthen the l i f e of the D E A E cellulose. Another suggestion of Mercier was to recrystallize the urea before using i t in the buffer. Several runs were attempted without following this step but proved, to be unsuccessful. The recrystallization procedure used was as follows: . o 600 grams of Fischer reagent gre.de urea was dissolved at 70 C in 600 ml of 9592 ethanol and 216 ml of deionized water. This was f i l t e r e d through 'Whatman Mo, 1 f i l t e r paper and stored in the cold room for 24 hours. The recrystallized urea was col-lected on a large Buehner funnel and the f i l t r a t e was stored in the deep freeze for 24-72 hours after which time more urea, had crystallized. This again xvas collected on a Buehner funnel and. washed with cold absolute ethanol. Suction was continued u n t i l almost a l l of the liquid, had passed through the urea, and. the recrystallized urea was placed in t i n f o i l at room temperature and allowed to dry. The yie l d of urea was 90-95%. Preliminary studies were carried out on a column 14 cm x 2.0 cm in size prepared as follows: A one-holed rubber stopper of appropriate size was placed on the bottom end of the glass tubing. One layer of cheesecloth was then placed on the bottom followed by a small layer of glass wool, a one-half inch layer of aquarium gravel and one thickness of Whatman No. 3 f i l t e r paper cut to f i t the tubing. De-gassed buffer or water was plaoed to a level of approximately one third of the length of the tube and to this was added the total amount of de-gassed cellulose as would be required to f i l l the column. One thickness of Whatman No. 3 f i l t e r pa.per was placed on the top of the resin. The cellulose was then washed with successive amounts of the above materials in proportion to the size of Mer-cier' s column.' Thus for a 14 cm x 2.0 cm column the following amounts of the four materials were used: 100-150 ml of 0.25N NaOH 170-240 ml of 0.1N HCl/25% NaCl 240-400 ml of 0.02M imidazole buffer, pH 7.0 Approximately 200 ml of complete buffer, pH 7.0. When f i r s t using the dry, sifted D E A E cellulose, an appro-priate amount i s suspended in a solution of 0.1N HCl/25% NaCl for 30 minutes, washed thoroughly with water and then placed in imida-zole buffer, pH ?,0., Following preliminary studies with the small column, a large column (20 cm x 7 cm) wa.s packed. The bottom of the column contained a sintered glass f i l t e r disc and was joined with-a very heavy clamp to the upper part o the column. One thickness of Whatman No. 3 f i l t e r paper 'was placed on top o the sintered glass. This was followed by a •§• inch layer of aquarium gravel and either a small piece of glass wool or another layer of Whatman No, 3 f i l ter paper. The column was packed using the same precautions as mentioned above, Samples were prepared as follows: fc-casein (approximately 1%) was dis-solved in the complete buffer and nitrogen was bubbled into the solution for 30 minutes. Then the 2-mercaptoethanol was added in the same concentra-tion as that in the whole elution buffer (3 ml/litre) and the sample stirred a further 1 5 minutes with bubbling nitrogen gas. The samples ( 0 , 3 to L.O gram of protein) were placed on the small column with a 5 ml long t i p delivery pipette altered so that the t i p was shaped like a fishhook so as not to disturb the top of the cellulose when the sample was added. The sample (5-10 grams of protein) was pumped onto the large column at a rate of 2 0 0 ml/hour. After applying the sample to the large column, the l a t t e r was washed with 1 l i t r e of complete buffer containing 0 . 0 2 5 M NaCl. The linear gradient gradient 0 . 0 2 5 to 0.1.35H NaCl was then set up by connecting two 2 , 5 0 0 ml bottles in an open system. The buffer which was pumped directly to the column contained the 0 . 0 2 5M NaCl and was stirred with a magnetic s t i r r e r throughout the operation. The other bottle contained buffer with 0 . 1 3 5 M NaCl, Both bottles were of exactly the same size and shape and were level with each other. Preliminary experiments were carried out to see what system would give a suitable gradient. The chloride concentration of every f i f t h tube was measured by the following procedure: One half m i l l i l i t r e of sample was diluted to 20 ml with deionized water. Three drops of 1 0 ^ J^Cr-pO^ was added and the mixture titrated with 0.1M AgNO to the f i r s t appearance of the reddish-brown precipitate of s i l v e r chromate. Series three Fractions obtained from the f i r s t chromatographic run were rechromao-graphed on the small columns described above. Two hundred milligrams of sample was applied and treated exactly as above. Several attempts were made to separate very impure fractions by re-chromatography. A l l fractions collected were dialyzed against running tap water over-night followed by ?2 hours against d i s t i l l e d water. They were then per-vaporated to one-half the volume and freeze-dried. hh RESULTS AND DISCUSSION S e r i e s one The f i r s t s e r i e s of experiments gave s u c c e s s f u l s e p 3.rfl.tion but not c o n s i s t e n t l y . S e r i e s two Figure 4 shows p a r t of a s u c c e s s f u l run of whole c a s e i n . The =S-casein obtained, from t h i s and s e v e r a l other runs was used f o r s t a -b i l i z i n g a b i l i t y t e s t s of K - c a s e i n , Figure 7 shows one of the f i r s t runs of K-casein. I t i s f e l t t h a t the poor separation evident i n t h i s run emphasised the importance of uniform column packing. A l l of f r a c t i o n s I, I I and I I I denoted by the arrows be-longed to s u b f r a c t i o n number one as shown by polya.cryla.mide g e l e l e c t r o -p h o r e s i s . F r a c t i o n IV was a mixture of s u b f r a c t i o n s two and. t h r e e , and the r e s t o f the f r a c t i o n s were mixtures. F r a c t i o n VI and the r e s t contained /$ -casein which was expected s i n c e the p u r i f i c a t i o n of tc-casein was carried, out only to the a.mmonium s u l f a t e step. Figure 8 shows one of s e v e r a l s u c c e s s f u l runs of whole /f-casein, 8,8 grams was a p p l i e d to the column and the f o l l o w i n g y i e l d s were obtained. KA1,502 mg; KA2 .343.5 mg; KA3 .565.5 mg: KA.4,32? mg; KA .5 ,56.7 mg; and KA6 .60 mg. Figure 9 shows a run of whole «r-casein where the g r a d i e n t was allowed to run by g r a v i t y onto the column. The s e p a r a t i o n was poor and i t i s be-l i e v e d t h a t the g r a d i e n t system was the cause f o r this. Even though separa-t i o n was not good, rechromatography of the shaded area gave 60 mg of pure s u b f r a c t i o n number one. S e r i e s three The three f r a c t i o n s denoted by the arrows i n Figure 7 were combined and rechromatcgraphed, with the r e s u l t s shown i n Figure 1 0 . The shape of the 45-Figure 6> Part of a successful chromatographic fractionation of whole casein on DEAE cellulose using the method described by Mercier. 45a M o l a r c o n c e n t r a t i o n o f NaCl CM O um 082 1* "(TO Figure 7. Elution profile of one of the f i r s t attempts to fractionate K-casein on DEAF cellulose using the modifications of Mercier et. a l . F i g u r e 13. S u c c e s s f u l f r a c t i o n a t i o n o f K - c a s e i n on DEAE c e l l u l o s e . Figure'10. Rechromatography of shaded areas of Figure 7. ////</ *3L MO 20 40 60 80 LOO 120 140 160 180 curve i s almost exactly the same as the rechromatograph of subtraction one shown by Mercier in 1968 (28). Better results were obtained when the frac-tions of Figure 8 were rechromatographed. The rechromatographs of KA1 and KA2 are shown in Figure 1 1 . It was noticed that a small band just before subfraction one persisted even after rechromatography. This i s in agree-ment with earl i e r work (62) and with that of Nakai ( 3 2 ) . SUMMARY The results of this work supported the work of Mercier in that highly pure subf ractions were obtained. Certain observations which a.re important are: (1) The DEAE cellulose must be sifted; this affords more uniform column packing by removing a large amount of small and large material. In a typical batch of Whatman DEAE cellulose ap-proximately 8 0 % was of the 1 2 0 - 2 0 0 mesh size. Thus there i s the possibility of large porosity gradients throughout a particular batch, ( 2 ) The urea must be recrystallized or purified by ion exchange. Several runs (results not shown) on a column which gave good separation with recrystallized urea fa i l e d to give good separation with unrecrystallized urea. In recrystallizing urea i t i s suggested that small amounts be used in l i e u of very large batches because the recrystallization procedure i s more readily controlled. ( 3 ) The DEAE cellulose must not be l e f t in strong base any longer than prescribed in the above method, Old DEAE cellulose was found to have an exchange capacity of 0 , 6 5 whereas a newly purchased one had an exchange capacity of 0 . 9 5 mil]iequivalents of Cl" per gram. 52 The urea b u f f e r must be f i l t e r e d j u s t before use. I f i o n exchange columns are used to t r e a t urea then f i l t r a t i o n i s not necessary p r o v i d i n g the b u f f e r i s used r i g h t away. At the end of the f i r s t run the column w i l l have dark brown o r black m a t e r i a l on the top of i t . This does not present a problem. F o l l o w i n g the wash with 0,2$11 NaOH most of the d i s c o l o r a t i o n disappears. 'Dark y e l l o w to brown m a t e r i a l remains even a f t e r complete re g e n e r a t i o n , but t h i s i s not a problem. The next run can be c a r r i e d out on the column. Once i t i s e s t a b l i s h e d t h a t a pa.rticu-l a r column gives good s e p a r a t i o n then i t i s recommended, tha t t h i s column be used f o r s e v e r a l more runs. I f a l l the runs are s u c c e s s f u l then t h i s column can be used a l s o f o r the rechromatography of a combination of one su.bfraction from s e v e r a l runs. CHAPTER V Modification of Histidine Residues with PDA and PMA As mentioned in the Literature Survey several workers obtained evidence that supports the contention that histidine residues are im-portant for the stabilizing a b i l i t y of K-casein. Nakai ( 3 2 ) alkylat K-casein at pH 6 . 6 and 7 , 5 with 2-phenyl-].,4-dibromoacetoin and ob-tained a decrease of 1 . 5 to 2 out of a total of 4 histidine residues per molecular weight 28,000, accompanied by a decrease in stabilizing a b i l i t y of 55%« Z i t t l e ( 6 7 ) photooxidized /<-casein and concluded that the resulting decrease in stabilizing a b i l i t y occurred as a result of destroying the histidine residues and therefore reasoned that histidine i s an important functional amino a.cid in K-casein. Neither of these workers tested the speci f i c i t y of the modifications. The purpose of the present work was to modify whole Hf-casein with PDA and to test the sp e c i f i c i t y of the reaction and also the effect of the reaction. KA1 and KA2 were also reacted with PDA, Early in these studies i t was found that PDA caused aggregation of. K-casein and of KA2, It was postulated that aggregation occurred by the PDA cross-linking with single histidine residues on two molecules of Hr-casein. For this reason the monobromo derivative (PMA) was pre-pared and reacted with whole K-casein and with KA1 and KA2. The spe c i f i c i t y and the effect of this reagent on the proteins were then tested. At the same time, the reaction conditions were changed to see i f a more usable derivative of PDA- K-casein could be obtained 5k MATERIALS k-Casein, s u b f r a c t i o n s one and two and -casein were prepared as d e s c r i b e d i n chapters I I and IV of t h i s t h e s i s . The s u l p h e n y l sulphonate d e r i v a t i v e s of K-casein and the two sub-f r a c t i o n s were prepared i n the f o l l o w i n g way: One hundred t h i r t y mg of p r o t e i n was d i s s o l v e d i n 14 . 0 ml of 0.02 M phosphate b u f f e r pH 6 , 7 , 8 M urea and 0 . 0 0 1 M EDTA (the urea was f r e s h l y r e c r y s t a l l i z e d ) . Nitrogen was bubbled i n t o the s o l u t i o n f o r 15 minutes, then 2 0 u l of 2-mercaptoethanol was added and the r e a c t i o n mixture i n c u -bated at 37°C f o r 30 minutes a f t e r which 2 0 0 mg of sodium tetra.thionate (NagS^O^.SPLjO) was added. A f t e r 5 minutes the pH" was adjusted to 6 . 7 w i t h IN NaOH and the p r o t e i n was then d e s a l t e d by passage through a 2.4 x 52 cm Sephadex G-25 column e q u i l i b r a t e d w i t h 0 . 0 0 0 8 M phosphate b u f f e r , pK 7 . 6 , PDA was prepared a c c o r d i n g to the method of Ruggli (44) by Dr. S. Nakai. PMA was prepared from PDA by the f o l l o w i n g method: Two grams of PDA was d i s s o l v e d i n 2 5 ml of 95% ethanol and 2 ml of d i s t i l l e d water. Two grams of aluminium amalgum (20-40 mesh) was added and the mixture was r e f l u x e d at 6 0 - 7 0 C f o r 5 hours. The aluminium amalgum was removed by c e n t r i f u g a t i o n and the supernatant was placed i n 100 ml of i c e water and allowed t o c r y s t a l l i z e i n the cold. room. The white m a t e r i a l which r e s u l t e d d u r i n g the c r y s t a l l i z a t i o n was s t o r e d at 4°C i n the mother l i q u o r and c o l l e c t e d and d r i e d before use. The aluminium amalgum was obtained from F i s c h e r S c i e n t i f i c Company and was t r e a t e d i n the f o l l o w i n g way before use. 10 grams alumium granule, 20 mesh, was washed w i t h IN NaOH, then w i t h water. I t was then soaked i n 100 ml of 0.5% mercuric c h l o r i d e f o r 2 minutes 55 and washed once or twine with water. The IN NaOH wash was then repeated and followed by another water wash. It was soaked again in 0.5% mercuric chloride for 1.5 minutes, a.nd washed successively with water, alcohol, anhydrous ether, and stored in Petroleum ether. METHODS Amino acid, analysis of the native and modified proteins was carried out in a Phoenix Model M68O0 Moore-Stein system ( 3 2 ) , three runs each after 24, 7 2 , 96 hour hydrolyses with gla s s - d i s t i l l e d 6N HCl. Nitrogen determinations were carried out by the micro-kjeldahl method (33). Stabilizing a b i l i t y tests were carried out by the centrifugation method (33). Preliminary experiments involved the reaction of PDA with k-casein as follows: 50 mg of K-casein was dissolved in 1 6 . 0 ml of 0.01M phosphate buffer, pH 6 . 6 , A solution containing 18 mg of PDA in 20 ml of absolute methanol was ad.ded and. the reaction allowed, to proceed in the dark for 16 hours at room temperature. At the end of the 16 hours the reaction mixture was either dialyzed against a total of 80 l i t r e s of d i s t i l l e d water for 72 hours in the cold room or placed on a Sephadex G-25 column equilibrated with 0.0008M phosphate buffer, pH 6.8 and eluted with the same buf-fer at room temperature. This i s the same treatment used earlier by Nakai ( 3 2 ) . The desalted modified protein was then frozen and freeze dried and analyzed by electrophoresis, amino acid analysis and for st a b i l i z i n g a b i l i t y against /c-casein. During the f i r s t reaction a negative control was run during which no PDA was added to the casein solution. Preliminary studies of the reaction of /c-casein, Al with PMA were done as follows: 10 mg of protein was dissolved in 4,0 ml of 0.02M phosphate buffer, pH 6.8, To t h i s was added 3 . 5 n>£ of PMA d i s s o l v e d i n 1 . 5 ml of absolute methanol. The r e a c t i o n was allowed to prodeed i n the dark at room temperature f o r v a r i o u s times up to 48 hours, Be-f o r e a n a l y s i s the mixtures were d e s a l t e d e i t h e r by Sephadex G-25 chromatography as d e s c r i b e d above o r by d i a l y s i s at 4°C f o r 48 hours a g a i n s t s e v e r a l changes of demineralized. water. E l e c t r o p h o r e s i s was c a r r i e d out acc o r d i n g to the method described, i n Chapter I I I where the g e l s were e q u i l i b r a t e d f o r 24 hours by running the e l e c t r o p h o r e s i s overnight without samples, f o l l o w e d by e l e c t r o p h o r e s i s of the samples f o r 24 hours with a cur r e n t of 20-2 5 mA and a voltage c f 100 v o l t s through the g e l . RESULTS AND DISCUSSION A. E f f e c t of PDA a l k y l a t i o n of *r-casein Table I I I g i v e s the r e s u l t s of amino acid, a n a l y s i s o f A'-casein reacted at pH 6.6 w i t h a t h i r t y f o l d excess o f PEA f o r 16 and .24 hours. The s t a -t i s t i c s of the analyses were based, on work done by Nakai ( 3 3 ) wM ch showed t h a t c e r t a i n amino a c i d s remain constant d u r i n g PDA a l k y l a t i o n of whole r^-casein. Thus, the c a l c u l a t i o n of the b a s i c amino a c i d s was based on a constant a r g i n i n e value of 5 . 1 r e s i d u e s per molecular weight 20,000 and tha t o f the neutral, and a c i d i c amino a c i d s was based on a constant l e u c i n e value of 9 . 3 r e s i d u e s per molecular weight'20,000, The r e s u l t s c o n f i r m Nakai's work ( 3 3 ) i n th a t a l k y l a t i o n of K - c a s e i n w i t h PDA f o r In hours at pH 6,6 r e s u l t e d i n a decrease i n h i s t i d i n e o f 0.60 residue and i n l y s i n e of 0.20 residue per molecular weight 20,000. Treat-ment under the same c o n d i t i o n s f o r 24 hours r e s u l t e d i n r e s p e c t i v e decreases of 0.86 and 0.20 i n h i s t i d i n e and l y s i n e residues. No new peak was seen on the a n a l y z e r as a r e s u l t o f the a l k y l a t i o n . Studies on the mechanisms of PDA m o d i f i c a t i o n are d e s c r i b e d i n the next chapter. Nakai a l s o found, t h a t a l k y l a t i o n a t pH 7 . 5 f o r 16 hours caused a 5? Table III. Amino acid analysis of K-casein and PDA K-casein. Amino Acid K-Casein PDA- K -Casein (pH 6.6) PDA-K-Casein (pH 6.6) 16 hour 24 hour Residues per 20,000 g Aspartic acid 14.9 14.9 14.9 Threonine 16.5 16.5 16.5 Serine 14.6 14.9 14.6 Aspartic acid 31.0 31.0 31.0 Proline 22.6 22.6 22.6 Glycine 2.9 2.9 2.9 Alanine 16.6 16.6 16.6 -§- Cystine 2.5 2.5 2.5 Valine 12.9 12.9 12.9 Methionine 2.0 2.0 2.0 Isoleucine 13.9 13.9 13.9 Leucine 10.4 10.4 10.4 Tyrosine 8.4 8.4 8.4 Phenylalanine • k.5 k.5 k.5 Lysine 9.6 9.k 9.k Histidine 3.0 2.4 2.2 Arginine 5.1 5.1 5.1 decrease of 3 l y s i n e , 0,8 r e s i d u e of t y r o s i n e along w i t h 0.5 residue of h i s t i d i n e per molecular weight 20,000'. The l o s s i n s t a b i l i z i n g a b i l i t y of the * - c a s e i n modified a t pH ?,5 was not r e v e r s i b l e , whereas t h a t at pH 6.6 was r e s t o r e d by treatment with urea o r 0.2N NaOH. Tt was postu- • l a t e d by these f i n d i n g s tha.t the decrease'in l y s i n e i n combination w i t h h i s t i d i n e might be r e s p o n s i b l e f o r the decrease i n s t a b i l i z i n g a b i l i t y of the /r-casein. At the same time the p o s s i b i l i t y of d i s u l f i d e l i n k a g e s p l a y i n g a. r o l e i n the s t a b i l i z i n g a b i l i t y r e s t o r a t i o n phenomenon cou l d not be r u l e d out. Experiments to t e s t the l a t t e r were c a r r i e d out by usi n g fr-casein s u b f r a c t i o n s A l and A2 and w i l l be des c r i b e d l a t e r i n t h i s chapter. S p e c i a l steps had. to be taken to d i s s o l v e the modified, p r o t e i n . One method was to suspend the dry m a t e r i a l i n water and r a i s e the pH to 12.0 w i t h 0.1N NaOH wh i l e m a i n t a i n i n g a low temperature by c a r r y i n g out a l l operations i n an i c e bath. 'The d i s s o l v e d p r o t e i n was then immediately n e u t r a l i z e d by the a d d i t i o n o f 0.1N HCl. An a l t e r n a t e method was to d i s -s o l v e the p r o t e i n i n 0.001N NaOH (pH 10.0) and then n e u t r a l i z e by the ad-d i t i o n of 0.001N H01. Figure 12 shows the e l e c t r o p h o r e s i s of /^-casein and PDA modified k - c a s e i n . The r e s u l t s confirm Nakai's work i n t h a t a smeared band was obtained. B. A l k y l a t i o n of /c-casein A l and A2 I t was p o s t u l a t e d t h a t PDA caused aggregation through c r o s s - l i n k a s e of two h i s t i d i n e s on separate /c-casein molecules. One way i n which t h i s was t e s t e d was by p r e p a r i n g the mono-bromo d e r i v a t i v e , 2-phenyl—4—bromo-a c e t o i n (PMA). This was then reacted w i t h s u b f r a c t i o n s KA1 and KA2 as described i n the methods. r o Figure 1 2 . E l e c t r o p h o r e s i s of -casein and PDA- K - c a s e i n . F i g u r e 13 shows the decrea.se i n h i s t i d i n e residues w i t h r e a c t i o n time. A t o t a l of 1 . 0 5 r e s i d u e s of h i s t i d i n e and 1 . 6 residues of l y s i n e were modi-f i e d a f t e r a r e a c t i o n time of 20 hours. F i g u r e lk shows t h a t the e l e c t r o -p h o r e t i c p a t t e r n of s u b f r a c t i o n KA1 was not changed at a l l d u r i n g the a l k y l a t i o n . Figure 15 shows t h a t the s t a b i l i z i n g a b i l i t y of PMA- /c-casein A l and PMA- K - c a s e i n A2 are the same as 'nat i v e ' K - c a s e i n and s u b f r a c t i o n s . SSS- K - c a s e i n and KA1 showed a s l i g h t l y h i g h e r s t a b i l i z i n g a b i l i t y , p o s s i b l y due to the i n c r e a s e d negative charge. These r e s u l t s support the theory t h a t PDA causes aggregation of K - c a s e i n by c r o s s - l i n k i n g and show t h a t the m o d i f i c a t i o n of one h i s t i d i n e residue does not cause a decrease i n s t a b i l i z i n g a b i l i t y . This was a l s o found by Nakai when he modifi e d one residue i n s u b f r a c t i o n KA2 w i t h the reagent N-bromo-a c e t y l - l - a . r s l n i n e methyl e s t e r (PAA) ( 3 3 ) ; I t appears from these r e s u l t s t h a t the reagent 2-phenyl - 4-bromoacetoin (PMA) r e a c t s more r a p i d l y w i t h l y s i n e i n K - c a s e i n A l and A2 than does PDA, as 1 .6 residues was modified by r e a c t i o n f o r 20 hours w i t h the former, whereas only 0,2 residue was de-creased by the l a t t e r , C. T e s t i n g the r o l e of SS-SS Interchange i n the l o s s of s t a b i l i z i n g  a b i l i t y due to Reaction o f K - c a s e i n and S u b f r a c t i o n s with PDA Nakai found t h a t the a l k y l a t i o n of whole K - c a s e i n w i t h PDA f o r 16 hours at pH 6 . 6 r e s u l t e d i n a decrease of 0.2 residue of c y s t i n e and th a t the decreased s t a b i l i z i n g a b i l i t y was r e s t o r e d by d i s s o c i a t i o n w i t h 0.2N NaOH. There i s a p o s s i b i l i t y of involvement of the SS-SS interchange to e x p l a i n the decreased s t a b i l i z i n g a b i l i t y due to aggregation. The normal procedure used to remove the b l o c k i n g group, S - s u l f e n y l -s u l f o n a t e , a f t e r a l k y l a t i o n was to add 22 ml of 0.1M c y s t e i n e s o l u t i o n i n 0.1M phosphate, pH 7 .6 and to incubate the mixture at 3?°C f o r 20 minutes. 61 Figure 13, Change in histidine residues in KA1 with time or reaction with 2-phenyl~4-bromoacetoin (PMA). Reaction Time (Hrs.) 62 Figure 14, Electrophoresis of KA1 and. PMA-KA1. I fyHrt. $#rS, / * / & y . H^<x. 2**#'t, ®f'%*>\ M&£$$&* M&®&i4 i ^ & S a & A «2UV&tia«A i£&5&3& J^esA 63 Figure 15. Stabilizing a b i l i t y of KA1, KA2, PMA-KA1, K-casein and SSS- K-casein. (Closed triangles - KA1 and KA2; Closed squares - P.MA-KA1; Open circles - K-casein; Open squares - SSS- ^-casein) 100 c G) r-c •<-t 0) a CJ 1 ^ -0.04 0.08 0.12 0.16 0.20 * A s -casein ratio 64 Table IV, Amino acid analysis of /^-casein and PMA-K-casein. Amino Acid K-Ca.sein PMA- K-Casein (pH 6 , 8 ) PMA--Casein (pH 6 . 8 ) 24 hour ^8 hour Residues per 2 0 , 0 0 0 g Aspartic acid 14.9 14.9 14.9 Threonine 1 6 . 5 1 6 . 5 1 6 . 5 Serine 14 .6 14.6 14 .6 Glutamic acid 3 1 . 0 3 1 . 0 3 1 . 0 Proline 2 2 . 6 2 2 . 6 2 2 . 6 Glycine 2.9 2.9 2 . 9 Alanine 1 6 . 6 1 6 . 6 1 6 . 6 •j Cystine 2 . 5 2 . 5 2 . 5 Valine 12.9 12.9 . 12.9 Methionine 2 . 0 2 . 0 2 . 0 Isoleucine 1 3 . 9 1 3 . 9 1 3 . 9 Leucine 10.4 10.4 10.4 Tyrosine 8.4 8.4 8.4 Phenylalanine 4 . 5 4 . 5 Lysine 9 . 6 8 .6 8 .6 Histidine 3 . 0 2 . 7 2 . 7 Arginine 5.1 5 . 1 5 . 1 To test the role of the SS-SS interchange reaction in the decrease in stab i l i z i n g a b i l i t y due to aggregation, the blocked SH groups of KA1 were not reduced after alkylation with PDA, thus leaving the modified protein in i t s S-sulfenylsulfonate form. The stabilizing a b i l i t y of this protein was the same as the reduced modified protein, thus indicating that the role of the sidulfide interchange was remote. D. The Reaction of -PHA with V/hole /c-casein Several experiments were carried out to see i f the reagent 2-phenyl-4-bromoacetoin reacted with K-casein in the same way as i t did with the sub-fractions KA1 and KA2. Table TV shows that 1 .0 lysine residue and only 0 . 3 histidine residues were modified after a 24 hour reaction time. Increasing the reaction time to 48 hours (Table IV) did not show any increase in reactivity. There was no change in sta b i l i z i n g a b i l i t y or electrophoretic pattern. These results could be due to the poss i b i l i t y that the subfractions of ic-casein were less aggregated than was the whole protein and support the statement made above that studies on the role of important amino acids in the stabilizing a b i l i t y of K-casein should be carried out on the sub-fractions for these are probably closer to the monomer unit of <<-casein than i s the heterogeneous molecule. The results again show a possible change in selectivity of the reagent PDA from histidine to lysine as one of the bromines i s removed. It may be possible to use this reagent for the specific modification of lysine in other proteins. Another aspect of this work i s that i t does not support the results of Waugh and von Hippel ( 5 0 ) who reported that the modification of one lysine residue by acetic anhydride and six other reagents caused a decrease in stab i l i z i n g a b i l i t y of fc-casein. This could be due both to the different 66 reagents used and again due to the heterogeneity of the d i f f e r e n t prepara-t i o n s of W-oasein. A l s o , the s p e c i f i c i t y of t h e i r r e a c t i o n was not checked. E. A l t e r a t i o n s i n the r e a c t i o n of PDA with Jc'-casein To see i f a more workable d e r i v a t i v e of PDA.- Af-casein could be ob-tained , the r e a c t i o n mixture was changed 'mainly by decreasing the i o n i c s t r e n g t h of the b u f f e r used i n the r e a c t i o n mixture. Instead of u s i n g 0.1M phosphate b u f f e r , the f o l l o w i n g b u f f e r was used: 0.01M imidazole-NaCl, pP 6 . 6 , The r e a c t i o n was allowed t o proceed f o r 16 hours i n the dark and was then t e s t e d f o r s t a b i l i z i n g a b i l i t y and amino a c i d a n a l y s i s . Table V shows shows t h a t 0 , 6 residue of h i s t i d i n e was modified. Figure 16 shows t h a t the s t a b i l i z i n g a b i l i t y of the modified p r o t e i n was the same as the unmodified p r o t e i n , This work was repeated u s i n g another p r e p a r a t i o n of /^-casein. I t wa.s found, t h a t the r e a c t i o n of PDA w i t h t h i s p r e p a r a t i o n f o r 24 hours at the low i o n i c s t r e n g t h r e s u l t e d i n complete aggregation and p r e c i p i t a t i o n of the p r o t e i n . The pH was 6 , 6 through the r e a c t i o n . Time d i d not permit f u r t h e r i n v e s t i g a t i o n of t h i s phenomenon but c e r t a i n p o s s i b i l i t i e s should be men-t i o n e d . 1. C l a r k e (?) concluded t h a t i n c r e a s i n g the i o n i c s t r e n g t h of s o l u -t i o n s of ^ - c a s e i n r e s u l t e d i n i n c r e a s e d compactness of the molecule, Thus the r e a c t i o n of t h i s p r o t e i n w i t h s p e c i f i c modifying reagents c o u l d be dependent upon the i o n i c s t r e n g t h of the r e a c t i o n mixture. I t would appear from Clarke's r e s u l t s that the lower the i o n i c s t r e n g t h the e a s i e r w i l l . c e r t a i n reagents combine w i t h s p e c i f i c amino a c i d s , A decrease i n s e l e c t i v i t y f o r c e r t a i n amino a c i d s could accompany the increased, reac-t i v i t y o f the reagent(s) w i t h d e c r e a s i n g i o n i c s t r e n g t h , Nakai ( 3 3 ) found t h a t PDA became l e s s s p e c i f i c when i t was reacted w i t h Ar-casein i n the presence of i n c r e a s i n g concentrations of urea, A s i m i l a r dependencv 6.7 Table V. Amino a c i d a n a l y s i s of PDA- ^"-casein reacted i n low i o n i c s t r e n g t h b u f f e r Amino A c i d ^ - C a s e i n PDA-K -Casein (pH 6 , 8 ) 16 hour Residues per 2 0 , 0 0 0 g A s p a r t i c a c i d 14.9 14 . 9 Threonine •16 .5 I 6 . 5 Serine 14 . 6 14 . 6 Glutamic a c i d 3 1 . 0 3 1 . 0 P r o l i n e 2 2 . 6 2 2 . 6 G l y c i n e 2 . 9 2 . 9 A l a n i n e 1 6 . 6 1 6 . 6 -§- Cystine 2 . 5 2 . 5 V a l i n e 1 2 . 9 1 2 . 9 Methionine 2 . 0 2 . 0 I s o l e u c i n e 1 3 - 9 1 3 . 9 Leucine 10.4 10.4 Tyrosine 8.4 8.4 Phen y l a l a n i n e k.5 4 . 5 L y s i n e 9 . 6 9 . ^ H i s t i d i n e 3 . 0 2.4 A r g i n i n e 5 . 1 5 . 1 Figure 16. Stabilizing a b i l i t y of untreated K-casein and PDA- <-casein reacted in low ionic strength buffer. (Closed circles - Untreated K-casein- Open squares low ionic strength PDA- k-casein) 100 ,f 80 i K \ i 60 I 4o: 20 i 0 £ 0.04 0.61 / .<, -casein ratio 69 i s possible with the ionic strength of the reaction mixture, and this factor should be taken into account when modifications of specific amino acids in k-casein are attempted. ?.. The d i f f i c u l t y in obtaining the same results with two different preparations of k-casein could accentuate the problem mentioned in Chap-ter II of this thesis, that i s , Z i t t l e ' s method i s a harsh method which must be standardized, especially when large amounts are orepa.red at one time, 3« If PDA caused aggregation of K-casein by cross-linking and the structure of K-casein opens up with decreasing ionic strength, then PDA should cause more rapid cross-linking a.s the ionic strength i s lowered. The results with one preparation of K-casein corroborate this hypothesis whereas those with another do not, A sample of the former was examined in the spectrofluorometer and found to have a higher fluorescent peak than did the K-casein which did not completely aggregate during a 2k hour reaction with PDA, It was also found that the former had a lower stabilizing a b i l i t y (82>t) and solubility than the lat t e r . This indicated that the k-casein prepara-tion which did precipitate was in a more aggregated state before reac-tion with PDA. Lowering of the ionic strength of a solution of aggregated k-casein could serve to push the aggregation to completion once PDA i s reacted with the protein. Thus, the state of aggregation of the native protein could be a determining factor in making use of possible higher reactiv-i t y of the protein in decreasing ionic strength toward modifying re-agents. 70 SUMMARY The finding that a PDA-k -casein derivative could be prepared which dissolved normally and also showed the same stabilizing a b i l i t y as untreated K-casein indicates that changing one histidine residue does not cause a decrease in st a b i l i z i n g a b i l i t y . This work and that with PMA which modified 1.05 residues of histidine and. 1 , 6 0 residues of lysine in KA1 and KA2 with-out any change in stabilizing a b i l i t y does not support the hypothesis that histidine i s an important amino acid for maintaining the stabilizing a b i l i t y of /<-casein and supports the contention that the decrease in s t a b i l i z i n g a b i l i t y observed earlier ( 3 2 ) resulted from aggregation caused possibly by cross-linking, the extent of which could be dependent upon ionic strength. Subtracting one bromine from 2-phen.yl-l,4-dibromoacetoin (PDA) to produce 2-phenyl-4-bromoacetoin (PMA) seems to result in a transfer of selectivity from histidine to lysine. The reagent PMA does not react with whole fc-casein in the same way as i t does with KA1 and KA2 and this could be due to the l a t t e r being more homogeneous proteins than the former. This finding supports the contention that future work on the modification of amino acids in K-casein and studies on the interaction of this protein with J~s -casein should be carried out on the subfractions rather than on the whole protein. 71 CHAPTER VI Studies to determine the mechanism of action of PDA with histidine Nakai digested PDA-alkylated -casein with the enzyme pronase and ran the mixture on T.L.C. plates in order to identify the modified histidine residue. It was postulated that PDA might react with histidine in -casein in a similar way as. does N-bromoacetyl-l-arginine methyl ester in that car-boxymethyl histidine derivatives could be formed. This was supported by an increase in absorbance at 280 nm of -casein after alkylation. However, due to two bromines in PDA the hydrolysis products could be different and more complicated. The loss of one histidine residue after alkylation of -casein with PDA could not be totally accounted for by the 3-carboxymethyl histidine peak on the amino acid analyser during analysis of the acid-hydrolyzed PDA--casein. A d i f f i c u l t y in identifying carboxymethyl histidine derivatives i s that several derivatives overlap with several of the amino acids present in -casein. The attempts to purify the modified amino acid by pronase d i -gestion followed by thin layer chromatography were not successful. There-fore, to simplify the experiments, Dr. Nakai reacted PDA with histidine and compared the derivatives with CM-histidine derivatives prepared according to the method of Moore, Crestfield and Stein ( 3 3 ) . Several experiments which Nakai performed are as follows: 1. Preliminary Experiments Four hundred and f i f t y milligrams of 1-histidine hydrochloride monohydrate (Mann Research Laboratory) dissolved in 90 ml of 0 . 1 M phos-phate, pH 7 . 5 , were alkylated with 1 , 3 5 g of PDA dissolved in 110 ml of methanol and freeze-dried. after 16 hours in the dark. Thirty m i l l i l i t r e s of water and 15 ml 0.2 M sodium citrate buffer, pH 3 . 2 5 . were added and the pH was adjusted to 1 . 6 with.HCl. The residue of the extraction was ?2 dissolved in 15 ml of methanol. The aqueous extract was then chromato-graphy on a column 4 x 32 cm of Dowex 50 x 8 , 1 0 0 - 2 0 0 mesh washed with 0 . 2 N NaOH and equilibrated with citrate buffer, pH 3 . 2 5 . The effluent was analyzed in 0 . 1 ml amounts by a ninhydrin method. The fractions were concentrated by rotary evaporation and the concentrates transferred to test tubes were cooled, at 4°C. The supernatant after crystallization of buffer salts was used for identification, N-Acetyl-1-histid.ine (Cal-biochem) 4 5 0 mg in 6 5 ml of the phosphate buffer, was also used instead of histidine to avoid alkylation of o(-amino groups. The derivatives ^ produced were hydrolyzed to remove the acetyl group by boiling for 6 hours under reflux after adding 1 5 5 ml of 8.5N HCl. The HCl was removed by rotary evaporation, Nakai obtained five fractions by chromatography of both the PDA-histidine and the PDA-N-acetyl-l-histidine reaction mixtures, two of which appeared to be the same derivative from both reactions, The respective T.L.C, Rf values of the Dowex 50 peaks were 0 . 2 5 , 0 . 4 9 , and 0 . 6 2 for peak ( i ) , peaks (II and IV) and peaks ( i l l and V). These were a l l higher than three derivatives of carboxymethyl-histidine. As the derivatives, especially those derived from histidine, pro-duced an odour similar to that of acetophenone after warming in the presence of 1strong bases, i t was reasoned that they may contain the phenylketone group, Since i t was known that the PDA decomposes and produces acetophenone by warming with bases, Nakai tested for the pres-ence of this group by a modification of the method of Tikhonova ( 3 3 ) . Acetophenone and PDA yielded a yellow colour reaction while acetone produced a pink colour. Fractions. ( l l ) and ( i l l ) of PDA derivatives from histidine indicated a distinct yellow colour. Fractions (IV) of the derivatives from acetyl-histidine reacted positively but with a decreased 73 i n t e n s i t y . The r e a c t i o n of f r a c t i o n s d ) and. (V) of the d e r i v a t i v e s w a s very f a i n t . Carboxymethyl-hist.id.ine d i d not r e a c t . I t was reasoned from these r e s u l t s t h a t the d e r i v a t i v e s are pos-s i b l y a d d i t i o n products and t h a t production of acetophenone i n the presence of bases may be a s i g n of r e l e a s e of a ohenylketone grout) attached t o h i s t i d i n e and may p a r t i a l l y e x p l a i n why the s t a b i l i z i n g . a b i l i t y of the PDA-alkylated whole - c a s e i n at pH 6 . 6 was r e s t o r e d by the a c t i o n of base ( 3 2 ) . The methanol e x t r a c t from the p r e p a r a t i o n of PDA-alkylated h i s t i -d i ne produced a f a i n t spot of h i s t i d i n e by T.L.C. 2 . I s o l a t i o n of PDA-Alkylated H i s t i d i n e The r e a c t i o n of PDA on h i s t i d i n e i s so m i l d t h a t the y i e l d without f u r t h e r p u r i f i c a t i o n was approximately 0 . 2 to 0.3?&. R a i s i n g the reac-t i o n pH above 7 . 5 i n c r e a s e d the r e a c t i o n r a t e but at the same time de-composed, the products to form c a r b o x y m e t h y l - h i s t i d i n e s which were con-fir m e d by T.L.C. Using Dowex 1 and g e l a t i n to remove bromine from the r e a c t i o n products i n c r e a s e d the y i e l d , without f u r t h e r p u r i f i c a t i o n to 2%. R a i s i n g the temperature of the r e a c t i o n beyond 2 5 C- was not e f f e c t i v e a t a l l . Ion exchange r e s i n s were u t i l i z e d f o r d e s a l t i n g without success because of r e s t r i c t i o n of the o p e r a t i n g pH range. Use of the m i l d con-d i t i o n of Dowex 2 e q u i l i b r a t e d w i t h p y r i d i n e - a c e t a t e b u f f e r , pH 6 . 0 , and e l u t i o n w i t h IN a c e t i c a c i d r e s u l t e d i n conversion of the d e r i v a -t i v e s t o carboxymethyl h i s t i d i n e s . The r e c r y s t a l l i z a t i o n of the de-r i v a t i v e s from ethanol hydrolyzed them as w e l l , Thus, i n the f i n a l experiments Nakai used a chromatography on Dowex 50 x 8 to separate the f i v e f r a c t i o n s , f o l l o w e d by c e l l u l o s e chromatography to d e s a l t each f r a c t i o n . 74 3. Final Experiments Ten grams (64.4 mM) of 1-histidine free base were dissolved in 1.4 l i t r e s of water at 25°C in which 21 g of gelatin was dissolved by warming to 70°C. Thirty grams of PDA ( 9 3 . 2 nM) dissolved in 2 . 3 1 of methanol were added slowly to the histidine solution, followed by 420 g of wet Dowex 1 x 4, OH-form. The pH of the solution was 7.2 to 7 . 3 . The solution was stored in the dark for 5 days while the pH was maintained at 7.2 to 7 . 3 with 2N NaOH. The solution was f i l t e r e d on Whatman No. 1, washed with N acetic acid, flash-evaporated to 4 5 ml. Nine hundred ml of absolute methanol were added, and the solution was centrifuged at 8,000 x g for 30 minutes at -15°C; this precipitated most of the gelatin. The supernatant was evaporated to 60 ml and desalted on a Sephadex G-50 column, 3 . 6 x 60 cm, equilibrated with 0.1N acetic acid. Effluents eluted with 0.1N acetic acid were analyzed by the ninhydrin reaction for 10 ul aliquots from each test tube containing 20 ml frac-tions. The ninhydrin-positive fractions were evaporated to 60 ml, cen-trifuged at 3»600 x g for 20 minutes and evaporated further to dryness. Approximately 20 ml of water was added and the solution redried. The residues were dissolved in 2 5 ml of water and the pH adjusted to 2.2 with 6N NaOH. The solution was centrifuged again at 3 . 6 0 0 x g for 20 minutes and chromatographed on a, Dowex 50.x 8, 200-400 mesh column, 2 x 90 cm, with a 0.2 M sodium acetate buffer at pH 3.2 5 . The effluent of 10 ml fractions was analyzed by the ninhydrin method for 100 ul from alternate test tubes. Usually three derivatives were separated between tubes 120 and 220, The combined fractions for each derivative were evaporated to approximately 20 ml, cooled to 4°G overnight to crystal-l i z e most of the buffer salts, centrifuged, the supernatant adjusted to pH 3.0 with HCl and freeze-dried. The residues were dissolved in 20 ml of the solvent, isopropanol-formic acid-water 2 0 : 1 : 5 , and chro-matographed with the sa.me solvent on a cellulose column, 2 x 90 cm, in which 100 g of Cellex-N-1 (BioRed Laboratories, Richmond, Califor-nia) were packed with the same solvent. Aliquots of 150 ul from alter-nate 5 ml fractions were analyzed by the ninhydrin method. The ninhydrin-positive fractions were combined, 50 to 100 ul of which was used for amino acid analysis, evaporated after adding 2 drops of HCl and f i n a l l y freeze-dried. The highest yield obtained was Fraction ( l l ) , ? 0 mg, which had a salt content of approximately 90%. It was found that the Sephadex 0 - 5 0 treatment described above did not desalt the derivatives and in fact resulted in a large decrease in total yield. The purpose of this chapter i s to report further work done In an at-tempt to elucidate the mechanism of reaction between PDA and histidine. The objectives were as follows: (a) to test the possibility of purifying the three derivatives by a one step procedure using cellex chromatography; (b) to test several basic factors of the reaction for the purpose of increasing the yield; (c) to test the p o s s i b i l i t y of changes in the products caused by treatment of proteins for amino acid analysis; and (d) to test whether each product was a histidine derivative. 76 THEORY .Based on the work done by Crestfield et al on the preparation of car-boxymethyl-histidine derivative (33), i t i s predicted that the following products (A,B Sr. C) could result from a combination between PDA (i) and histidine ( l l ) . l ' HC — <l - CHx.~- <LU- COO U Br C/tx — C Ii CO — C i\ ,3, j j , X % ** ty Vii HC — ( \ A • C -i "Hi. i C t> ~CoOt4 c \0i C f i i C - C c ; W \ C ' u o X c. 1 I n It i s f e l t that the 4-bromine i s the most reactive of PDA because of i t s proximity to the carbonyl group. Nakai has proven that the ^-amino gx-oup of histidine i s not a main participant and also that treatment with the reaction products of PDA and histidine with base gives rise to GM-histidine. 77 If the reaction between PDA and histidine acts in the proposed way then the ratios of the three (A), (B) and (C) should be close to 1:1:2 in line with the work of Crestfield et al on carboxymethyl histidine derivatives. One problem in predicting the reaction i s that PDA i s an optically active compound and no attempt has been made to resolve i t (4). Another d i f f i c u l t y with this compound i s that acetoins are readily hydrolyzed in water and this could account for the very low yields obtained by Nakai, Other solvents such as formamide should be attempted as suggested by Dr. Bose (4), Also the concentration of reactants may play a role in the type and ratio of products. The replacement of the hydrogen atoms on histidine and the removal of bromines from PDA should result in a decrease in pH through the formation of HBr, Thus there i s a possi b i l i t y that this could be used to monitor the ef-fect of changing the conditions of the reaction. Several changes in the reaction were carried out following preliminary attempts to purify the products by chromatography on cellex-N-1 monionic cellulose. Experimental Plan Series one The f i r s t series of experiments were designed to test the possibility of eliminating the Dowex 50 x 8 step by carrying out an immediate fractiona-tion of a reaction mixture after removal of the gelatin and concentration of the mixture by flash evaporation. Also attempted was the purification of fractions by both thin layer and paper chromatography. Series two Several basic factors of the reaction were studied, including varying the concentration of reactants, changing the solvent system, and running the reaction in the presence of aluminium amalgum and refluxing in an attempt to 78 more rapidly push the reaction to completion. Series three The reaction mixture was treated in boiling 6N HCl in the same way as are proteins in preparation for amino acid analysis. This was done to see i f this treatment caused changes in the products which would explain the d i f f i c u l t y of not being able to account for the modification product during amino acid analysis of whole PDA-K-casein. Also paper chromatography of the reaction mixture were stained with Pauley's reagent for histidine. MATERIALS PDA was prepared by Dr. Nakai according to the method, of Ruggli ( 4 4 ) . 1-Histidine hydrochloride free base was obtained from Nutritional Bio-chemicals Corporation. Cellulose--N-l nonionic precoated plastic plates were purchased, from. Sigma Chemical Company.. Whatman No, 3 f i l t e r paper was used for paper chromatography, Formamide was obtained from Nutritional Biochemical Company. Aluminium amalgum was obtained from Fischer Scientific Company and treated as described in Chapter VI, Hydrochloric acid was glass d i s t i l l e d , of which the f i r s t and the last 10c? of the d i s t i l l a t e was discarded, METHODS Series one Preliminary experiments designed to fractionate the products by cellex-N-l chromatography were carried out by reacting PDA with histidine following Nakai's method up to and including the point of removing the gelatin. The gelatin-free concentrate was then flash evaporated to dryness and the resi-due dissolved in the solvent isopropanol-formic acid-water (20:5:1) and chromatographed, using this solvent, on a short cellex column ( 15 x 0 .9 cm). The reactions were carried out using small amounts of reactants for these preliminary experiments. One hundred milligrams of 1-histidine was dissolved in 14,0 ml of water in which 0 .21 g of gelatin had been dissolved by heating to 70°C. To this was added 300 mg of PDA dissolved in 2 3 ml of absolute methanol. Finally 0.42 gr of wet Dowex 1 x 4 (OH-form) was added and the pH of the mixture was raised to 7.2 with 2!T NaOH. The reaction was allowed to run in the dark for 24 hours. When the reactions were run longer than 24 hours the pH was readjusted to 7.2 daily. Purification of fractions by paper chromatography was carried out by placing a strip of concentrated reaction mixture dissolved in the solvent mentioned above along the fold at the top of an 8 inch wide strip of 'Whatman No. 3 f i l t e r paper and allowing the chromatographs to run for 16-18 hours in descending fashion in the same solvent. After the runs were completed a -§ inch strip was cut off the right side of the paper and stained. This was then aligned with the remainder of the paper and the spots marked with pencil. The strips containing spots were then cut from the paper and eluted with the same solvent by placing them between glass slides in petri dishes and cutting the bottom ends of the strips to a point and placing this in the mouth of small beakers. When i t was found that the solvent would not move up the glass sides and that the spots were water-soluble, water was used to elute the spots. The liquid in the beakers was then freeze-dried and. the residue stored under vacuum in a desiccator. Series two Several basic factors involved, in the reaction were studied in an at-tempt to increase the yield. It was f i r s t of a l l suggested by Dr. Bose that PDA would possibly be bydrolyzed. in water and therefore the concentra-tion' of the l a t t e r should be kept to a minimum. The molar ratio of reactants used by Nakai was a 1 . 8 0 molar excess of PDA to histidine. Thus ratios of 0 . 1 to 30 were set up using total reaction volumes of 5 or 10 ml. Again the pH and the T.L.C. patterns were measured after reaction in the dark for 24 hours. The reaction was also carried out in the presence of formamide instead of water. This material was flash evaporated until no more liq u i d could be removed and then run on T.L.C, One other attempt taken to increase the yield was to reflux 100 mg of histidine dissolved in 1.4 ml of water with 300 mg of PDA dissolved in 2 3 ml of absolute methanol in the presence of aluminium amalgum prepared as de-scribed earlier. One ml aliquots were withdrawn at intervals over a three hour period, centrifuged and 10 Lil spotted on cellulose plates for thin layer chromatography, Seri es three Ten milligrams of the freeze-dried water-soluble products of the re-action mixture was dissolved in 5*0 ml of 6K HCl and boiled under vacuum for 24 hours. During the f i r s t experiment in this series the residue of solvent-soluble material was treated in this way, .The material which was not soluble in the HCl after boiling was removed by centrifugation. The clear solution of HCl. was then evapoxated to dryness with 4 washes of dis-t i l l e d water and the residue taken up in 1 . 0 ml of water. Ten microlitres of sample was applied to T.L.C. plates and run in Jones' solvent for five hours followed by staining with ninhydrin spray. The directions for the preparation and use of Pauley's reagent are as follows: 81 Solution A - dissolve 1 gram of sulphanilic acid in 100 ml of N HCl Solution B - prepare just before using a 0-.?% solution, of NaNO^  Solution C - prepare a 10% aqueous solution of NaHCO-^  Carefully mix solutions A and B in a ratio of 1 : 1 . Spray the paper with this mixture and dry the paper in a stream of a i r . Soray again l i g h t l y with solution C. It i s important to spray the paper l i g h t l y , otherwise large red spots appear. Care must be taken in the staining of precoated thin layer plates because of the pos s i b i l i t y of l i q u i d running down.the plates. RESULTS AND DISCUSSION Series one Fractionation of Reaction Products of PDA-Histidine by Cellex Chromatography Reactions involving 100 mg of histidine and 300 mg of PDA in a total volume of 37 ml were carried out for 24 hours; the gelatin removed; the re-action mixture was dried down; and the residue was dissolved in 5 . 0 ml of solvent. Various amounts were applied to a 1 5 . 0 x 0 . 9 cm cellex column pre-viously equilibrated with the same solvent (isopropanol-formic acid-wa.ter, 20 : 1 : 5 ) . After eluting the mixture with this solvent 1 . 0 ml fractions were collected and 2 50 ul aliquots of every second fraction analyzed by the ninhydrin reaction ( 3 3 ) . Figure 2 3 shows the results of a successful fractionation of the mixture for the whole reaction mixture in 0 . 5 ml of solvent. The chromatographic fractionation of larger volumes of sample was unsuccessful. It w i l l be noted from Figure 2 3 that a l l of the peaks contained traces of CM-histidine-like material. Fraction Cj of two runs was .combined, dried down and taken up in 1,0 ml of solvent and rechromatographed with the results shown in Figure 24. These experiments showed that i t is possible to obtain the three main products of the reaction mixture by cellex chromatography. There was, however, a trace of CM-his.tidine-like substance in a l l fractions. Figure 17, Fractionation on cellex of 0.5 ml of reaction mixture of PDA and histidine. Figure 18-. Rechromatography on cellex of several preparations Fraction C T shown in Figure 17. 84 It was then decided to scale up the experiment by using a large cellex column (60 x 2.4 cm), Also, since i t was apparent that whenever the reaction mixture was dissolved in solvent and then chromatographed, every fraction contained CM-histidine-like material, the freeze-dried reaction mixture was extracted with water and the insoluble material discarded. Figure 2 5 shows the fractionation of 1 , 5 ml of solvent containing 80 mg of water-soluble material on a large cellex column. Fraction I contained the Rf 0 . 6 2 pink spot and a small amount of the Rf 0 . 5 9 material. Fraction II contained the Rf 0 . 5 0 and 0 . ^ 8 spots, and Fraction III contained mainly the 0.48 spots. Fractions IV-VII a l l contained histidine. A l l of the f i r s t three spots con-tained CM-histidine-like material. It was found that washing the cellex with approximately 100 ml of for-mic acid followed by several washings in solvent was an effective method of equilibration, Figure 26A shows the fractionation of 80 mg of water-soluble components dissolved in 3«0 m l °f solvent and chromatographed on a 60 x 2 , 4 cm cellex column which had been equilibrated by washing with 300 ml of solvent. Frac-tion I (Tubes 31-42) contained a large a.mount of Rf 0.62 spot and CM-histi-dine. Fraction II (Tubes 43-48) contained a mixture of Rf 0 . 4 9 and 0 . 4 ? spots and traces of CM-histidine. Fraction III (Tubes 4 9 - 5 7 ) contained a mixture of a l l fractions except histidine. Fraction IV contained only h i s t i -dine and a trace amount of Rf 0 . 4 7 spot. The tubes spanning Fraction I were analyzed with the results shown in Figure 26B, The last three tubes con-tained a mixture of 0 . 6 2 spot and more CM-histidine than the f i r s t three tubes. The fractions rich in Rf 0 . 6 2 spot were combined. Figure 27 shows a rechromatography on the small cellex column of 1 ml of solution of this material gathered from several cellex runs as well as several paper chromato-graphic runs. Fraction I and several individual tubes of the second fraction 8-5 Figure 19. Fractionation of 1 . 5 ml of solvent containing 80 mg of reaction mixture of PDA and histidine on a large cellex column. Figure 20A. Fractionation of 3*0 ml of solvent containing 80 mg of reaction mixture and histidine on a large cellex colum 87 Figure 20B, Thin layer chromatography of the tubes spanning Fraction I of Figure 20A, Figure 21. Rechromatography of 1 ml of solvent containing material rich in Rf 0.62 substance, 883 0 S * !v £ 4 F /f: ^  4 O ft a 9 ftayfwc t** m t ? £ ft 89 were tested. Fraction I contained mainly the Rf 0 . 6 2 spot, tubes 1 0 9 and 111 a mixture of the Rf 0 . 6 2 and 0 . 5 9 , and tube 115 a l l three. Tubes 119 and 1 2 5 contained, only the Rf 0.48 spot. By repeating this type of procedure the total amount of Rf 0 . 6 2 , 0 . 5 3 , and 0.48 material purified was 16 mg, 10 mg., and 5 . 0 mg. The actual weight of dry material was much larger but the salt content of each fraction was approximately 90?& and thus there was not enough material for a proper chemi-cal analysis, One serious d i f f i c u l t y which was found to happen with a l l three fractions was that when they were stored in water they broke down to the unfractionated reaction mixture. These results show that i t i s possible to fractionate the reaction mix-ture of PDA-histidine on cellex columns using the standard Jones solvent for amino acid analysis by thin layer chromatography. The low yields, however, did not permit the second phase of this work being done, that i s , attempting to desalt on a cellex column using -formic acid-water ( l : 5 ) a s the solvent. Also these studies emphasized the in s t a b i l i t y of the reaction products in water. Series two A* The Effect of varying the Concentration of Reactants Table VI. shows the effect on pF. of the reaction mixture after 24 hours and 72 hours of varying the concentration of reactants, One great d i f f i c u l t y in carrying this experiment longer than 24 hours was that the methanol evaporated and had to be replenished daily. It i s clear from Table ".VI, however, that increasing the molecular ratio of PDA to histidine led to a larger decrease in pK after a reaction time of 24 hours. Raising the ratio beyond 10 did not seem to result in further decreases in pH after a 24 hour reaction time, nor did i t result in a further pH decrease when the reaction was allowed to go for 72 hours. It was then decided to set up Table VI. The effect of concentration of reactants on the pK of the reaction mixture (PDA + histidine) after ?A and 72 hours. Molar Ratio  PDA: Histidine 1:2 1 : 5 1 : 1 0 1 : 2 0 1 : 3 0 pH of Reaction Mixture 2k hour 72 hour 6 . 2 2 6 . 2 2 5 . 9 9 5 . 9 9 5 . 2 0 5 . 2 0 5 . 2 0 5 . 1 5 5 . 2 0 5 - 1 5 91 a s m a l l s c a l e r e a c t i o n mixture using a. t e n f o l d excess of PDA and to f r e e z e -dry, e x t r a c t , and f r a c t i o n a t e t h i s mixture. Figure 22 shows the r e s u l t s of t h i s experiment and seems to i n d i c a t e that more r e a c t i o n products were present than was the case with the r e a c t i o n s i n v o l v i n g 1 , 8 molar excess of PDA, I t a l s o appears that the Pa 0 . 6 2 m a t e r i a l c o n s t i t u t e s more of the product. Figure 2 3 i s a T.L.C, of 10 u l of r e a c t i o n mixtures set up usin g va.ri.ous molar r a t i o s of PDA to h i s t i d i n e . The sol v e n t was allowed to ran o f f the p l a t e i n order to o b t a i n maximum se p a r a t i o n . No obvious diffe-rsr.ce was detected by t h i n l a y e r chromatography, B. The e f f e c t of c a r r y i n g out the r e a c t i o n i n the presence of Formamide The same co n c e n t r a t i o n of rea.cta.nts as used by Na.kai was used f o r t h i s experiment, and water was repl a c e d by formamide. The r e a c t i o n was allowed to go f o r 24 hours, f r e e z e - d r i e d and extra.cted with water. I t was found that the s o l u t i o n turned from c l e a r to a dark brown at the end of 2 hours r e a c t i o n . The mixture a l s o could not be i reeze-dried. to complete dryness. Attempts to e x t r a c t w i t h water r e s u l t e d i n no pre-c i p i t a t i o n o f unreacted PDA whatsoever. Nakai found only a s l i g h t i n c r e a s e i n y i e l d and recommended th a t no f u r t h e r attempts be made wi t h t h i s r e -a c t i o n . C. The e f f e c t of r e f l u x i n g PDA and H i s t i d i n e i n the presence of .Aluminium Amalgum Fig u r e 24 shows the T.L.C, of 10 u l of the r e a c t i o n mixture set up by r e f l u x i n g f o r 6 hours at 6 5 ° 0 2 grams of PDA d i s s o l v e d i n 2 3 ml of absolute methanol w i t h 300 mg of h i s t i d i n e d i s s o l v e d i n 3 , 0 ml of water at 65°C. I t seemed th a t t h i s r e a c t i o n d i d not g i v e the same products as d i d the r e a c t i o n a t room temperature and f o r t h i s reason no f u r t h e r work was done i n t h i s area. Figure 22, Fractionation on cellex- of PDA and histidine reaction mixture containing a .10 molar excess of PDA, 93 F i g u r e 23. Thin Layer chroma, to grams of r e a c t i o n mixtures of PDA and h i s t i d i n e c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s of reactive-s. r a t i o 9 4 Figure 24. Thin Layer Chromatography of reaction mixtures of PDA and histidine refluxed in the presence of Aluminium Ama]gum. 9 5 Series three A. The effect of refluxing the Reaction Mixture in 6N HCl for 1 hour Figure 2 5 shows the results of T.L.C. of 10 ^1 of the reaction mixture refluxed in 6N HCl in the same way as i s done in the preparation of pro-teins for amino acid analysis. No difference was apparent indicating that no change takes place in PDA-histidine during hydrolysis of the modified protein. B. The reaction of spots of the Reaction Mixture to Pauley's Reagent A l l were cherry red, which indicates that a l l spots contained, the imida-zole ring. This indicates that the reaction of PDA with histidine results in addition products and one method, which might be used to give a preliminary indication of the chemical composition i s to compare the absorbance at 220 nm. One d i f f i c u l t y in doing this i s the low yields of products. SUMMARY Cellex chromatography in Jones* solvent can be used to separate the products of the reaction between,PDA and histidine. Paper chromatography i s also an effective method of purifying the individual fractions. Using a 10 molar excess of PDA seems to increase the amount of product ha.ving an Rf of 0 . 6 2 on thin layer chromatography plates. Refluxing PDA and h i s t i -dine at 65°C in the presence of aluminium amalgum for 6 hours did not lead to an increase in yield. Carrying out the reaction in the presence of for-mamide resulted in only a slight increase in yield and no advantage since the formamide could not be easily removed. The products were not damaged by the normal method of preparing proteins for amino acid analysis. A l l of the products were Pauley reagent-positive, which indicates that h i s t i -dine addition products were formed. ^7 96 Figure 2 5 . Thin Layer chromatography of reaction mixtures of PDA and histidine refluxed in 6N HCl for i and 2k hour.-.. / 0 ( ) V J $,»4«'4<V*t ft/nh^iM 9 ? CHAPTER VIT SUMMARY AND CONCLUSIONS During preliminary studies in this thesis observations were noted in detail regarding the practical aspects of large scale production cf 'Zittle's K-casein. Temperature was found to be a c r i t i c a l factor throughout the procedure. Skimming was more efficient when cool milk was warmed to 45"C; the yield, of casein was higher when the temperature of the milk was raised to 30°C{ the mixture of the <*s - and 4-casein precipitate and K-casein su-pemate had to be held at room temperature during centrifugation because the *s-casein was soluble at k°C. During alcohol fractionation s t i r r i n g had to be minimal, otherwise aggregation occurred. Electrophoretic analyses at various steps in the procedure showed that fractionations were successfully carried out even though the amounts of material used were at least ten times that in Z i t t l e ' s original work. The lowest yield cf K-casein obtained in these studies was 6,0% and the highest was 7.5%- The amino acid composition of two preparations of the K-casein resembled that published in a recent review by McKenzie ( 2 3 ) . Electrophoretic analysis showed that the K-ca.sein was heterogeneous. The subfractions could not be seen and studies were then carried out to improve the.electrophoresis technique. Four modifications which gave promising results were described in deta i l . They involved increasing the voltage through the gel, equilibrating the system by carrying out preliminary runs without samples, increasing the concentration of dissociating agents in the gel by equilibration in 0 . 0 & 7 5 M Tris-glycine buffer pH 9 . 1 containing 8M urea and 0 . 0 0 1 M 2-mercaptoethanol, and decreasing the ionic strength of the system. The subfractions were clearly seen when methods incorporating these principles were applied and 98 were purified by DEAE cellulose chromatography during which several sugges-tions of Mercier (28) had. to be closely followed, The steps in Mercier's method, which proved to be the most important included s i f t i n g the cellulose and using only that having a. mesh size of 100-200 u, recrystallyzing the urea, and using low molarity NaOH and HCl for regenerating the cellulose. Subfractions 1 and 2 were purified and used., along with K-casein, in reactions with PDA and PMA, It was found that these reactions did not support the hypothesis that histidine plays an important role in the st a b i l i z i n g a b i l i t y of K-casein because a derivative of PDA- K-casein was prepared in low ionic strength buffer which had 0 , 6 residue of histidine less than untreated K-casein but had the same stabilizing a b i l i t y as untreated K-casein. Moreover, PMA-KA1 and FMA-KA2, which had 1 , 0 5 histidine and 1 . 6 0 lysine residues less than the corresponding untreated proteins had the same stab i l i z i n g a b i l i t y as KA1 and KA2, These results support the theory that the mechanism by which PDA causes K-casein to aggregate is cross-linking between histidine r e s i -dues on separate K-casein molecules. Studies using the blocking grcuo, sodium sulphenyl sulfonate, indicated that the role of disulfide bonds in the process of aggregation of PDA- fc-casein i s remote. An interesting phenomenon which was not investigated further was that a preparation of K-casein whose state of aggregation was higher than that used in the preparation of the low ionic strength PDA- k-casein, precipi-tated when i t was reacted for 24 hours with PDA in the low ionic strength buffer. This indicates that the degree of cross-linking could be dependent upon the state of aggregation of the protein and the ionic strength of the reaction. Also; i t may be conjectured that i f PDA acts more rapidly with K-casein when the ionic strength i s low, i t s selectivity might also de-crease. Removing 1 bromine from PDA to make PHA seemed to result in a change of selectivity from histidine to lysine in that 1 .6 out of 9 . 6 lysine r e s i -dues per molecular weight 20,000 in KA1 and KA2 were modified by PMA whereas only 0.2 lysine residue was modified by PDA in these proteins. It might be possible to use PMA to modify lysine in other proteins. Incidentally, these studies involving the modification of lysine did not support those of Talbot and Waugh ( 5 0 ) who reported that decreasing 1.0 lysine residue in *"-casein led. to a decrease in stab i l i z i n g a b i l i t y . One other approach taken to explain the mechanism of action of PDA was to react i t with the amino acid histidine. The reaction was very slow and low yields were obtained. Three main products having Rf values of 0 . 6 2 , O . 58 , and 0.40 on thin layer plates were purified by paper and cellex chromatog-raphy using Jones' solvent ( 3 3 ) for the chromatographic separation of amino acids. The derivatives were histidine addition products as judged, by their positive reaction to Pauley's reagent. Because of the low yields this ap-proach has not led to an explanation of the mechanism of action of PDA. However, the products break down to carboxymethyl histidine-like products when reacted in base and this may help to explain the restoration of sta-b i l i z i n g a b i l i t y obtained when Nakai ( 3 2 ) treated PDA- K-casein in 0.1N NaOH. 100 BIBLIOGRAPHY 1 . Aschaffenburg, R. ( 1 9 6 4 ) Biochim. Biophys. Acta 82:188. 2 . Beveridge, H. J. T. (1968) B.S.A. Thesis, University of British Columbia 3 . Bingham, S. W. ( 1 9 7 1 ) .1. Dairy Sci. 5 4 ( 7 ) : 1 0 7 7 - 1 0 8 0 . 4 . Bose, R. ( 1 9 7 1 ) Personal communication. 5. Cherbuliez, E. and P. Baudet ( 1 9 5 0 a ) Helv. Chim. Acta 3 3 : 3 9 8 . 6 . \ ( 1 9 5 0 b ) Helv. Chim. Acta 3 3 : 1 6 7 3 . 7 . Clarke, R, ( 1 9 7 1 ) Personal communication. 8 . Crestfield, A. M., W. H. Stein and S. Moore ( 1 9 6 3 ) J. Biol. 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