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Investigations into the constitution of casein Hummel, Brian Christopher Warren 1950

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fffo  Mis'  INVESTIGATIONS into THE CONSTITUTION OF CASEIN  by Brian Christopher Warren Hummel  A thesis submitted i n partial fulfilment of the requirements for the degree of  MASTER OF ARTS  i n the Department of* CHEMISTRY  THE UNIVERSITY OF BRITISH COLUMBIA August, 1950  ABSTRACT  A comparison of the products obtained by two methods of fractionation of casein indicate that the methods do not give the same results. An electrophoretically homogeneous fraction yielded, upon hydrolysis with pepsin, at least two phosphopeptones. The one having an N / P ratio of 6.6 was shown by f i l t e r paper partition chromatography and qualitative colour tests to be composed of serine, cystine, glutamic acid, alanine, one or more of the leucines, tyrosine, and possibly valine and arginine.  ACKNOWLEDGMENT  I -would l i k e t o express my sincere appreciation to Dr. B. Eagles f o r i n s p i r a t i o n and encouragement, t o Dr. S.Zbarsky f o r valuable c r i t i c i s m and advice regarding both experimental and l i t e r a r y aspects of t h i s work, and to Dr. 0. Bluh f o r the use o f h i s T i s e l i u s apparatus and advice concerning the l a t t e r . I would also l i k e t o thank Mr. D. Townsley f o r generously loaning h i s apparatus f o r paper p a r t i t i o n chromatography, as w e l l as giving invaluable p r a c t i c a l i n s t r u c t i o n i n i t s use, and Mr. Werner f o r advice and apparatus for.the p u r i f i c a t i o n of reagents.  Ill  TABLE OF CONTENTS  Page Introduction  1  Historical A. The products o f hydrolysis o f casein by B.  pepsin and t r y p s i n  3  The f r a c t i o n a t i o n o f casein  7  Experimental A. Preparation of casein from milk  13  B. Fractionation o f casein  14  1. Using 5% ammonium chloride and acetone 2. Using acetone, d i l u t e a c i d and d i l u t e base i n the c o l d C. Examination of the casein f r a c t i o n s  19  1. Electrophoresis 2. Phosphorous determinations D. Peptic hydrolysis of casein  22  1. Preliminary experiments with commercial casein 2. Hydrolysis of alpha casein E. P u r i f i c a t i o n of the hydrolysis products  30  F. Examination o f the peptones  31  1. Nitrogen and phosphorous determinations 2. Preparation o f the a c i d hydrolysates of the peptones  32  IV  Page 3. F i l t e r - p a p e r p a r t i t i o n chromatography  32  4. Q u a l i t a t i v e c o l o u r t e s t s  42  Discussion Summary  48  Bibliography  50  1  INTRODUCTION  The action o f the p r o t e o l y t i c enzymes, pepsin and t r y p s i n , upon bovine casein has been the subject o f investigations by many workers, among whom Posternak ( I ) , Rimington ( 2 ) , Levene and H i l l (3), Damodaran and Ramachandran (4) have made s i g n i f i c a n t contributions. Diterest has been directed mainly toward three aspects of t h i s problem: (a) the rate of hydrolysis, (b) the extent o f hydrolysis, and (c) the nature o f the products of h y d r o l y s i s .  With regard to the t h i r d aspect,  i t has been observed repeatedly that both pepsin and t r y p s i n are capable o f hydrolysing casein t o a l i m i t e d extent only, unhydrolysed peptones remaining a f t e r prolonged peptic or t r y p t i c hydrolysis.  Sever-  a l investigators, each using a d i f f e r e n t procedure, have i s o l a t e d one or more such peptones, but the reported amino a c i d compositions o f these peptones exhibit, unfortunately, only a q u a l i t a t i v e s i m i l a r i t y . They have, on the other hand, been consistently observed t o possess a considerably higher phosphorous content than the caseins from which they were d e r i v e d . I t was with a view t o resolving these c o n f l i c t i n g reports on the compositions o f casein phospho-peptones that t h i s work was  undertaken.  Svedberg and h i s co-workers  (5,6),  using the u l t r a - c e n t r i f u g e ,  Warner ( 7 ) , using the T i s e l i u s electrophoresis apparatus, and other workers (14,23-28), employing various organic solvents, inorganic s a l t s , acids and bases, have accumulated convincing evidence that native  bovine casein i s not homogeneous, but can be resolved i n t o several components d i f f e r i n g i n s o l u b i l i t y , amino a c i d composition and phosphorous content.  I t i s apparent that the inhomogeneity of casein may  account f o r the inconsistencies previously mentioned.  I t was thus  considered e s s e n t i a l i n the present work to study the enzymic hydrolysis of a single component o f casein, rather than an i n d e f i n i t e mixture of components, as appears to have been done heretofor.  HISTORICAL  A.  The products of hydrolysis o f casein by pepsin and t r y p s i n ; I t appears that the f i r s t i n v e s t i g a t o r to study the action  of a p r o t e o l y t i c enzyme on casein was Lubavin (8) who noted, i n 1871, that g a s t r i c j u i c e did not completely dissolve casein, and that the residue contained a higher proportion of phosphorous than the o r i g i n a l casein.  Later workers observed s i m i l a r r e s u l t s i n the case of pan-  c r e a t i c extracts.  During the period  1871-1927,  many investigators repeated  these experiments, on the whole confirming the o r i g i n a l r e s u l t s . Seve r a l attempts were made t o i s o l a t e products r e s u l t i n g from the digestion of casein by these crude extracts.  Salkowski (9) claimed t o have  i s o l a t e d the uranium s a l t of a phospho-peptone having a nitrogen to phosphorous r a t i o o f 3.1. D i e t r i c h ( 1 0 ) l a t e r i s o l a t e d four s i m i l a r products with N/P r a t i o s o f 1.00, 3.07, 2.59 and 3.87.  Posternak ( I )  prepared the calcium s a l t of a phospho-peptone obtained by the pancreat i c digestion o f casein, followed by p r e c i p i t a t i o n by a lead s a l t . Analysis o f his product indicated an N/P r a t i o o f 5.3*  During t h i s  early period the various products reported showed marked d i s s i m i l a r i t i e s , as i l l u s t r a t e d by the fact that the reported phosphorous contents varied from  0.88$ to 6.8$. I t i s appropriate, perhaps to point out at t h i s juncture that  t h i s d i v e r s i t y o f r e s u l t s may be e n t i r e l y explicable i n view o f the  possible variations i n (a) the o r i g i n a l milk from which each casein was prepared, (b) the methods of p u r i f i c a t i o n o f the caseins, (c) the imp u r i t i e s present i n the enzyme preparations used, (d) the conditions under which hydrolyses were carried out (factors being pH, time, temperature, presence of s a l t s , etc.), and (e) the extent o f b a c t e r i a l a c t i v i t y .  The f i r s t serious attempt to i s o l a t e a product the composition of which d i d not change on exhaustive p u r i f i c a t i o n , and the f i r s t complete amino a c i d determination on such a product were carried out i n 1927  by Rimingtbn  (2),  He subjected casein to t r y p t i c digestion and  p u r i f i e d the r e s u l t i n g phospho-peptone by means of i t s copper  salt.  Following t h i s work, the i s o l a t i o n of the products formed on t r y p t i c digestion of casein has been carried out by numerous i n v e s t i g a t o r s , each one of whom apparently obtained s l i g h t l y d i f f e r e n t r e s u l t s .  Those re-  sults which give information concerning the amino acids present or t h e i r r e l a t i v e proportions i n the various phospho-peptones are set f o r t h i n Table I.  An attempt has been made to arrange the data i n such a manner  that the simplest products (having only a few amino acids and a low N/P r a t i o ) are followed by the apparently more complex.  The purpose of  t h i s i s to lend support to the suggestion that the various products l i s t e d represent progressive stages i n the hydrolysis o f casein.  A  possible reason why t h i s theory i s not e n t i r e l y supported by the evidence for the products do not present a continuous s e r i e s - may be that the caseins employed by these workers were not, i n a l l p r o b a b i l i t y , prepared by a uniform method. no means i d e n t i c a l .  The s t a r t i n g materials i n each case were thus by  TABLE I  THE  COMPOSITION OF VARIOUS PHOSPHO-PEPTONES ISOLATED  FROM AMONG THE ENZYMIC HYDROLYSIS PRODUCTS OF CASEIN  Investigator  Amino acids & Phosphoric acid reported i n peptone (proporEnzymes tions indicated where known) used Gluta- Ser- Iso- Aspar- Phosin phorhydrolysis mic ine l e u - t i c ic cine acid acid acid  Posternak & Pollaczek(l6) trypsin  Total ho. of amino acid residues  N/P ratio  P  P  P  7  1  2  3  I  2  7  3.5  II  2  3  3  P  8  ?  II  5 5  4 4  I  3 3  9 10  3*0 3.3  3  4  3  3  10  3.3  Posternak & trypsin Pollaczec(l6)  P  P  P  P  P  Posternak (I)  3 ?  7  1  1  4 ?  ?  4 ? ?  Nicollet & Shinn ( 1 7 ) * "  ti  Lowndes et a l . (18) Rimington (19) Damodaran & Ramachandran ( 4 )  *  ^P  it  trypsin & pepsin  II  •  1  ?  10 or 11 15 16 18  •  3.8 1  ?  These workers also reported the presence of one additional amino acid of undetermined constitution. Indicates that t h i s amino a c i d was reported present but that i t s proportion was not determined.  6  I t w i l l be noted that a f t e r Rimington (2) the workers mentioned thus f a r , with the exception o f Damodaran and Ramachandran (4), used t r y p s i n e x c l u s i v e l y .  The study of the peptic hydrolysis o f casein  has received comparatively l i t t l e attention, quantitative r e s u l t s being meagre.  Holter et a l . ( l l ) found that on prolonged peptic digestion  of casein, a "paranuclein" o r phospho-peptone remained undigested and possessed an N/P r a t i o o f 7.1.  S t i r l i n g and Wishart (12) also observed  the formation o f products i n s o l u b l e i n t r i c h l o r o a c e t i c acid by the action of both pepsin and t r y p s i n .  In the former case, starting with casein  of N/P r a t i o 20, they found an acid-insoluble product o f N/P r a t i o 12,8.  Jones and Gersdorff (13) also i s o l a t e d from among the products  of peptic hydrolysis o f casein, two paranucleins having N/P r a t i o s o f  13.2 and 13.8  ( o r i g i n a l casein was  35.3).  Both these products con-  tained l y s i n e and tryptophane but no cystine.  Linderstrom-Lang (14)  supported t h i s finding i n part by h i s observation that those f r a c t i o n s of the peptic hydrolysate containing the most phosphorous, also contained the most tryptophane, tyrosine, h i s t i d i n e and arginine. (15) observed the formation o f a  Utkin  substance of N/P r a t i o 7 during the  f i r s t stages o f peptic hydrolysis of casein, and l a t e r i n the hydrolys i s , of a substance of N/P 3.5.  Miss Herd (20) found evidence f o r  the presence o f at l e a s t two components i n a paranuclein which she prepared;  these components d i f f e r e d i n s o l u b i l i t y , amino acid com-  position and iodine-binding capacity.  Winnick (21) studied the pro-  ducts formed by the action on casein of pepsin, t r y p s i n , and other enzymes, and found, on the average, that the non-protein molecules contained 5 t o 7 amino acid residues and possessed a molecular weight of  450  to 600.  Bo  The f r a c t i o n a t i o n o f casein; The early workers who  studied the enzymic degradation products  of casein t a c i t l y assumed that the l a t t e r could be t r e a t e d as a single chemical e n t i t y .  Among the f i r s t experimenters to attempt a resolution  of casein i n t o f r a c t i o n s were Linderstrom-Lang and Kodama (22)  who  stu-  died the s o l u b i l i t y of t h i s substance i n hydrochloric acid, and d i s covered that i t was  separable into two components, one more soluble  and the other l e s s soluble than the o r i g i n a l casein. was,  Neither component  however, claimed by them to be homogeneous because the  solubility  of each f r a c t i o n varied with the quantity of p r e c i p i t a t e present. Differences i n phosphorous and nitrogen contents were detected between the two f r a c t i o n s .  Several years l a t e r , Linderstrom-Lang (14) c a r r i e d out f u r ther f r a c t i o n a t i o n experiments and concluded that i n native casein there are several kinds of c o l l o i d a l molecules which i n t e r a c t , forming a co-precipitating system, and hence are not r e a d i l y separable  or  distinguishable.  He was  able to obtain three constituents, character-  ized as follows:  (a) basic, alcohol-soluble and phosphorous-free,  (b) combination o f (a) with one containing phosphorous and soluble i n the presence of calcium s a l t s , (c) containing more phosphorous than (b) and which i s p r e c i p i t a t e d by calcium  salts.  The molecular weight of casein, as prepared by Hammarsten, was  studied by Svedberg, Carpenter and Carpenter (5)  c e n t r i f u g a l sedimentation v e l o c i t y method, and was of a mixture of molecules of d i f f e r e n t weights.  by means of the found to consist  Serologic studies on the proteins found i n casein were c a r r i e d out by Carpenter and Hucker ( 2 3 ) . By extracting casein with 7 0 $ a c i d i f i e d alcohol and f r a c t i o n a t i n g with potassium oxalate, they i s o l a t e d three constituents. weights of 9 8 , 0 0 0 ,  188,000  These were found to have molecular  and 3 7 5 , 0 0 0 ,  and were reported to be c l e a r l y  distinguishable by serologic reactions.  After several preliminary papers by Cherbuliez and h i s coworkers ( 2 4 , 2 5 ) on the s o l u b i l i t y o f casein i n various s a l t  solutions  and organic solvents, there appeared a paper by Cherbuliez and Jeannerat ( 2 6 ) giving a detailed procedure f o r the f r a c t i o n a t i o n of casein by means of ammonium chloride and acetone.  The y i e l d s of the three  f i n a l fractions were reported as: alpha (II)  32.8$,  gamma  48.7$  and  delta 3 . 5 $ . Each of these was claimed t o be non-resolvable i n t o either of the others, but was not further characterized.  These workers  state that t h e i r r e s u l t s were not influenced by the presence o f calcium ions, a finding which appears t o be completely a t variance with that o f Linderstrom-Lang ( 1 4 ) , mentioned e a r l i e r .  Groh et a l . ( 2 7 ) also reported the f r a c t i o n a t i o n o f casein by three d i f f e r e n t methods: _  (a) by the addition of'absolute  ethyl alcohol to  solutions of casein i n 40$ urea, (b)  by the addition o f absolute ethyl alcohol to casein dissolved i n anhydrous molten phenol at  (c)  70°  C.  by the addition o f d i l u t e hydrochloric  acid  to a solution of casein i n d i l u t e ammonia containing 70$ e t h y l a l c o h o l . Two  fractions were thus obtained i n equal proportions, containing  and 3.9$ tyrosine, and 1.6 and 1.1$ tryptophane respectively.  7.8  It  was  maintained that these fractions were not the products of hydrolysis or denaturation  since casein with i t s o r i g i n a l properties was  i n each case by complete p r e c i p i t a t i o n or by mixing the two  obtained separated  fractions. Another method of f r a c t i o n a t i o n was attempted by McKee and Gould (28) who  determined the s o l u b i l i t y of casein i n sodium cymene  sulphonate, potassium thiocyanate and sodium benzene sulphonate at various temperatures at a pH of 4.6. as'a'rule, 'directly with temperature. f r a c t i o n s was  S o l u b i l i t y was found to increase, Separation of casein i n t o two  effected by the f i r s t and t h i r d of the above reagents,  but the second was  found to be i n e f f e c t i v e .  to d i f f e r i n the following respects: formaldehyde absorbing  (a)  The fractions were found phosphorous content,  (b)  capacity ( i n d i c a t i n g unlike numbers of amino  groups), and (c) s o l u b i l i t y i n pyridine ( i n d i c a t i n g differences i n a c i d i c groups).  In the f i r s t electrophoretic studies on casein, Mellander (29)  found that i t consisted of three sharply separable f r a c t i o n s , the  f a s t e s t moving one of which contained a greater proportion of phosphorous than the o r i g i n a l casein, and the slowest-moving one l e s s . ing  Follow-  t h i s up, Warner ( 7 ) , guided by electrophoretic patterns, developed  a procedure by which he was able to obtain "alpha" and "beta" f r a c t i o n s  10  (containing 0 . 9 9 and 0 , 6 1 $ phosphorous respectively) whose e l e c t r o phoretic peaks corresponded to those o f the o r i g i n a l casein. bination o f the f r a c t i o n s i n the proper proportions, was restored.  By recom-  the o r i g i n a l pattern  I t was claimed that each i s a d i s t i n c t f r a c t i o n , although  not e l e c t r o p h o r e t i c a l l y homogeneous at a l l pH values, and free of a l l traces o f the other.  The method employed i s based on p r e c i p i t a t i o n at  c a r e f u l l y controlled pH by acetone, very d i l u t e a c i d and a l k a l i a t 2°C. and at room temperature.  Warner's fractions have recently been  examined with regard to amino a c i d composition by Gordon et a l . ( 3 0 ) . The most s i g n i f i c a n t differences are shown i n the following t a b l e :  TABLE I I SIGNIFICANT DIFFERENCES IN AMINO ACID COMPOSITION BETWEEN THE CASEBf FRACTIONS OBTAINED BY WARNER ( 7 )  Amino a c i d  % i n alphacasein  % i n betacasein  Alanine  3.7  1.7  Valine  6.3  10.2  Leucine  7.9  11.6  Proline  8.2  16.0  Cystine  0.43  0 . 0 to 0 . 1  Tryptophane  1.6  0*65  Aspartic a c i d  8.4  4.9  Tyrosine  8.1  3.2  11  The above data concerning tryptophane and tyrosine may be compared with the corresponding findings by Groh et a l . (27), mentioned previously.  The values obtained by them are s i m i l a r enough to suggest  that they may have obtained fractions of approximately the same cons t i t u t i o n as those of Warner.  From the foregoing b r i e f h i s t o r i c a l account, i t i s clear that, to the present time, investigators concerned with the enzymic hydrolysis of casein have completely overlooked the findings of those concerned with the f r a c t i o n a t i o n of t h i s substance. sistency i n the r e s u l t s of the former group may  The lack of con-  thus be due to the  f a c t that casein i s , i n r e a l i t y , a mixture of very s i m i l a r proteins which, i n spite of t h e i r s u p e r f i c i a l s i m i l a r i t y , may not y i e l d products of i d e n t i c a l composition when hydrolysed by pepsin or t r y p s i n . To c l a r i f y t h i s s i t u a t i o n , i t i s c l e a r l y necessary to study the enzymic hydrolysis of each component of casein i n d i v i d u a l l y .  The question  which immediately arose was to decide which group of fractions reported i n the l i t e r a t u r e most c l o s e l y represented single protein constituents. Two groups, that of Cherbuliez and Jeannerat (26) and of Warner ( 7 ) , were selected. The former was considered more r e l i a b l e than most others for a number of reasons:  (a) the conditions o f f r a c t i o n a t i o n were  r e l a t i v e l y mild, the reagent being d i l u t e ammonium chloride and acetonej consequently the p o s s i b i l i t y of appreciable denaturation was considered negligible,  (b) there had been extensive preliminary studies of the  s o l u b i l i t y of casein i n various reagents, and (c) each f r a c t i o n re-fractionated repeatedly u n t i l free of the others.  The  was  procedure  12  of Warner ( 7 ) was also selected f o r s i m i l a r reasons, v i z . : (a) the use of acetone, d i l u t e a c i d and a l k a l i was not considered t o cause denaturation, was  and (b) the extremely s e n s i t i v e electrophoretic procedure  employed as a guide f o r the progress of f r a c t i o n a t i o n and as a  check on the p u r i t y of the f i n a l products.  In the present work, the procedure of Cherbuliez and Jeannerat (26) was  employed, but i t was  found that the phosphorous contents of  the f r a c t i o n s obtained bore no close r e l a t i o n s h i p to those of Warner's fractions.  The l a t t e r appeared, on the basis of electrophoretic studies,  to be the truest a v a i l a b l e representation o f the i n d i v i d u a l protein constituents of casein. peptic hydrolysis.  Alpha-casein was prepared and subjected  to  From the products thus formed, were obtained two  peptones, one of which appeared to contain the amino acids mentioned i n table I .  In addition there were present other amino acids, some of  which were reported i n the products of Jones and Gersdorff (13) Linderstrom-Lang (14).  The N/P  and  r a t i o s of the products obtained from  alpha-casein were such as to suggest a simpler structure than that described by Jones and  Gersdorff.  EXPERIMENTAL  A.  Preparation of casein from milk; In an investigation o f the constitution of a protein, an  obvious pre-requisite f o r v a l i d results i s that the protein be i n the "native" state, that i s t o say, unchanged i n physical or chemical properties from what i t i s as found naturally occurring.  Since i t i s  next to impossible to study the chemical constitution of such a substance as a protein without a l t e r i n g i t i n some way, closely approached only i n the s t a r t i n g m a t e r i a l .  t h i s i d e a l can be  In selecting a  possible source o f starting m a t e r i a l f o r f r a c t i o n a t i o n of casein, the commercial preparations were rejected because the extent of denaturat i o n and the presence of impurities were uncertain.  Of the methods  of p u r i f i c a t i o n reported i n the l i t e r a t u r e , that of Warner ( 7 ) seems to minimize denaturation and to r e s u l t i n a more nearly pure product than was obtainable by the other methods.  I t was p a r t i c u l a r l y appro-  p r i a t e to employ t h i s method because Warner's fractionation procedure was made use o f i n the present work.  The following, operations,  e s s e n t i a l l y those of the above mentioned investigator, were c a r r i e d out.  Five l i t e r s of cow's milk, c o l l e c t e d at the time o f milking, were immediately cooled t o 40°F. and placed i n an i c e bath.  After  centrifuging, the skim milk was siphoned o f f and kept at 0 - 5°C., a l i t t l e toluene being added to i n h i b i t b a c t e r i a l a c t i v i t y .  The pH  was  a d j u s t e d t o 4.6  by t h e a d d i t i o n o f 0.1  p r e c i p i t a t e which formed was  0.1  N) was  and b r i n g t h e s o l u t i o n t o pH 6.5.  S u f f i c i e n t sodium h y d r o x i d e  added t o d i s s o l v e t h e p r e c i p i t a t e S i n c e o p e r a t i o n s had  i l y d i s c o n t i n u e d a t t h i s p o i n t , t h e s o l u t i o n was time.  The  s o l i d mass was  t h e n thawed out and f a t was The  extracted from i t extracted  d i l u t e d t o a p r o t e i n c o n c e n t r a t i o n o f a p p r o x i m a t e l y 0.5%,  t e d on t h e a s s u m p t i o n t h a t t h e o r i g i n a l m i l k c o n t a i n e d H y d r o c h l o r i c a c i d was  added t o b r i n g t h e pH t o 4.6,  b e i n g made w i t h a s m a l l amount o f 0.01 The  t o be t e m p o r a r -  f r o z e n s o l i d f o r some  by s h a k i n g w i t h e t h e r , f o l l o w e d by c e n t r i f u g i n g . was  p r e c i p i t a t e was  s u p e r n a t a n t was  3%  etherj  liquid  calcula-  casein.  f i n a l adjustment  N sodium h y d r o x i d e s o l u t i o n .  allowed to s e t t l e i n the c o l d , a f t e r which the  siphoned o f f .  The p r e c i p i t a t e was  washed t h r e e t i m e s  w i t h an e q u a l volume o f i c e - w a t e r , and f i n a l l y c e n t r i f u g e d . o f t h e wet  The  s e p a r a t e d by c e n t r i f u g i n g and washed s i x  t i m e s w i t h an e q u a l volume o f i c e w a t e r . s o l u t i o n (approximately  N hydrochloric acid.  c a s e i n was  d r i e d t o constant  f r o m t h i s i t was  An a l i q u o t  w e i g h t by means o f a l c o h o l  c a l c u l a t e d t h a t a p p r o x i m a t e l y 170 gm.  of  c a s e i n (on a d r y b a s i s ) had been o b t a i n e d .  The wet  s u b j e c t e d t o f r a c t i o n a t i o n (see p a r t B . I ) .  A f u r t h e r 5 1. o f raw  was  worked up as above ( e x c e p t  t h e wet  B.  c a s e i n was  F r a c t i o n a t i o n of the I.  p r o d u c t was  milk and  B.2).  casein:  U s i n g 5$ ammonium c h l o r i d e and  acetone:  T h i s p r o c e d u r e i s t h a t o f C h e r b u l i e z and J e a n n e r a t who  then  f o r f r e e z i n g the s o l u t i o n s o l i d ) ,  a l s o subjected t o f r a c t i o n a t i o n (see p a r t  and  (26)  had d e v e l o p e d t h e i r method a f t e r e x t e n s i v e p r e l i m i n a r y i n v e s t i g a -  t i o n s , during the, course of which they discovered that casein i s more soluble i n ammonium chloride s o l u t i o n than i n a number o f other s a l t solutions, and that a 5% concentration o f t h i s s a l t was optimum. The casein, according to t h e i r method, was brought i n t o solution by means of 5$ ammonium chloride and 0 . 1 N sodium hydroxide solutions, a f t e r which the f i r s t f r a c t i o n was obtained by the a d d i t i o n o f 0 . 1 N hydrochloric acid.  Upon adding more o f the acid, a second f r a c t i o n was  precipitatedj  from the supernatant two other f r a c t i o n s were obtained  by the a d d i t i o n o f acetone.  Each f r a c t i o n was repeatedly r e - f r a c t i o n -  ated i n the same manner, with the result that the second p r e c i p i t a t e mentioned above was resolved i n t o the others, thus leaving three f i n a l products, each o f which could not be resolved i n t o the others. d e t a i l e d account o f these operations i s given i n Figure I .  A more  FIGURE I SCHEME FOR THE FRACTIONATION OF CASEIN (ACCORDING TO CHERBULIEZ AND JEANNERAT (26) ) PURIFIED CASEIN I (suspend. 1 5 gm. i n 1 1 4 0 ml. o f 5 $ NH.C1  plus 90 ml. of 2 0 $ NH.C1, then add 1 4 0 ml. o f 0 . 1 N NaOH) [CASEIN SOLUTION (at pH 6.5)1 (add 140 ml. o f 0.1 N HC1 and bring t o pH 4.8, then 5 ml. more to bring t o pH 4.6; centrifuge & wash ppt, with water) IMPURE ALPHA FRACTION. (kept under acetone)  SUPERNATANT CONTAINING! OTHER FRACTIONS  MPHA ( I I ) FRACTION .  SUPERNATANT CONTAINING JAMMA & D E L T A FRACTIONS!  (add 1/5 volume of acetone; centrifuge & wash ppt.) 16.7$ acetone  (add 0.1 N HC1 u n t i l pH 3.8 reached) IMPURE GAMMA FRACTION  SUPERNATANT CONTAINING! DELTA FRACTION (add 0.1 N NaOH t o pH 5.3; add one volume of acetone & l e t stand overnight; add one volume more)  IMPURE DELTA! FRACTION  NOTE:  DISCARDED LIQUID FROM WHICH ACETONE IS RECOVERED  Each f r a c t i o n obtained as above i s subjected to t h i s f r a c t i o n a t i o n repeatedly.  72.5$ acetone  In the present work, the f r a c t i o n a t i o n proceeded as expected at f i r s t , the alpha ( I I ) f r a c t i o n i n i t i a l l y obtained being gradually resolved i n t o alpha (I) and gamma f r a c t i o n s .  On attempting t o further  p u r i f y the gamma f r a c t i o n , however, about f o u r - f i f t h s o f the l a t t e r p r e c i p i t a t e d under conditions o f alpha ( I I ) p r e c i p i t a t i o n .  This new  alpha ( I I ) f r a c t i o n remained, despite repeated attempts t o resolve i t , only very small amounts o f alpha ( I ) and gamma f r a c t i o n s being obtainable from i t .  These operations were performed at room temperature but  products were stored i n the cold.  The four fractions were dried by  washing with alcohol and ether, followed by evaporation i n a vacuum dessicator.  2.  Using acetone, d i l u t e acid and base i n the cold:  The method used here i s that o f Warner (7), and based upon the i s o e l e c t r i c p r e c i p i t a t i o n a t a given pH a t 2°C. o f the f i r s t f r a c t i o n from a very d i l u t e a c i d solution of casein, followed by the p r e c i p i t a t i o n of the second f r a c t i o n a t a s l i g h t l y higher pH a t room temperature.  The novelty of t h i s procedure l i e s i n p r e c i p i t a t i o n by  approaching the i s o e l e c t r i c point from the a c i d side.  Since i t i s  extremely d i f f i c u l t to d i s s o l v e casein i n a c i d , the procedure f o r preparing a solution o f t h i s substance at pH 3.5 deserves p a r t i c u l a r attention.  Further d e t a i l s are given i n figure I I .  Only that part of the  procedure concerning alpha casein i s given because the present work deals with the peptic hydrolysis o f t h i s f r a c t i o n only.  The casein  from 5 l i t e r s of raw milk y i e l d e d about 50 gm. (dry weight) o f alpha casein.  FIGURE I I SCHEME FOR THE FRACTIONATION OF CASEIN (ACCORDING TO WARNER ( 7 ) ) |PURIFIED CASEINI  1.  (dissolve i n IOO volumes of water containing 0.65 m. equ. of NaOH per gm. q f casein to give a 1% solution a t pH 6.5)  ICASEIN SOLUTION] 2.  ( s t i r vigorously with motor s t i r r e r & add 0.05 N HCI (0.85 m.equ. per gm. casein) as r a p i d l y as possible to give solution of pH 3.5)  ICASEIN SOLUTION)  3.  ( c h i l l t o 2°C. and add ice-water to give a 0.2 to 0,3% protein solution)  DILUTE CASEIN SOLUTION!  4.  ( s t i r with motor s t i r r e r and add 0.01 N NaOH dropwise u n t i l pH 4.2 reached and ppt. forms; continue addition u n t i l c l e a r supernatant obtained on centrifuging (pH 4.4 to 4.5)  PRECIPITATE OF IMPURE ALPHA CASEIN 1st. three or four f i l t r a t e s  MOST OF THE BETA CASEIN  (centrifuge o f f ppt. and subject i t to procedures 1,2,3 and 4 i n order f i v e times t o remove a l l beta casein)  ALPHA CASEIN PRECIPITATE (dissolve i n NaOH to give a 0,2% solution; chill to 2° C. and add HCI t o give pH 4«5j wash ppt. thoroughly with water) PURE ALPHA  3ASEIN FOR HYDROLYSIS  C.  Examination of the casein fractions; 1.  Electrophoresis:  The alpha casein and the raw casein  from which i t was derived were each examined e l e c t r o p h o r e t i c a l l y i n order to determine whether the f r a c t i o n obtained was i d e n t i c a l to that described by Warner ( 7 ) . ed by Moore and White ( 3 1 ) ,  The apparatus employed was that develop-  and i s a modification of t h e - o r i g i n a l  T i s e l i u s apparatus incorporating the Toepler schlieren scanning improvements made by Longsworth ( 3 2 ) . tus,  (The manufacturers o f t h i s appara-  the Perkin-Elmer Corporation, Glenbrook, Conn., U. S. A., provide  a manual of comprehensive i n s t r u c t i o n s f o r operation).  The alpha  casein showed only a s i n g l e , very sharp boundary, whereas the o r i g i n a l casein showed two rounded'but d i s t i n c t peaks. carried out i n phosphate buffer at pH 7.7.  This examination was  The r e s u l t s obtained,  although q u a l i t a t i v e , were judged to be e s s e n t i a l l y i d e n t i c a l with those of Warner.  Drawings of the photographic patterns obtained are  shown i n figures I l i a and  Illb.  FIGURE I I I DRAWINGS OF PHOTOGRAPBIC PATTERNS OBTAINED BY THE ELECTROPHORESIS OF NATIVE BOVINE CASEIN (a) AND ALPHA CASEIN (b) DERIVED FROM IT BY THE METHOD OF WARNER . ( 7 )  a  2  b  Phosphorous determinations:  The various f r a c t i o n s of casein obtained by the two procedures were analysed f o r phosphorous content by means o f modified versions o f the Fiske - Subbarow colourimetric method (45)- The alpha ( I ) , alpha ( I I ) , gamma and delta f r a c t i o n s were treated by the method of King (33) who used I-amino-2-naphthol-4-sulphonic  a c i d as  the reducing agent, whereas the alpha casein was treated according to the method of A l l e n (34) who used 2,4-diaminophenol.  The l a t t e r  reagent, i t i s claimed, gives more consistent results than other reducing reagents, but i t was not a v a i l a b l e at the time the alpha ( I ) , alpha ( I I ) , gamma and d e l t a fractions were analysed. shown i n tables I I I and IV.  The results are  TABLE I I I PHOSPHOROUS CONTENT OF FRACTIONS AT PROGRESSIVE STAGES OF THE FRACTIONATION OF CASEIN BY THE METHOD OF CHERBULLEZ AND JEANNERAT ( 2 6 ) % Phosphorous i n : Alpha ( I ) fraction  Alpha ( I I ) Gamma fraction fraction  0.784  0.763  0.691  0.764  0.680  0.750  0.761  0.674  0.753  0.814  0.733 Original/casein:  Delta fraction 0.728  0.741$ phosphorous  The figures i n each column of the above Table correspond to samples taken a t successive stages of p u r i f i c a t i o n of each f r a c t i o n . TABLE IV PHOSPHOROUS CONTENT OF CASEIN AND ALPHA CASEIN PREPARED BY THE METHOD OF WARNER ( 7 )  Substance  % P Reported  % P Found  Original casein  0.86  0.83  Alpha casein  0.99  0.99  From the r e s u l t s o f table I I I i t i s apparent that there i s , i n each f r a c t i o n , a v a r i a t i o n o f phosphorous content, but that i t i s only i n the case of the gamma f r a c t i o n that t h i s v a r i a t i o n i s i n  one d i r e c t i o n a l o n e .  None o f t h e f r a c t i o n s f i n a l l y o b t a i n e d  possessed  a phosphorous c o n t e n t s u f f i c i e n t l y s i m i l a r t o e i t h e r o f Warner's f r a c t i o n s t o suggest t h e i r i d e n t i t y w i t h the The a s s u m p t i o n t h a t the a l p h a  latter.  casein obtained  i n the p r e s e n t  work i s i d e n t i c a l w i t h t h a t d e s c r i b e d by Warner, i s s u p p o r t e d s t r o n g l y by t h e r e s u l t s shown i n t a b l e  IV.  Since Warner's.fractions  and t h e c a s e i n f r o m w h i c h t h e y  were d e r i v e d showed o n l y m i n o r d i f f e r e n c e s i n n i t r o g e n c o n t e n t , l a t t e r was  not.considered  o f v a l u e , i n t h e p r e s e n t work, f o r p u r p o s e s  o f c h a r a c t e r i z a t i o n and c o m p a r i s o n , and hence was  D.  the  not  determined.  Peptic hydrolysis of casein: 1.  P r e l i m i n a r y experiments w i t h commercially prepared  casein: P r e l i m i n a r y e x p e r i m e n t s u s i n g p e p s i n and c a s e i n were c a r r i e d out f o r s e v e r a l r e a s o n s :  ( a ) t o t e s t and p r a c t i s e t h e f o r m o l  titra-  t i o n t e c h n i q u e as a p p l i e d t o c a s e i n h y d r o l y s i s , (b) t o f o l l o w t h e c o u r s e o f p e p t i c h y d r o l y s i s o f c a s e i n under t h e c o n d i t i o n s used by Damodaran and Ramachandran (4), w i t h a view t o reducing  (c) t o modify the c o n d i t i o n s i n  (b)  f o r t h e sake o f c o n v e n i e n c e , t h e t i m e r e q u i r e d  t o r e a c h t h e p o i n t where t h e l i b e r a t i o n o f t i t r a t a b l e c a r b o x y l groups p r o c e e d e d a t a v e r y s l o w r a t e compared w i t h t h e i n i t i a l r a t e , and t o d e t e r m i n e whether, under t h e s e new  conditions, appreciable  hydroly-  s i s o f t h e c a s e i n c o u l d be a t t r i b u t e d t o f a c t o r s o t h e r t h a n t h e presence o f  pepsin.  (d)  The method of formol t i t r a t i o n adopted was found to be a quick, convenient and consistent one f o r following the course of hydrolysis.  The rate o f hydrolysis i n the f i r s t run ( f i g u r e I I I ) was con-  sidered too slow f o r p r a c t i c a l purposes, and gave time, i t was thought, for b a c t e r i a l action t o begin despite the presence o f toluene. conditions f i n a l l y adopted were the following:  a  The  substrate/enzyme  r a t i o o f 4.0, an enzyme concentration of 8.3 gm./liter of approximately 0. I N sulphuric a c i d , a pH of 1.0 and a temperature of 50 - 52°C, These conditions were such that the rate o f hydrolysis was reduced to l / 2 0 t h the i n i t i a l rate a f t e r about two hours.  The removal of the  products o f digestion by washing the residue, reduced the rate o f pept i d e re-synthesis, i t was thought, and allowed the second digestion to proceed r e l a t i v e l y uninhibited by these reverse reactions.  Twenty grams o f dry, finely-powdered, commercially-prepared casein ( N u t r i t i o n a l Biochemicals Corporation) were s t i r r e d f o r an hour with approximately 1 l i t e r of 0.05 N sodium hydroxide s o l u t i o n .  Un-  dissolved p a r t i c l e s , including impurities, were removed by f i l t e r i n g through glass wool.  The f i l t r a t e was brought to pH 4.6 by the gradual  addition of 0.05 N sulphuric a c i d , and the f l o c c u l e n t , white p r e c i p i tate was recovered by c e n t r i f u g i n g .  The p r e c i p i t a t e was then suspended  i n 300 ml. of 0.1 N sulphuric a c i d , and the mixture was brought to pH 1.8 by the addition of 5 N sulphuric a c i d .  Half a gram o f pepsin  (Difco 1:10,000) was dissolved i n 2 ml. o f 0. I N added t o the casein suspension. 40 hours at 35 - 39°C.  sulphuric a c i d and  The mixture was then incubated f o r  Due to unequal rate o f l i b e r a t i o n of amino and  carboxyl groups during hydrolysis, the pH rose to 2.4, and was reduced  to 1.6  by the addition of 5 N sulphuric a c i d .  pepsin (dissolved i n 2 ml. of 0.1 cubation was  continued as before.  A further 0.5  gram of  N sulphuric acid) was added and i n The progress of enzymic hydrolysis  was followed by formal t i t r a t i o n s carried out under conditions which were considered to be i n accordance with the recommendations of Levy,  (35).  The procedure employed was as follows:  At i n t e r -  vals, 10 ml. samples of the incubating mixture were removed and fuged.  centri-  Two ml. aliquots of each sample were placed i n small beakers  and t i t r a t e d to the f i r s t f a i n t pink of phenolphthalein with 0.02 sodium hydroxide solution. approximately  The volume o f base was noted, and  N  then  5 ml. of f i l t e r e d formalin s o l u t i o n , which had been  previously adjusted to pH 7.0, to the second end point.  were added.  T i t r a t i o n was  continued  The volume of formalin added i n each t i t r a -  t i o n was c a r e f u l l y adjusted so that the f i n a l concentration of formaldehyde was 6 to 9$.  The r e s u l t s , plotted g r a p h i c a l l y i n Figure I I I ,  indicate a slowly progressing hydrolysis, the rate of which i s almost constant.  I t was thought that the time required for hydrolysis to reach  completion at t h i s rate would have been too great f o r p r a c t i c a l purposes.  This i s supported by the work of Damodaran and Ramachandran  (4) who allowed the hydrolysis of casein to continue f o r a week under conditions very similar to those mentioned above, before the amino nitrogen content of the hydrolysate ceased to increase.  Hydrolysis was  repeated with f u r t h e r 20 gm. l o t s of casein,  but t h i s time the temperature was raised to  50 - 53°<>.,  the pepsin  concentration was increased by a factor of ten ( i . e . to 5.0 300 ml. of 0.1 N sulphuric a c i d ) , and the mixture was t i n u a l l y by a motor stirrer,,  gm.  per  s t i r r e d con-  The object i n making these changes i n  conditions was to increase the rate of hydrolysis to a more convenient level, and to reduce the time during which bacterial action might begin.  The results are plotted graphically i n figure IV, and show  that, under these new conditions, hydrolysis of casein proceeds very much more rapidly than under the conditions used i n the f i r s t hydrolysis (Figure III). During the f i r s t hour, the rate of hydrolysis was 20 times that during the following 70 hours. The above mentioned hydrolyses were carried out i n beakers covered by watch glasses.  It was thought that the increase i n acidity  might, i n part at least, be due to a slow evaporation of water and concentration of the acid. The hydrolyses were therefore repeated as i n the second run, except that a glass-stoppered was used to prevent evaporation of water, the pH was made 1.0, and occasional swirling of the flask was substituted for continual stirring.  Formol titration  results are plotted i n Figure Va., and show that, using a closed vessel for hydrolysis, the results obtained are very similar to those obtained from a beaker covered with a watch glass. There was found, as before, a rapid, i n i t i a l rise i n titratable carboxyl groups, f o l lowed by a relatively slow r i s e .  Since the hydrolysis flask was  stoppered, the slower rise cannot be attributed to concentration of the acid by evaporation of the water.  The residue remaining after  this hydrolysis lasting approximately 17 hours, was separated by centrifuging and washed with 0.1 N sulphuric acid.  It was then sub-  jected to further hydrolysis under the same conditions as was the casein from which i t was derived.  The results obtained, plotted i n  Figure'Vb., show that this residue underwent further hydrolysis, but  26  20 1  LO  0  C  3<3 i  J±  mn  H s  •  ?1  23  y  1  Vi  An  AD 1r  I  0 J.  ilJ  IV  L ".  j  1  , r, H — — — — 1 — — — — —— — \ A  OF  r 22  m O  T3  JJ.  TEMPS  )1  1.6 •  2 1T>' c p E P D i  OF 31 CUBA1  _  n  15 CA •  •1  50  /]  J  n  v- 0 * 0  N  O  21  g §H  s  e O  CNi O O <H  •  O  /  / 19  18  10  20  "^ime i n hours  50  60  70  27  FIGURE PEP  V  HYDROLYSIS OF COMMERCIAL  1  CASEIN  i n glass-stoppered flask to prevent evaporation)  nv- MM  15  WEIGHT ASEIN: 20 gm. IT OF OF CCASE; CONCENTRATION O F P E P S I N : 15 gm./l. TEMPERATURE OF INCUBATION: 50 - 53 C.  fill  [lid j  (curve a.: f i r s t hydrolysis curv.e b : second hydrolysis; washed residue)  13  e  11  13  TEMPERATURE OF INCUBATION: 50 CONCENTRATION O F A C I D : O.I VOLUME OF A C I D : 600 ml.  12  11  0  20  40  S 36 G R A P H 6 & E R . SMI-  time i n hours  80  S O N  a WRI  100  51°C  EPTIC  HYDROLYSIS  OF  ALPHA  CASEIN  WEIC EIGHT OF ALPHA O i o M U ImjAi CONCENTRATION OF PEPSIN: 8.3 gm./l. TEMPERATURE OF INCUBATION: 50 - 52°C pH: 1.0 curve a.: hydrolysis of alpha casein cruve b.: hydrolysis of residue from a.  T  time i n hours  T7T  TZT  —  29  to a much l e s s e r extent and a t a slower rate than the o r i g i n a l casein.  A control experiment was run under the same conditions as above (Figure V), omitting the pepsin, to determine the rate o f hydrolysis by a c i d alone.  Twenty grams o f casein, a f t e r r e - p r e c i p i t a t i o n as  before, were suspended i n 600 ml. of 0. I N sulphuric a c i d and incubated a t 50 - 51°C. Changes i n formol t i t r e were followed as before (see Figure VI).  From the graph i t i s apparent that casein i s hydro-  lyzed s l i g h t l y by d i l u t e sulphuric a c i d alone under these conditions, but that the rate i s only one tenth as great as when pepsin i s also present, as shown by a comparison of Figures V and VI. The progress of hydrolysis by d i l u t e a c i d appears, however, t o come to a v i r t u a l stop a f t e r about 18 hours, possibly due to a r e l a t i v e l y l i m i t e d number of peptide bonds susceptible to hydrolysis under these conditions.  2.  Hydrolysis of alpha casein:  Two hundred grams of wet alpha casein (40 gm. dry weight) were suspended i n 1200 ml. of 0.1 N sulphuric acid a t 50°C. and adjusted to pH 1.0 with 5 N sulphuric a c i d .  Ten grams o f pepsin, d i s -  solved i n 0.1 N sulphuric a c i d , were added with s t i r r i n g , and two equal portions of the mixture were placed i n I I . Erlenmeyer, glass, stoppered f l a s k s .  Incubation was carried out at 50 - 52°C. with  occasional shaking f o r 23 hours. i n t e r v a l s as before.  Formol t i t r a t i o n s were performed at  The r e s u l t s are plotted i n Figure V i l a .  The  residue which remained was recovered by centrifuging and was washed s i x times with 200 ml. portions of d i s t i l l e d water, u n t i l the supernatant was free of sulphate i o n (no p r e c i p i t a t e with barium c h l o r i d e ) .  30  The wet p r e c i p i t a t e was then suspended i n 240 ml, of 0.1 N sulphuric a c i d , the pH was adjusted to 1.0 with 5 N sulphuric acid, and d i g e s t i o n with pepsin continued as before (see Figure V l l b ) .  After 47 hours,  the residue was recovered by centrifuging and washed as described above.  The r e s u l t s shown i n Figure VII  indicate that the hydrolysis  of alpha casein proceeded i n a manner similar to that of commercial casein as previously described. Hydrolysis of the residue appeared to have ceased a f t e r approximately 6 hours.  E. P u r i f i c a t i o n o f the hydrolysis products: The wet residue (from D.2 above) was suspended i n 150 of d i s t i l l e d water.  ml.  Slowly and with s t i r r i n g , I N sodium hydroxide  was added u n t i l the residue had almost completely dissolved and the solution had attained a pH of approximately 8.  Undissolved p a r t i c l e s  were removed by f i l t r a t i o n through glass wool, a f t e r which the f i l t r a t e was brought to a pH of 1 - 2 (as indicated by pH paper) by means of 0.1 N sulphuric a c i d .  The f i n e , l i g h t brown p r e c i p i t a t e which formed  was recovered by centrifuging.  The supernatant was c o l l o i d a l and gave  a voluminous p r e c i p i t a t e with tannic acid solution when a small sample was t e s t e d . To the supernatant were added 4 volumes o f ethanol.  The  white p r e c i p i t a t e which formed was allowed t o s e t t l e overnight i n the r e f r i g e r a t o r and was then recovered by centrifuging. was washed three times with 50 ml. of 50% ethanol.  The p r e c i p i t a t e By washing the  f i r s t (brown) p r e c i p i t a t e with water and adding 4 volumes of ethanol to the washings, a l i t t l e more of the white p r e c i p i t a t e was obtained. Both precipitates were washed 4 times with ethanol, a f t e r which the  l a t t e r was removed by washing twice with ether. - The ether was  evapora-  ted i n a stream of dry a i r , and the p r e c i p i t a t e s placed i n a vacuum dessicator f o r 24 hours.  The brown p r e c i p i t a t e w i l l hereafter be  termed "alpha phosphopeptone I" and the white one "alpha phosphopeptone I I " .  F.  Examination of the peptones; I.  Nitrogen and phosphorous determinations:  At t h i s stage i n the work, i t was o f i n t e r e s t to compare with respect to the N/P r a t i o , the alpha phosphopeptones mentioned above with one another and with the products s i m i l a r l y derived from casein by other workers.  As has been observed by S t i r l i n g and Wishart  (12), the hydrolysis of casein by pepsin r e s u l t s i n the production of two groups of products, one o f gradually increasing and the other of gradually diminishing N/P r a t i o . r a t i o p a r a l l e l s , i n a general way,  That the magnitude of the  N/P  the complexity of the peptone, i s  indicated by the data of Table I.  T o t a l nitrogen was determined by the micro-Kjeldahl method and t o t a l phosphorous by the method of A l l e n (34) • given i n Table V.  The r e s u l t s are  TABLE V NITROGEN AND PHOSPHOROUS CONTENTS OF ALPHA PHOSPHOPEPTONES Alpha Phosphopeptone  I AND I I  % Nitrogen  % Phosphorous  I  11.5  2.8  9.1  11  11.7  3.9  6.6  2.  N/P r a t i o (atomic)  Preparation of the acid hydrolysates o f the peptones:  In preparation f o r the next stage of examination o f peptones by chromatography (see section 3 below), i t was necessary to decompose these substances into t h e i r constituent amino a c i d s .  The procedure  employed i n order to effect t h i s was that o f Townsley ( 3 6 ) , and i s as follows:  Ninety to 100 MGM. o f each peptone were dissolved i n 3 ml.  of 6 N hydrochloric a c i d .  The yellowish solutions were sealed i n  small v i a l s ( 2 by % inches) and placed i n the bottom o f a 4 1. E r l e n meyer f l a s k containing water t o the depth o f an i n c h .  The water was  b o i l e d under a r e f l u x condenser f o r 24 hours. A l l v i a l s then contained a yellowish hydrolysate, but those i n which the brown alpha phosphopeptone I had been placed, contained also a dark sediment, whereas the other v i a l s contained none.  3.  F i l t e r paper p a r t i t i o n chromatography:  In view o f the small y i e l d o f phosphopeptones obtained and the d e s i r a b i l i t y o f gaining some knowledge o f the amino acid c o n s t i t uents o f the substances, i t was decided t o employ the technique o f f i l t e r paper p a r t i t i i o n chromatography.  This ingeneous method of  protein analysis was extensively developed by Consden, Gordon and  33  Martin ( 3 7 ) , and has become an immensely popular tool for several reasons:  (a)  i t i s possible to detect the presence of every amino  acid i n a single experiment, (b) the technique i s truly micro, the quantity of material required for analysis being a fraction of a milligram, (c) the procedure i s exceedingly convenient, the simplest of apparatus being required, and (d) the time necessary for a complete determination i s not more than 48 hours.  The procedures and techniques  employed i n the present work are based largely on a modification of those of Consden et a l . by Williams and Kirby (38) and by Townsley ( 3 9 ) , and are as described below. It was f i r s t of a l l necessary to prepare standard samples of amino acids which, having had practically the same pre-treatment as the peptone hydrolysates, could be validly compared with the latter. Each of nineteen amino acids was dissolved i n water to make a solution containing approximately 2 mgm.  per ml.  Two drops of each solution  were placed i n the depressions of a spot plate, and to each was added an equal volume of concentrated hydrochloric acid.  In other depres-  sions were placed two drops of alpha phosphopeptone II hydrolysate and some of the unhydrolysed peptone (to be checked f o r free amino acids). acid.  To the latter only were added 4 drops of 6 N hydrochloric  The spot plate was then placed i n a vacuum dessicator over  sodium hydroxide pellets u n t i l a l l the liquid had evaporated.  Two  drops of water were added to each depression, and evaporation repeated. This evaporation was repeated twice more, the object being to quantitatively remove a l l free hydrochloric acid.  By this means the  standards, peptone and hydrolysate were prepared for chromatography  under comparable conditions o f hydrochloric acid pre-treatment.  The solvents required were then prepared.  Phenol was d i s -  t i l l e d over zinc dust i n an a l l - g l a s s apparatus i n order t o remove impurities, p a r t i c u l a r l y those giving the pink colour. While  still  l i q u i d , the phenol was thoroughly shaken with a small excess of water, thus producing a f i n e emulsion. cool to room temperature  The l a t t e r was allowed t o  and was then centrifuged. Of the two clear  layers which formed, the bottom one (phenol saturated with water) was c a r e f u l l y separated and kept i n the dark u n t i l used.  Commercial 2,4,  6 - c o l l i d i n e ( l i g h t amber colour) was f r a c t i o n a l l y d i s t i l l e d under vacuum i n an a l l - g l a s s apparatus, the f r a c t i o n passing over a t 55 to 60°C. at a pressure of 10 to 15 mm. of mercury being c o l l e c t e d .  This  colourless c o l l i d i n e was shaken with d i s t i l l e d water at room temperature u n t i l an emulsion formed.  The l a t t e r was centrifuged, and o f the  two layers which formed, the upper one ( c o l l i d i n e saturated with water) was kept f o r chromatography.  A large sheet o f Whatman f i l t e r paper no^I (approximately 18 by 22 inches) was selected and a p e n c i l l i n e drawn across i t s l e s s e r dimension at l e a s t one inch from the edge.  Care was takento  avoid touching the paper except where necessary, with clean, dry fingers.  Along the p e n c i l l i n e , a t i n t e r v a l s o f 3/4 o f an inch, were  placed small drops of the amino acid solutions, peptone and hydrolysati by means o f a c a p i l l a r y tube. were dried by warm a i r .  The wet spots, about 3 nun. i n diameter,  The sheet of f i l t e r paper was formed into a  cylinder of 18 inches circumference around a supporting glass frame.  35  S p i k e s a l o n g t h e upper r i m o f t h e l a t t e r secured t h e t o p end o f t h e p a p e r , w h i l e t h e end h a v i n g t h e amino a c i d s p o t s hung f r e e .  The whole  was p l a c e d u p r i g h t i n a p y r e x p i e p l a t e , 6 i n c h e s i n d i a m e t e r ,  resting  upon a l a r g e sheet o f p l a t e - g l a s s . The p h e n o l s a t u r a t e d w i t h w a t e r , p r e p a r e d a s d e s c r i b e d above, was p i p e t t e d c a r e f u l l y i n t o t h e p i e p l a t e t o a depth o f about 5 mm,  A s m a l l b e a k e r o f w a t e r was p l a c e d b e s i d e  t h e p i e p l a t e and t h e s e , t o g e t h e r w i t h t h e sheet and r a c k , were enclosed w i t h i n a l a r g e , inverted glass c y l i n d e r .  The c r a c k between t h e  c y l i n d e r and sheet o f g l a s s was s e a l e d w i t h p l a s t i c i n e . hours t h e s o l v e n t f r o n t had r i s e n 27 cm.  A f t e r 21+  The c y l i n d e r was t h e n removed,  t h e wet sheet c a r e f u l l y d e t a c h e d f r o m t h e r a c k , and suspended i n a n e l e c t r i c a l l y heated oven m a i n t a i n e d a t 100 t o 110° C,  The s h e e t ,  having  become d r y a f t e r about 20 m i n u t e s , was removed from t h e oven and s p r a y ed w i t h a 0,25$ s o l u t i o n o f n i n h y d r i n ( t r i k e t o h y d r i n d e n e h y d r a t e ) i n w a t e r - s a t u r a t e d n - b u t a n o l by means o f a n a t o m i z e r .  J u s t enough s o l u -  t i o n was sprayed on t o make t h e p a p e r u n i f o r m l y damp, b u t w i t h no excess b e i n g a l l o w e d t o r u n o v e r t h e s u r f a c e .  The p a p e r was t h e n d r i e d  a t 85 t o 90° C. f o r 10 m i n u t e s t o d e v e l o p t h e c o l o u r s .  The p h e n o l  f r o n t was marked and t h e c o l o u r e d a r e a s o u t l i n e d i n p e n c i l .  In the  case o f c o l o u r e d a r e a s o f u n i f o r m i n t e n s i t y , a p e n c i l d o t was p l a c e d at the geometrical center;  i n t h e case o f a r e a s o f n o n - u n i f o r m i n -  t e n s i t y , , o h t h e o t h e r hand, a p e n c i l d o t was p l a c e d a t t h e c e n t e r o f greatest i n t e n s i t y o f colour.  The d i s t a n c e o f each p e n c i l d o t from  t h e base l i n e , d i v i d e d b y t h e d i s t a n c e o f t h e p h e n o l f r o n t f r o m t h e base l i n e was d e t e r m i n e d .  T h i s r a t i o was termed b y Consden e t a l .  (37) t h e "Rp v a l u e " , and i s c h a r a c t e r i s t i c , w i t h i n c e r t a i n l i m i t s , f o r  36  each amino a c i d .  Another chromatogram was made i n the same way, and Rp  values determined, A p a i r of two-dimensional chromatograms were then prepared as follows:  Two sheets o f Whatman no.I paper 11 inches square were cut  from a large sheet.  At a point 1 inch from the bottom, left-hand corner  of each sheet was placed a p e n c i l dot.  To one dot was applied a small  drop o f peptone hydrolysate, while t o the other was applied a small drop containing a mixture o f 19 amino acids prepared exactly as described previously, but t h i s time used as a mixture.  Each sheet was clipped  along two edges by means of a stapler i n such a way that a cylinder was formed having a gap o f a few millimeters between the clipped edges. These cylinders were dried a t 110°C. i n the oven f o r a few minutes and then treated with phenol as previously described, except that the cyl i n d e r s could stand alone, no glass support being needed.  After 10  hours, the phenol fronts had r i s e n nearly to t h e tops o f the c y l i n d e r s . The l a t t e r were then dried at 100 t o 110°G. f o r 20 minutes i n the oven. The c l i p s were removed, the phenol front marked i n p e n c i l , and the sheets were clipped along the other p a r a l l e l edges to form new cylinders having the amino acids and hydrolysate d i s t r i b u t e d around the bottoms. The cylinders were placed upright i n pie dishes containing 2 , 4 , 6 - c o l l i dine (saturated with water) to a depth o f a few m i l l i m e t e r s .  Each  cylinder and pie plate, together with a small beaker of water, was sealed under a glass tank as before.  The c o l l i d i n e was thus allowed  to ascend at r i g h t angles to the d i r e c t i o n i n which the phenol had ascended.  A f t e r 11 hours, the paper cylinders were removed, dried  at 100 to 110° C. f o r 20 minutes and treated with ninhydrin as  described previously. The coloured front (taken to be the c o l l i d i n e front) and coloured areas were outlined and marked as before, the  R  F  values f o r c o l l i d i n e being measured at right angles to those f o r phenol.  The results obtained are shown i n Tables VI and VII.  The data o f Table VI show a reasonable consistency.in Rp values found, although these are, on the average, approximately higher than those of Williams and Kirby (38).  0.04  This discrepancy i s  probably due to the s l i g h t differences i n experimental conditions, such as q u a l i t y of paper, quantity of amino acid and nature of solvent. Serine and glutamic acid, although showing considerably higher Rp values than those of Williams and Kirby, are, nevertheless, s e l f ^ con-, sistent.  Satisfactory r e s u l t s f o r tyrosine, however, were not obtained  only a long, f a i n t streak appeared on the chromatogram.  This may have  been due to the i n s o l u b i l i t y of tyrosine i n the solvents used, to i t s low concentration i n the controls, or to the faintness of i t s ninhyd r i n colour r e a c t i o n .  (  TABLE VI Rp VALUES FOR AMINO ACID STANDARDS AS DETERMINED BY THE ASCENDING METHOD OF FILTER PAPER PARTITION CHROMATOGRAPHY OF WILLIAMS AND KIRBY . (38) USING PHENOL.AS THE SOLVENT Amino Acid  Rp Values (found) 1st run  2nd run  3rd run  Rp Values (reported by Williams and Kirby)  X*  0.36  0.37  0.30  0.28  0.28  0.31  0.23  Leucine  X  0.86  0.80  0.80  Norleucine  X.  0.88  0.82  0.83.  Threonine  X  0*48  0.43  0.43  Alanine  0.58  0.59  0.55  0.55  Glycine  0.41  om.**  0.41  0.36  Serine Glutamic acid  Histidine  X  X  0.62  0.62  Methionine  X  0.86f  0.78  0.74  Phenylalanine  X  0.90f  0.89  0.83  Arginine  X  0.53  0.58  0.54  Proline  X  X  0.88  0.88  Tyrosine  X  X  l.s.  0.55  Hydroxyproline -  X.  X  0.66  (not given)  Tryptophane  x •  X  0.76  0.71  Iso-leucine  X  0.89  0*83  0.83  Cystine  X  X  0.28  0.24  Valine  X  0.79  om.  0.72  Aspartic acid  X  om.  om.  0.22  v  •* This i n d i c a t e s no spot appeared.  Omitted,  l . s . denotes the appearance o f a long streak, f denotes that the spot was f a i n t .  TABLE V I I R VALUES FOUND FOR THE HYDROLYSATE OF ALPHA CASEIN PHOSPHOPEPTONE I I BY THE ASCENDING METHOD OF FILTER PAPER PARTITION CHROMATOGRAPHY OF WILLIAMS AND KIRBY (38), USING PHENOL AND COLLIDINE AS SOLVENTS . F  Phenol R 2nd Run  3rd Run  F  Values Two dimensional  C o l l i d i n e Rp Values Two dimensional  0.00 0.089f 0.25  0.27f  0.27  0.28  0.34  0.35 0.47 0.55 0.75  0.82  . . 0.55f  f denotes that the spot was f a i n t .  The Rp values i n Table VII also show a f a i r degree of s e l f consistency, several values being repeated three times.  The  second  (phenol) run showed fewer components than the t h i r d , presumably because there was i n s u f f i c i e n t hydrolysate used i n the former case.  In a l l  three phenol runs a spot with an Rp value of 0,27 was observed and appears to correspond to the values found f o r glutamic a c i d and cystine (see t a b l e VI).  On comparing the corresponding c o l l i d i n e Rp value  ( i . e . 0.10) with the c o l l i d i n e Rp values reported by Williams and Kirby for glutamic acid and .cystine, i t i s apparent that the value f o r the l a t t e r corresponds more c l o s e l y to the value found than does the former. The presence of cystine i n the hydrolysate i s thus i n f e r r e d .  The set of Rp values 0.32  (phenol) and 0.26  given i n Table VII may now be considered.  (collidine),  Of the amino acids l i s t e d  i n Table VI, glutamic acid alone has a phenol Rp value ( i . e .  0.31)  comparable with that of the unknown spot. The c o l l i d i n e Rp value f o r glutamic acid i s , furthermore, reported (38) t o be 0.27,  a figure  which i s very s i m i l a r t o the c o l l i d i n e Rp value of the unknown c o n s t i tuent under consideration. That the l a t t e r i s glutamic a c i d i s , therefore, strongly indicated.  The set of Rp values 0.35  (phenol) and 0.15  (collidine)  which characterizes another unknown spot on the chromatogram of the peptone hydrolysate are now to be examined. ;  Other than glutamic a c i d ,  serine i s the only amino a c i d having a phenol Rp value approximating 0.35.  The c o l l i d i n e Rp value f o r t h i s compound as reported i n the  l i t e r a t u r e (38), agrees, moreover, with that obtained i n the case of  the unknown, i . e . , 0.15.  I t i s reasonably c e r t a i n from these data,  therefore, that the peptone hydrolysate contained serine. The occurrence of the next set o f two dimensional Rp values i n Table VII, i . e . 0.54 (phenol) and 0.28 ( c o l l i d i n e ) , can reasonably be attributed to the presence o f alanine i n the peptone hydrolysate for the following reasons:  Of the control amino acids (Table VI),  only alanine and arginine have phenol Rp values - 0.55 and 0.58 resp e c t i v e l y - which are comparable t o the phenol Rp value - 0.54 - o f the chromatogram spot now being considered.  Of the c o l l i d i n e Rp values  reported (38) f o r alanine and arginine, that o f alanine corresponds f a i r l y c l o s e l y with the c o l l i d i n e Rp value o f the unknown spot. I t i s to be concluded from t h i s that alanine was a constituent o f the peptone hydrolysate.  Returning to the data of Table VII, we may now consider the set o f Rp values 0.72 (phenol) and 0.39 ( c o l l i d i n e ) , corresponding to another constituent o f the peptone hydrolysate.  The phenol Rp values  found i n the neighbourhood o f 0.72 (see Table VI) are tryptophane  (0.76), methionine (0.78) and valine (0.79). Of these three amino acids, only valine i s reported (38) to have a c o l l i d i n e Rp value comparable with the c o l l i d i n e Rp value, i . e . 0.39, found f o r the unknown spot.  (Evidence f o r the absence of tryptophane i s presented  below i n section 4.)  The single c o n t r o l determination o f the phenol Rp  value of valine (Table VI) d i f f e r s by approximately 10$ from that reported by Williams and Kirby, and hence i t i s not considered to be a dependable c r i t e r i o n .  The conclusion that valine was present i n the  peptone hydrolysate i s , . f o r t h i s reason, only t e n t a t i v e . The remaining spot of an i n t e n s i t y comparable to those already mentioned, was observed to have Rp values of 0.80 (collidine);  0.50  (phenol) and  i t was therefore considered to be due t o the presence of  leucine, iso-leucine, norleucine or perhaps a l l three.  The  similarity  i n Rp values of these amino acids i s such that i t i s d i f f i c u l t to decide, from the a v a i l a b l e data, which ones were-present i n the peptone hydrolysate.  ••  There remain to be discussed four f a i n t spots. one, having R  F  values 0.88  (phenol) and 0.55  ,  ••  The upper  ( c o l l i d i n e ) , i s thought  to be due to p r o l i n e because of the correspondence  of i t s phenol Rp  value with that of the proline standard (see t a b l e VI), and of i t s two dimensional values with those of Williams and Kirby.  The other f a i n t  spots do not appear to correspond to any amino acids tested or reported. They may be due to.small amounts of unhydrolysed peptone or to phosphorylated serine. The l a t t e r p o s s i b i l i t y i s suggested by the work of Lipmann (40) who  i s o l a t e d serine phosphoric a c i d from among the pro-  ducts of hydrolysis of casein by hot, .2.5 N hydrochloric a c i d f o r 10 to 12 hours. 4.  Qualitative colour t e s t s :  The hydrolysate of alpha phosphopeptone I I was tested by Ehrlich's reagent.  An intense orange colour was produced, i n d i c a t i n g  the presence of tyrosine, h i s t i d i n e , or both.  On reduction with  zinc dust, followed by n e u t r a l i z a t i o n with ammonia, only a f a i n t colour remained i n the s o l u t i o n .  This was taken to indicate the  straw  presence o f tyrosine, and the absence of h i s t i d i n e ( A l ) . The same hydrolysate was tested with a solution of ammonium thiocyanate, whereupon an intense, blood-red colour formed, i n d i c a t i n g the presence o f i r o n .  This impurity was thought to have come acciden-  t a l l y from the s t i r r i n g motor used during p u r i f i c a t i o n and f r a c t i o n a t i o n of the casein.  The presence o f i r o n i n the hydrolysate was not con-  sidered to invalidate the r e s u l t s obtained by chromatography because i t r e a d i l y combined with phenol which was present i n large excess.  Both the alpha casein and the hydrolysate mentioned above were tested with p-dimethylaminobenzaldehyde ( 4 2 ) . The hydrolysate gave a deep yellow colour, which f i n d i n g was taken to i n d i c a t e the absence of tryptophane, whereas the alpha casein gave a purple colour, as was expected,  since alpha casein i s reported t o contain t r y p t o -  phane (see Table I I ) . The alpha casein and i t s peptone hydrolysate I I were then subjected t o the Sakaguchi t e s t ( 4 3 ) , the former giving a b r i g h t red colour, changing to yellowish-pink a f t e r a few minutes, and the l a t t e r giving a transient bright pink colour, changing to yellow.  These r e -  s u l t s were taken to i n d i c a t e the presence o f arginine i n the alpha casein, and i t s possible presence i n the hydrolysate.  Considering the lack of s a t i s f a c t o r y r e s u l t s with regard t o tyrosine i n paper p a r t i t i o n chromatography, i t was t o be expected that the presence of t h i s amino acid i n the hydrolysate o f alpha phosphopeptone I I might not be indicated by t h i s method as described above, was therefore resorted t o .  0  A specific test,  The presence o f t y r o s i n e  lends support to the findings of Linderstrom-Lang earlier;  (14) mentioned  the same may be said f o r arginine.  DISCUSSION The f i r s t problem which arose i n the present work, namely the possible i d e n t i t y of Warner's fractions with those of Cherbuliez and Jeannerat, has been solved with reasonable c e r t a i n t y .  Since •  Warner's alpha and beta caseins are reported t o show large differences i n phosphorous content  (0.99$ and 0.61$ r e s p e c t i v e l y ) , t h e i r i d e n t i t y  with or close s i m i l a r i t y t o the fractions of Cherbuliez and  Jeannerat  would have immediately become apparent upon determination of t h i s element.  Such, however, was by no means the case, as i s c l e a r l y shown  by the results of Tables I I I and IV.  An electrophoretic  examination  of the fractions obtained by the method of Cherbuliez and Jeannerat  was  not undertaken because the simpler and more d i r e c t approach o f phosphorous determination had f a i l e d to show any s i m i l a r i t y between these fractions and those of Warner which were claimed to be electrophoret i c a l l y homogeneous.  In a l l p r o b a b i l i t y the alpha (1), alpha  (11),  gamma and delta fractions are mixtures of alpha and beta caseins.  Turning now to the preparation of alpha casein, i t can be stated that the electrophoretic examination of the product and the determination of i t s phosphorous content demonstrated, beyond a reasonable doubt, that an alpha casein s i m i l a r to that described by Warner had indeed been prepared.  The preliminary experimental hydrolysis of commercially prepared casein was merely an attempt to establish conditions for conveniently rapid and sufficiently exhaustive hydrolysis of alpha casein, while conserving the limited quantity of the latter.  It was not assumed  that the course of hydrolysis of the former would necessarily be ident i c a l with that of the latter, although i t was later discovered that there was l i t t l e difference between the two i n this respect.  The  titration curves following peptic hydrolysis of alpha casein showed that the hydrolysis eventually came to a stop, under the conditions employed, as far as could be determined by the formol titrations. There remained, after the above-mentioned hydrolysis of alpha casein, an insoluble residue which was resolved into two products of differing phosphorous content. This result could be taken to indicate that alpha casein i s actually a mixture of two or more electrophoret i c a l l y indistinguishable phosphoproteins. Such a possibility seems highly unlikely, considering the great resolving power of the Tiselius apparatus and the improbability of the existence of two proteins having precisely the same electrophoretic mobility.  It i s possible  that part of the organically bound phosphorous of alpha casein was liberated by the acid during the course of peptic hydrolysis.  The  work of Lipmann (46) shows, however, that the serine ester of phosphoric acid i s resistant to hydrolysis by acid of higher concentration >  "  -  and temperature than that employed i n the present work. On the other hand, not a l l the organically bound phosphorous i s definitely known to be attached to serine residues;  a part, therefore, may  be  susceptible to hydrolysis by 0 . 1 N sulphuric a c i d .  The extent o f  l i b e r a t i o n of inorganic phosphorous under conditions s i m i l a r to those employed i n the present work f o r the hydrolysis of alpha casein, was reported by Damodaran and Ramachandran (4) to be n e g l i g i b l e .  We now  a r r i v e at the conclusion that the production o f at l e a s t two phosphopeptones from alpha casein was probably not a r e s u l t of the a c t i o n of a c i d on the l a t t e r , but rather a r e s u l t of the a c t i o n o f pepsin. Speculations concerning the structure of alpha casein i n an attempt to explain these findings are admittedly hazardous and l a c k i n g i n experimental foundation.  I t seems not unreasonable, nevertheless, to  suggest as a working hypothesis that phosphorous i s not d i s t r i b u t e d uniformly within the alpha casein molecule, but that at c e r t a i n places i t i s more concentrated than at others.  I f peptic hydrolysis occurred  between these l o c a l i t i e s of r e l a t i v e l y high phosphorous content, several different phosphorous-containing products might r e s u l t .  It  is  suggested that an examination of X-ray d i f f r a c t i o n patterns of alpha casein before and a f t e r treatment with sodium hydroxide (which would remove o r g a n i c a l l y bound phosphorous (2) ) might r e s u l t i n the d i s appearance of c e r t a i n p e r i o d i c i t i e s due to centers of phosphorous concentration.  Although highly speculative, the foregoing i s intended to  outline a possible d i r e c t i o n f o r further i n v e s t i g a t i o n .  Examination of the alpha phosphopeptone I I hydrolysate by f i l t e r paper p a r t i t i o n chromatography, demonstrated with reasonable c e r t a i n t y , the presence of serine, glutamic a c i d , cystine, v a l i n e , p r o l i n e and one o r more of the l e u c i n e s . phenol R  F  alanine,  In a l l cases the  values obtained with the controls were employed for purposes  o f comparison, even when t h e s e d i f f e r e d somewhat f r o m t h e v a l u e s r e p o r t e d i n t h e l i t e r a t u r e , as i n t h e case o f g l u t a m i c I n t h e two  d i m e n s i o n a l chromatograms, where two  a c i d and  serine.  p h e n o l Rp v a l u e s were  s i m i l a r , t h e c o r r e s p o n d i n g c o l l i d i n e Rp v a l u e s d i s t i n g u i s h e d t h e  spots.  I n such cases t h e c o l l i d i n e Rp v a l u e s o f W i l l i a m s and K i r b y were s u c c e s s f u l l y used as a check. I n a d d i t i o n t o t h e above-mentioned amino a c i d s , t h e s a t e was  shown t o c o n t a i n t y r o s i n e and p o s s i b l y a r g i n i n e .  can be drawn, however, r e g a r d i n g  No  conclusion  t h e r e l a t i v e amounts o f amino a c i d s  n o r t h e i r s p a t i a l arrangement i n t h e p e p t o n e . l y l a r g e N/P  hydroly-  I n view o f i t s r e l a t i v e -  r a t i o and t h e number o f amino a c i d s i t y i e l d s on  hydrolysis  t h e phosphopeptone examined a p p e a r s t o be more eomplex t h a n t h o s e l i s t e d i n Table I .  An i n s t r u c t i v e c o m p a r i s o n o f t h i s peptone w i t h  the  p r o d u c t s o f t r y p t i c h y d r o l y s i s o f c a s e i n i s , however, s c a r c e l y p o s s i b l e . There i s , on t h e o t h e r hand, f a i r l y good agreement w i t h t h e r e s u l t s o f workers u s i n g p e p s i n ( s e e p.3).  I t must be re-emphasized, i n c o n -  c l u s i o n , t h a t t h e p r e s e n t work i s t h e f i r s t , t o t h e a u t h o r ' s knowledge, to d e a l w i t h the products o f peptic h y d r o l y s i s of a s i n g l e casein constituent.  The  r e s u l t s p r e v i o u s l y r e p o r t e d by o t h e r w o r k e r s i n t h i s  f i e l d can t h u s be c o r r e l a t e d and  c l a r i f i e d only i n reference  to  the  p r e s e n t work and a n e c e s s a r y p a r a l l e l i n v e s t i g a t i o n o f b e t a c a s e i n .  SUMMARY 1  Casein has been fractionated by two methods reported i n the  literature:  those of Warner and of Cherbuliez and Jeannerat.  The  phosphorous content of the two sets of fractions obtained indicate that the two methods of.fractionation do not give the same results. 2  That the fraction obtained was that described by Warner  was shown by a similarity of phosphorous content.  Homogeneity of  the product was demonstrated by electrophoresis. 3  The alpha casein was subjected to rapid hydrolysis by pepsin  i n the presence of sulphuric acid of pH 1.0 at 50-52°C. The residue from this hydrolysis was exhaustively hydrolysed under the same conditions, the course of the reaction being followed by formol t i t rations.  After hydrolysis had apparently ceased, an insoluble pro-  duct remained. 4  This product was resolved,by precipitation with acid and  alcohol, into two components of differing phosphorous content. 5  The component having the greater phosphorous content was  hydrolysed by means of 6 N hydrochloric acid at 1C0°C.  The hydroly-  sate was examined qualitatively by the method of f i l t e r paper partition chromatography. The results indicate that the hydrolysate contained cystine, glutamic acid, serine, alanine, one or more of the leucines and possibly valine. present.  In addition, proline may have been  Three constituents, apparently present i n traces, were  not identified.  6  Qualitative colour tests indicated that the hydrolysate  contained i r o n (presumably from the apparatus used i n f r a c t i o n a t i o n ) , tyrosine and possibly  arginine.  BIBLIOGRAPHY 1  Posternak, S., U.S.Patent 1, 555, 517, Sept. 29, 1927.  2  Rimington, C , Biochem. J . ,  3  Levene and H i l l , J . B i o l . Chem..  4  Damodaran and Ramachandran, Biochem. J . ,  5  Svedberg, Carpenter and Carpenter, J . Am. Chem. S o c , 52, 241 &  701  21, 204, 1179, 1187 (1927).  35>122 (1941).  (1930).  6  Carpenter, J . B i o l . Chem.,  7  Warner, R.C., J . B i o l . Chem..  8  Lubavin  9  Salkowski, Z. Physiol. Chem.,  10  10. 711 (1933).  54,  1012  (1931).  66, 1725 (1944).  (1871), from Raudnitz, Ergeb. Physiol., 2.  D i e t r i c h , Biochem.Z.. 22,  193 (1903).  32, 245, (1901).  120 (1909).  11 Holter, Linderstrom-Lang and Funder, Compt. Rend. TravyLab. Carlsberg. 19, No.10  )1933).  12  S t i r l i n g and Wishart, Biochem. J . ,  13  Jones and Gersdorff, J . B i o l . Chem.,  14  Linderstrom-Lang, Compt. Rend. Trav. Lab. Carlsberg, 17, No. 9  (1929).  26, 1989 (1932). 106, 707 (1934).  '  '  15  Utkin, Biochem. Z.,  16  Posternak and Pollaczec, Helv. Chem. Acta, 24, 1190 (1941). N i c o l l e t and Shinn, Abstracts. Chicago Meeting, Am. Chem. S o c .  17  283, 233 (1936).  P20-B  (1946).  '  ~~  35, 315 (1941).  18  Lowndes, Macara and Pliramer, Biochem. J . ,  19  Rimington,  20  Herd, Biochem. J . .  21  Winnick, J . B i o l . Chem.,  22  Linderstrom-Lang and Kodama, Compt. rend, trav. Lab. Carlsberg,  Biochem. J . ;  35, 321 (1941).  30, 1743 (1936);  2i> 1478 (1937).  152, 465 (1944).  l6,.No.ll  (1925).  23  Carpenter and Hucker, J . Infect. P i s . , 4JL>  24  Cherbuliez and Schneider, Helv. Chem. Acta, 1£, 597  25  Cherbuliez and Meyer, Helv. Chem. Acta, 1 6 , 600  26  Cherbuliez and Jeannerat, Helv. Chem. Acta, 2 2 , 952  27  Groh, Kardos, Denes and Serenyi, Z. Physiol. Chem.. 2 2 6 , .  952  435  (1930). (1932).  (1933). (1939).  (1939).  28  McKee and Gould, J . Agr. Research,  29  Mellander, Biochem. Z., 300. 240  30  Gordon, Semett, Cable and Morris, J . Am. Chem. S o c , 2i,  3293 31  125  (1938).  (1939).  (1949).  Moore and White, Rev. S c i e n t i f i c Instruments, 1 9 , No.10, 700  (1948).  ~ ~  32  Longsworth, J . Am. Chem. S o c , 6 1 , 529  33  King, Biochem. J . , 26, 292  34  A l l e n , Biochem. J . , 3Jt,  (1939)'.  (1932).  858 ( 1 9 4 0 ) .  35 Levy, J . Biol.Chem., 105, 157 (1934) 36  Townsley, P.M., Bachelor's Thesis, Univ. o f B.C. ( 1 9 4 9 ) .  37  Consden, Gordon and Martin, Biochem. J . , 3 8 , 224  38  Williams and Kirby^ S c i e n c e , 1 0 7 , 4 8 1 ( 1 9 4 8 ) .  39  Townsley, D., Bachelor's Thesis, Univ. of B.C.  40  Lipmann, Biochem. Z.. 262, 3, 9  41  Totani, Biochem. J . , %  42  Lloyd, D.J., Chemistry of the Proteins, P. Blakiston and Co. 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