@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Chemistry, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Hummel, Brian Christopher Warren"@en ; dcterms:issued "2012-03-15T19:52:41Z"@en, "1950"@en ; vivo:relatedDegree "Master of Arts - MA"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "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 filter 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."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/41429?expand=metadata"@en ; skos:note "f f f o Mis' INVESTIGATIONS into THE CONSTITUTION OF CASEIN by Brian Christopher Warren Hummel A thesis submitted in partial fulfilment of the requirements for the degree of MASTER OF ARTS in 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 like to express my sincere appreciation to Dr. B. Eagles for inspiration and encouragement, to Dr. S.Zbarsky for valuable criticism and advice regarding both experimental and li t e r a r y aspects of this work, and to Dr. 0. Bluh for the use of his Tiselius apparatus and advice concerning the l a t t e r . I would also l i k e to thank Mr. D. Townsley for gener-ously loaning his apparatus for paper partition chromatography, as well as giving invaluable practical instruction i n i t s use, and Mr. Werner for advice and apparatus for.the purification of reagents. I l l TABLE OF CONTENTS Page Introduction 1 Historical A. The products of hydrolysis of casein by pepsin and trypsin 3 B. The fractionation of casein 7 Experimental A. Preparation of casein from milk 13 B. Fractionation of casein 14 1. Using 5% ammonium chloride and acetone 2. Using acetone, dilute acid and dilute base i n the cold C. Examination of the casein fractions 19 1. Electrophoresis 2. Phosphorous determinations D. Peptic hydrolysis of casein 22 1. Preliminary experiments with commercial casein 2. Hydrolysis of alpha casein E. Purification of the hydrolysis products 30 F. Examination of the peptones 31 1. Nitrogen and phosphorous determina-tions 2. Preparation of the acid hydrolysates of the peptones 32 I V 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 D i s c u s s i o n Summary 48 B i b l i o g r a p h y 50 1 INTRODUCTION The action of the proteolytic enzymes, pepsin and trypsin, upon bovine casein has been the subject of investigations by many workers, among whom Posternak (I), Rimington ( 2 ) , Levene and H i l l (3), Damodaran and Ramachandran (4) have made significant contributions. Diterest has been directed mainly toward three aspects of this problem: (a) the rate of hydrolysis, (b) the extent of hydrolysis, and (c) the nature of the products of hydrolysis. With regard to the third aspect, i t has been observed repeatedly that both pepsin and trypsin are capable of hydrolysing casein to a limited extent only, unhydrolysed peptones remaining after prolonged peptic or tryptic hydrolysis. Sever-a l investigators, each using a different procedure, have isolated one or more such peptones, but the reported amino acid compositions of these peptones exhibit, unfortunately, only a qualitative similarity. They have, on the other hand, been consistently observed to possess a considerably higher phosphorous content than the caseins from which they were derived. It was with a view to resolving these conflicting reports on the compositions of casein phospho-peptones that this work was undertaken. Svedberg and his co-workers ( 5 , 6 ) , using the ultra-centrifuge, Warner ( 7 ) , using the Tiselius electrophoresis apparatus, and other workers (14,23-28), employing various organic solvents, inorganic salts, acids and bases, have accumulated convincing evidence that native bovine casein i s not homogeneous, but can be resolved into several components differing i n solubility, amino acid composition and phos-phorous content. It i s apparent that the inhomogeneity of casein may account for the inconsistencies previously mentioned. It was thus considered essential i n the present work to study the enzymic hydrolysis of a single component of casein, rather than an indefinite mixture of components, as appears to have been done heretofor. HISTORICAL A. The products of hydrolysis of casein by pepsin and trypsin; It appears that the f i r s t investigator to study the action of a proteolytic enzyme on casein was Lubavin (8) who noted, i n 1871, that gastric juice did not completely dissolve casein, and that the residue contained a higher proportion of phosphorous than the original casein. Later workers observed similar results i n the case of pan-creatic extracts. During the period 1871-1927, many investigators repeated these experiments, on the whole confirming the original results. Sev-eral attempts were made to isolate products resulting from the digestion of casein by these crude extracts. Salkowski (9) claimed to have isolated the uranium salt of a phospho-peptone having a nitrogen to phosphorous ratio of 3.1. Dietrich (10) later isolated four similar products with N/P ratios of 1.00, 3.07, 2.59 and 3.87. Posternak (I) prepared the calcium salt of a phospho-peptone obtained by the pancrea-t i c digestion of casein, followed by precipitation by a lead salt. Analysis of his product indicated an N/P ratio of 5.3* During this early period the various products reported showed marked dissimilarities, 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$. It i s appropriate, perhaps to point out at this juncture that this diversity of results may be entirely explicable i n view of the possible variations i n (a) the original milk from which each casein was prepared, (b) the methods of purification of the caseins, (c) the im-purities present i n the enzyme preparations used, (d) the conditions under which hydrolyses were carried out (factors being pH, time, tempera-ture, presence of salts, etc.), and (e) the extent of bacterial a c t i v i t y . The f i r s t serious attempt to isolate a product the composition of which did not change on exhaustive purification, and the f i r s t com-plete amino acid determination on such a product were carried out i n 1927 by Rimingtbn (2), He subjected casein to tryptic digestion and purified the resulting phospho-peptone by means of i t s copper sa l t . Following this work, the isolation of the products formed on tryptic digestion of casein has been carried out by numerous investigators, each one of whom apparently obtained slightly different results. Those re-sults which give information concerning the amino acids present or their relative proportions i n the various phospho-peptones are set forth 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 ratio) are followed by the apparently more complex. The purpose of this 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 of casein. A possible reason why this theory i s not entirely supported by the evidence for the products do not present a continuous series - may be that the caseins employed by these workers were not, i n a l l probability, prepared by a uniform method. The starting materials i n each case were thus by no means identical. TABLE I THE COMPOSITION OF VARIOUS PHOSPHO-PEPTONES ISOLATED FROM AMONG THE ENZYMIC HYDROLYSIS PRODUCTS OF CASEIN Investigator Enzymes used Amino acids & Phosphoric acid reported i n peptone (propor-tions indicated where known) Total ho. of amino N/P in hydrolysis Gluta-mic acid Ser-ine Iso-leu-cine Aspar-t i c acid Phos-phor-i c acid acid resid-ues ratio Posternak & Pollaczek(l6) trypsin P P P 7 1 Nicollet & Shinn (17)*\" ti 2 3 I 2 7 3.5 Lowndes et a l . (18) II 2 3 3 P 8 ? Rimington (19) II it 5 5 4 4 I 3 3 9 10 3*0 3.3 Damodaran & Ramachan-dran (4) trypsin & pepsin 3 4 3 3 10 3.3 Posternak & Pollaczec(l6) trypsin P P P P P 10 or 11 • Posternak (I) II 3 ? 1 7 • ? 4 ? 1 1 ? 4 ? ? 15 16 18 3.8 1 ? * These workers also reported the presence of one additional amino acid of undetermined constitution. ^ P Indicates that this amino acid was reported present but that i t s proportion was not determined. 6 It w i l l be noted that after Rimington (2) the workers men-tioned thus far, with the exception of Damodaran and Ramachandran (4), used trypsin exclusively. The study of the peptic hydrolysis of casein has received comparatively l i t t l e attention, quantitative results being meagre. Holter et a l . ( l l ) found that on prolonged peptic digestion of casein, a \"paranuclein\" or phospho-peptone remained undigested and possessed an N/P ratio of 7.1. S t i r l i n g and Wishart (12) also observed the formation of products insoluble i n trichloroacetic acid by the action of both pepsin and trypsin. In the former case, starting with casein of N/P ratio 20, they found an acid-insoluble product of N/P ratio 12,8. Jones and Gersdorff (13) also isolated from among the products of peptic hydrolysis of casein, two paranucleins having N/P ratios of 13.2 and 13.8 (original casein was 35.3). Both these products con-tained lysine and tryptophane but no cystine. Linderstrom-Lang (14) supported this finding i n part by his observation that those fractions of the peptic hydrolysate containing the most phosphorous, also con-tained the most tryptophane, tyrosine, histidine and arginine. Utkin (15) observed the formation of a substance of N/P ratio 7 during the f i r s t stages of peptic hydrolysis of casein, and later i n the hydroly-s i s , of a substance of N/P 3.5. Miss Herd (20) found evidence for the presence of at least two components i n a paranuclein which she prepared; these components differed i n solubility, amino acid com-position and iodine-binding capacity. Winnick (21) studied the pro-ducts formed by the action on casein of pepsin, trypsin, and other enzymes, and found, on the average, that the non-protein molecules contained 5 to 7 amino acid residues and possessed a molecular weight of 450 to 600. Bo The fractionation of casein; The early workers who studied the enzymic degradation products of casein t a c i t l y assumed that the latter could be treated as a single chemical entity. Among the f i r s t experimenters to attempt a resolution of casein into fractions were Linderstrom-Lang and Kodama (22) who stu-died the so l u b i l i t y of this substance i n hydrochloric acid, and dis-covered that i t was separable into two components, one more soluble and the other less soluble than the original casein. Neither component was, however, claimed by them to be homogeneous because the solubility of each fraction varied with the quantity of precipitate present. Differences i n phosphorous and nitrogen contents were detected between the two fractions. Several years later, Linderstrom-Lang (14) carried out fur-ther fractionation experiments and concluded that i n native casein there are several kinds of colloidal molecules which interact, forming a co-precipitating system, and hence are not readily separable or distinguishable. He was able to obtain three constituents, character-ized as follows: (a) basic, alcohol-soluble and phosphorous-free, (b) combination of (a) with one containing phosphorous and soluble i n the presence of calcium salts, (c) containing more phosphorous than (b) and which i s precipitated by calcium salts. The molecular weight of casein, as prepared by Hammarsten, was studied by Svedberg, Carpenter and Carpenter (5) by means of the centrifugal sedimentation velocity method, and was found to consist of a mixture of molecules of different weights. Serologic studies on the proteins found i n casein were carried out by Carpenter and Hucker ( 2 3 ) . By extracting casein with 7 0 $ acidified alcohol and fractionating with potassium oxalate, they isolated three constituents. These were found to have molecular weights of 9 8 , 0 0 0 , 188 , 000 and 3 7 5 , 0 0 0 , and were reported to be clearly distinguishable by serologic reactions. After several preliminary papers by Cherbuliez and his co-workers ( 2 4 , 2 5 ) on the solubility of casein i n various salt solutions and organic solvents, there appeared a paper by Cherbuliez and Jean-nerat ( 2 6 ) giving a detailed procedure for the fractionation of casein by means of ammonium chloride and acetone. The yields of the three f i n a l fractions were reported as: alpha (II) 3 2 . 8 $ , gamma 4 8 . 7 $ and delta 3 . 5 $ . Each of these was claimed to be non-resolvable into either of the others, but was not further characterized. These workers state that their results were not influenced by the presence of calcium ions, a finding which appears to be completely at variance with that of Linderstrom-Lang ( 1 4 ) , mentioned earlier. Groh et a l . ( 2 7 ) also reported the fractionation of casein by three different methods: _ (a) by the addition of'absolute ethyl alcohol to solutions of casein i n 40$ urea, (b) by the addition of absolute ethyl alcohol to casein dissolved i n anhydrous molten phenol at 7 0 ° C. (c) by the addition of dilute hydrochloric acid to a solution of casein i n dilute ammonia containing 70$ ethyl alcohol. Two fractions were thus obtained i n equal proportions, containing 7.8 and 3.9$ tyrosine, and 1.6 and 1.1$ tryptophane respectively. It was maintained that these fractions were not the products of hydrolysis or denaturation since casein with i t s original properties was obtained i n each case by complete precipitation or by mixing the two separated fractions. Another method of fractionation was attempted by McKee and Gould (28) who determined the solubility of casein i n sodium cymene sulphonate, potassium thiocyanate and sodium benzene sulphonate at various temperatures at a pH of 4.6. Solubility was found to increase, as'a'rule, 'directly with temperature. Separation of casein into two fractions was effected by the f i r s t and third of the above reagents, but the second was found to be ineffective. The fractions were found to d i f f e r i n the following respects: (a) phosphorous content, (b) formaldehyde absorbing capacity (indicating unlike numbers of amino groups), and (c) solubility i n pyridine (indicating differences i n acidic groups). In the f i r s t electrophoretic studies on casein, Mellander (29) found that i t consisted of three sharply separable fractions, the fastest moving one of which contained a greater proportion of phosphor-ous than the original casein, and the slowest-moving one less. Follow-ing this up, Warner ( 7 ) , guided by electrophoretic patterns, developed a procedure by which he was able to obtain \"alpha\" and \"beta\" fractions 1 0 (containing 0 . 9 9 and 0 , 6 1 $ phosphorous respectively) whose electro-phoretic peaks corresponded to those of the original casein. By recom-bination of the fractions i n the proper proportions, the original pattern was restored. It was claimed that each i s a distinct fraction, although not electrophoretically homogeneous at a l l pH values, and free of a l l traces of the other. The method employed i s based on precipitation at carefully controlled pH by acetone, very dilute acid and a l k a l i at 2°C. and at room temperature. Warner's fractions have recently been examined with regard to amino acid composition by Gordon et a l . ( 3 0 ) . The most significant differences are shown i n the following table: TABLE II SIGNIFICANT DIFFERENCES IN AMINO ACID COMPOSITION BETWEEN THE CASEBf FRACTIONS OBTAINED BY WARNER ( 7 ) Amino acid % i n alpha- % i n beta-casein casein Alanine 3 . 7 1 .7 Valine 6 . 3 1 0 . 2 Leucine 7 . 9 1 1 . 6 Proline 8.2 1 6 . 0 Cystine 0 . 4 3 0 . 0 to 0.1 Tryptophane 1 .6 0*65 Aspartic acid 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 pre-viously. The values obtained by them are similar enough to suggest that they may have obtained fractions of approximately the same con-stitution as those of Warner. From the foregoing brief 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 fractionation of this substance. The lack of con-sistency i n the results of the former group may thus be due to the fact that casein i s , i n reality, a mixture of very similar proteins which, i n spite of their superficial similarity, may not yield products of identical composition when hydrolysed by pepsin or trypsin. To c l a r i f y this situation, i t i s clearly necessary to study the enzymic hydrolysis of each component of casein individually. The question which immediately arose was to decide which group of fractions reported i n the literature most closely represented single protein constituents. Two groups, that of Cherbuliez and Jeannerat (26) and of Warner (7), were selected. The former was considered more reliable than most others for a number of reasons: (a) the conditions of fractionation were relatively mild, the reagent being dilute 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 solubility of casein i n various reagents, and (c) each fraction was re-fractionated repeatedly u n t i l free of the others. The procedure 12 of Warner (7) was also selected for similar reasons, v i z . : (a) the use of acetone, dilute acid and a l k a l i was not considered to cause denaturation, and (b) the extremely sensitive electrophoretic procedure was employed as a guide for the progress of fractionation and as a check on the purity of the f i n a l products. In the present work, the procedure of Cherbuliez and Jeanner-at (26) was employed, but i t was found that the phosphorous contents of the fractions obtained bore no close relationship to those of Warner's fractions. The latter appeared, on the basis of electrophoretic studies, to be the truest available representation of the individual protein constituents of casein. Alpha-casein was prepared and subjected to peptic hydrolysis. 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) and Linderstrom-Lang (14). The N/P ratios 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 of the constitution of a protein, an obvious pre-requisite for valid results i s that the protein be i n the \"native\" state, that i s to 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 sub-stance as a protein without altering i t i n some way, this ideal can be closely approached only i n the starting material. In selecting a possible source of starting material for fractionation of casein, the commercial preparations were rejected because the extent of denatura-tion and the presence of impurities were uncertain. Of the methods of purification reported i n the literature, that of Warner (7) seems to minimize denaturation and to result i n a more nearly pure product than was obtainable by the other methods. It was particularly appro-priate to employ this method because Warner's fractionation procedure was made use of i n the present work. The following, operations, essentially those of the above mentioned investigator, were carried out. Five l i t e r s of cow's milk, collected at the time of milking, were immediately cooled to 40°F. and placed i n an ice bath. After centrifuging, the skim milk was siphoned off and kept at 0 - 5°C., a l i t t l e toluene being added to inhibit bacterial a c t i v i t y . The pH was adjusted t o 4.6 by the a d d i t i o n o f 0.1 N h y d r o c h l o r i c a c i d . The p r e c i p i t a t e which formed was separated by c e n t r i f u g i n g and washed s i x times w i t h an equal volume of i c e water. S u f f i c i e n t sodium hydroxide s o l u t i o n (approximately 0.1 N) was added t o d i s s o l v e the p r e c i p i t a t e and b r i n g the s o l u t i o n t o pH 6.5. Since operations had t o be temporar-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 , the s o l u t i o n was f r o z e n s o l i d f o r some ti m e . The s o l i d mass was then thawed out and f a t was e x t r a c t e d from i t by shaking w i t h ether, f o l l o w e d by c e n t r i f u g i n g . The e x t r a c t e d l i q u i d was d i l u t e d to a p r o t e i n c o n c e n t r a t i o n of approximately 0.5%, c a l c u l a -t e d on the assumption t h a t the o r i g i n a l m i l k contained 3% c a s e i n . 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 the pH t o 4.6, f i n a l adjustment being made wi t h a small amount of 0.01 N sodium hydroxide s o l u t i o n . The p r e c i p i t a t e was allowed to s e t t l e i n the c o l d , a f t e r which the supernatant was siphoned o f f . The p r e c i p i t a t e was washed three times w i t h an equal 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 . An a l i q u o t o f the wet c a s e i n was d r i e d t o constant weight by means of a l c o h o l and e t h e r j from t h i s i t was c a l c u l a t e d t h a t approximately 170 gm. o f c a s e i n (on a dry b a s i s ) had been obtained. The wet product was then subjected 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 m i l k was worked up as above (except 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 ) , and the wet c a s e i n was 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 B.2). B. F r a c t i o n a t i o n o f the c a s e i n : I . Using 5$ ammonium c h l o r i d e and acetone: This procedure i s t h a t o f Cherbuliez and Jeannerat (26) who had developed 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 -tions, during the, course of which they discovered that casein i s more soluble i n ammonium chloride solution than i n a number of other salt solutions, and that a 5% concentration of this salt was optimum. The casein, according to their method, was brought into solution by means of 5$ ammonium chloride and 0 . 1 N sodium hydroxide solutions, after which the f i r s t fraction was obtained by the addition of 0 . 1 N hydro-chloric acid. Upon adding more of the acid, a second fraction was precipitatedj from the supernatant two other fractions were obtained by the addition of acetone. Each fraction was repeatedly re-fraction-ated i n the same manner, with the result that the second precipitate mentioned above was resolved into the others, thus leaving three f i n a l products, each of which could not be resolved into the others. A more detailed account of these operations i s given i n Figure I. 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. of 5 $ NH.C1 plus 90 ml. of 2 0 $ NH .C1, then add 1 4 0 ml. of 0 . 1 N NaOH) [CASEIN SOLUTION (at pH 6.5)1 IMPURE ALPHA FRACTION. (kept under acetone) (add 140 ml. of 0.1 N HC1 and bring to pH 4.8, then 5 ml. more to bring to pH 4.6; centrifuge & wash ppt, with water) SUPERNATANT CONTAINING! OTHER FRACTIONS MPHA ( I I ) FRACTION . (add 1/5 volume of acetone; centri-fuge & wash ppt.) SUPERNATANT CONTAINING JAMMA & DELTA FRACTIONS! IMPURE GAMMA FRACTION (add 0.1 N HC1 u n t i l pH 3.8 reached) SUPERNATANT CONTAINING! DELTA FRACTION IMPURE DELTA! FRACTION 16.7$ acetone (add 0.1 N NaOH to pH 5.3; add one volume of acetone & l e t stand overnight; add one volume more) DISCARDED LIQUID FROM WHICH ACETONE IS RECOVERED 72.5$ acetone NOTE: Each fraction obtained as above i s subjected to this fractionation repeatedly. In the present work, the fractionation proceeded as expected at f i r s t , the alpha (II) fraction i n i t i a l l y obtained being gradually resolved into alpha (I) and gamma fractions. On attempting to further purify the gamma fraction, however, about four-fifths of the latter precipitated under conditions of alpha (II) precipitation. This new alpha (II) fraction remained, despite repeated attempts to resolve i t , only very small amounts of alpha (I) and gamma fractions being obtain-able 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, dilute acid and base i n the cold: The method used here i s that of Warner (7), and based upon the isoelectric precipitation at a given pH at 2°C. of the f i r s t fraction from a very dilute acid solution of casein, followed by the precipitation of the second fraction at a slightly higher pH at room temperature. The novelty of this procedure l i e s i n precipitation by approaching the isoelectric point from the acid side. Since i t i s extremely d i f f i c u l t to dissolve casein i n acid, the procedure for pre-paring a solution of this substance at pH 3.5 deserves particular atten-tion. Further details 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 of this fraction only. The casein from 5 l i t e r s of raw milk yielded about 50 gm. (dry weight) of alpha casein. FIGURE II 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. qf casein to give a 1% solution at 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 rapidly as possible to give solution of pH 3.5) ICASEIN SOLUTION) 3. ( c h i l l to 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 clear 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 off ppt. and subject i t to procedures 1,2,3 and 4 i n order five times to remove a l l beta casein) ALPHA CASEIN PRECIPITATE (dissolve i n NaOH to give a 0,2% solution; c h i l l to 2° C. and add HCI to 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 electrophoretically i n order to determine whether the fraction obtained was identical to that described by Warner ( 7 ) . The apparatus employed was that develop-ed by Moore and White ( 3 1 ) , and i s a modification of the-original Tiselius apparatus incorporating the Toepler schlieren scanning im-provements made by Longsworth (32). (The manufacturers of this appara-tus, the Perkin-Elmer Corporation, Glenbrook, Conn., U. S. A., provide a manual of comprehensive instructions for operation). The alpha casein showed only a single, very sharp boundary, whereas the original casein showed two rounded'but distinct peaks. This examination was carried out i n phosphate buffer at pH 7.7. The results obtained, although qualitative, were judged to be essentially identical with those of Warner. Drawings of the photographic patterns obtained are shown i n figures I l i a and I l l b . FIGURE III 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 b 2 Phosphorous determinations: The various fractions of casein obtained by the two pro-cedures were analysed for phosphorous content by means of modified versions of the Fiske - Subbarow colourimetric method (45)- The alpha (I), alpha (II), gamma and delta fractions were treated by the method of King (33) who used I-amino-2-naphthol-4-sulphonic acid as the reducing agent, whereas the alpha casein was treated according to the method of Allen (34) who used 2,4-diaminophenol. The latter reagent, i t i s claimed, gives more consistent results than other reducing reagents, but i t was not available at the time the alpha (I), alpha (II), gamma and delta fractions were analysed. The results are shown i n tables III and IV. TABLE III PHOSPHOROUS CONTENT OF FRACTIONS AT PROGRESSIVE STAGES OF THE FRACTIONATION OF CASEIN BY THE METHOD OF CHERBULLEZ AND JEANNERAT (26) % Phosphorous i n : Alpha (I) fraction Alpha (II) fraction Gamma fraction Delta fraction 0.784 0.763 0.691 0.728 0.764 0.680 0.750 0.761 0.674 0.753 0.814 0.733 Original/casein: 0.741$ phosphorous The figures i n each column of the above Table correspond to samples taken at successive stages of purification of each fraction. TABLE IV PHOSPHOROUS CONTENT OF CASEIN AND ALPHA CASEIN PREPARED BY THE METHOD OF WARNER (7) Substance % P % P Reported Found Original casein 0.86 0.83 Alpha casein 0.99 0.99 From the results of table III i t i s apparent that there i s , i n each fraction, a variation of phosphorous content, but that i t i s only i n the case of the gamma fraction that this variation i s i n one d i r e c t i o n alone. None o f the f r a c t i o n s f i n a l l y obtained possessed a phosphorous content 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 of 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 l a t t e r . The assumption t h a t the a l p h a c a s e i n obtained i n the present 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 supported s t r o n g l y by the r e s u l t s shown i n t a b l e IV. Since Warner's.fractions and the c a s e i n from which they were de r i v e d showed o n l y minor d i f f e r e n c e s i n n i t r o g e n content, t h e l a t t e r was not.considered of v a l u e , i n the present work, f o r purposes o f c h a r a c t e r i z a t i o n and comparison, and hence was not determined. D. P e p t i c h y d r o l y s i s of c a s e i n : 1. P r e l i m i n a r y experiments w i t h commercially prepared c a s e i n : P r e l i m i n a r y experiments u s i n g pepsin 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 reasons: (a) t o t e s t and p r a c t i s e the formol t i t r a -t i o n technique 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 the course 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 the c o n d i t i o n s used by Damodaran and Ramachandran ( 4 ) , (c) t o modify the c o n d i t i o n s i n (b) w i t h a view t o reducing f o r the sake of convenience, the time r e q u i r e d t o reach the p o i n t where the 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 proceeded at a v e r y slow r a t e compared w i t h the i n i t i a l r a t e , and (d) t o determine whether, under these new c o n d i t i o n s , a p p r e c i a b l e h y d r o l y -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 other than the presence o f pepsin. The method of formol t i t r a t i o n adopted was found to be a quick, convenient and consistent one for following the course of hydrol-y s i s . The rate of hydrolysis i n the f i r s t run (figure III) was con-sidered too slow for practical purposes, and gave time, i t was thought, for bacterial action to begin despite the presence of toluene. The conditions f i n a l l y adopted were the following: a substrate/enzyme ratio of 4.0, an enzyme concentration of 8.3 gm./liter of approximately 0. I N sulphuric acid, a pH of 1.0 and a temperature of 50 - 52°C, These conditions were such that the rate of hydrolysis was reduced to l / 2 0 t h the i n i t i a l rate after about two hours. The removal of the products of digestion by washing the residue, reduced the rate of pep-tide re-synthesis, i t was thought, and allowed the second digestion to proceed relatively uninhibited by these reverse reactions. Twenty grams of dry, finely-powdered, commercially-prepared casein (Nutritional Biochemicals Corporation) were stirred for an hour with approximately 1 l i t e r of 0.05 N sodium hydroxide solution. Un-dissolved particles, 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 acid, and the flocculent, white precipi-tate was recovered by centrifuging. The precipitate was then suspended i n 300 ml. of 0.1 N sulphuric acid, and the mixture was brought to pH 1.8 by the addition of 5 N sulphuric acid. Half a gram of pepsin (Difco 1:10,000) was dissolved i n 2 ml. of 0. I N sulphuric acid and added to the casein suspension. The mixture was then incubated for 40 hours at 35 - 39°C. Due to unequal rate of liberation 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 acid. A further 0.5 gram of pepsin (dissolved i n 2 ml. of 0.1 N sulphuric acid) was added and i n -cubation was continued as before. The progress of enzymic hydrolysis was followed by formal titrations 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 inter-vals, 10 ml. samples of the incubating mixture were removed and centri-fuged. Two ml. aliquots of each sample were placed i n small beakers and titrated to the f i r s t faint pink of phenolphthalein with 0.02 N sodium hydroxide solution. The volume of base was noted, and then approximately 5 ml. of f i l t e r e d formalin solution, which had been previously adjusted to pH 7.0, were added. Titration was continued to the second end point. The volume of formalin added i n each t i t r a -tion was carefully adjusted so that the f i n a l concentration of formal-dehyde was 6 to 9$. The results, plotted graphically i n Figure III, indicate a slowly progressing hydrolysis, the rate of which i s almost constant. It was thought that the time required for hydrolysis to reach completion at this rate would have been too great for practical pur-poses. This i s supported by the work of Damodaran and Ramachandran (4) who allowed the hydrolysis of casein to continue for 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 further 20 gm. lots of casein, but this time the temperature was raised to 50 - 53°<>., the pepsin concentration was increased by a factor of ten ( i . e . to 5.0 gm. per 300 ml. of 0.1 N sulphuric acid), and the mixture was stirred con-tinually by a motor stirrer,, 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 in figure IV, and show that, under these new conditions, hydrolysis of casein proceeds very much more rapidly than under the conditions used in the fi r s t hydroly-sis (Figure III). During the first hour, the rate of hydrolysis was 20 times that during the following 70 hours. The above mentioned hydrolyses were carried out in beakers covered by watch glasses. It was thought that the increase in acidity might, in part at least, be due to a slow evaporation of water and concentration of the acid. The hydrolyses were therefore repeated as in 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 in 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 in titratable carboxyl groups, f o l -lowed by a relatively slow rise. 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 in Figure'Vb., show that this residue underwent further hydrolysis, but 26 23 22 m O N T3 O g § H O s e CNi O O ' 1 5 n /] J J . i C A • J n TEMPS OF 31 CUBA1 • 1 50 v- 0 0 * 1.6 • )1 • / / 19 18 10 20 \"^ime in hours 50 60 70 27 PEP1 FIGURE V HYDROLYSIS OF COMMERCIAL CASEIN 15 i n glass-stoppered flask to prevent evaporation) IT OF CASE; nv- MM 13 11 13 WEIGH IN: 20 gm. CONCENTRATION OF PEPSIN: 15 gm./l. 50 -TEMPERATURE OF INCUBATION: fill [ l i d j (curve a.: f i r s t hydrolysis curv.e b e: second hydrolysis; washed residue) 53 C. TEMPERATURE OF INCUBATION: 50 CONCENTRATION OF ACID: O.I VOLUME OF ACID: 600 ml. 51°C 12 11 0 20 40 S 36 GRAPH 6 & E R . SMI-time i n hours 80 S O N a WRI 100 E P T I C H Y D R O L Y S I S OF A L P H A C A S E I N WEIC GHT 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. time in hours T7T T TZT — 29 to a much lesser extent and at a slower rate than the original casein. A control experiment was run under the same conditions as above (Figure V), omitting the pepsin, to determine the rate of hydrolysis by acid alone. Twenty grams of casein, after re-precipitation as before, were suspended i n 600 ml. of 0. I N sulphuric acid and incubat-ed at 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 slightly by dilute sulphuric acid 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 dilute acid appears, however, to come to a v i r t u a l stop after about 18 hours, possibly due to a relatively limited 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 at 50°C. and ad-justed to pH 1.0 with 5 N sulphuric acid. Ten grams of pepsin, dis-solved i n 0.1 N sulphuric acid, were added with st i r r i n g , and two equal portions of the mixture were placed i n II . Erlenmeyer, glass, stoppered flasks. Incubation was carried out at 50 - 52°C. with occasional shaking for 23 hours. Formol titrations were performed at intervals as before. The results are plotted i n Figure V i l a . The residue which remained was recovered by centrifuging and was washed six times with 200 ml. portions of d i s t i l l e d water, u n t i l the super-natant was free of sulphate ion (no precipitate with barium chloride). 30 The wet precipitate was then suspended i n 240 ml, of 0.1 N sulphuric acid, the pH was adjusted to 1.0 with 5 N sulphuric acid, and digestion 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 results 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 after approximately 6 hours. E. Purification of the hydrolysis products: The wet residue (from D.2 above) was suspended i n 150 ml. of d i s t i l l e d water. 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 particles were removed by f i l t r a t i o n through glass wool, after 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 acid. The fine, l i g h t brown precipitate which formed was recovered by centrifuging. The supernatant was colloidal and gave a voluminous precipitate with tannic acid solution when a small sample was tested. To the supernatant were added 4 volumes of ethanol. The white precipitate which formed was allowed to settle overnight i n the refrigerator and was then recovered by centrifuging. The precipitate was washed three times with 50 ml. of 50% ethanol. By washing the f i r s t (brown) precipitate with water and adding 4 volumes of ethanol to the washings, a l i t t l e more of the white precipitate was obtained. Both precipitates were washed 4 times with ethanol, after 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 precipitates placed i n a vacuum dessicator for 24 hours. The brown precipitate w i l l hereafter be termed \"alpha phosphopeptone I\" and the white one \"alpha phosphopep-tone II\". F. Examination of the peptones; I. Nitrogen and phosphorous determinations: At this stage i n the work, i t was of interest to compare with respect to the N/P ratio, the alpha phosphopeptones mentioned above with one another and with the products similarly 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 results i n the production of two groups of products, one of gradually increasing and the other of gradually diminishing N/P ratio. That the magnitude of the N/P ratio parallels, i n a general way, the complexity of the peptone, i s indicated by the data of Table I. Total nitrogen was determined by the micro-Kjeldahl method and t o t a l phosphorous by the method of Allen (34) • The results are given i n Table V. TABLE V NITROGEN AND PHOSPHOROUS CONTENTS OF ALPHA PHOSPHOPEPTONES I AND II Alpha Phos- % Nitrogen % Phos- N/P ratio phopeptone phorous (atomic) I 11.5 2.8 9.1 11 11.7 3.9 6.6 2. Preparation of the acid hydrolysates of the peptones: In preparation for the next stage of examination of peptones by chromatography (see section 3 below), i t was necessary to decompose these substances into their constituent amino acids. The procedure employed i n order to effect this was that of Townsley (36), and i s as follows: Ninety to 100 MGM. of each peptone were dissolved i n 3 ml. of 6 N hydrochloric acid. The yellowish solutions were sealed i n small vials (2 by % inches) and placed i n the bottom of a 4 1. Erlen-meyer flask containing water to the depth of an inch. The water was boiled under a reflux condenser for 24 hours. A l l vials then contained a yellowish hydrolysate, but those i n which the brown alpha phospho-peptone I had been placed, contained also a dark sediment, whereas the other vials contained none. 3. F i l t e r paper partition chromatography: In view of the small y i e l d of phosphopeptones obtained and the desirability of gaining some knowledge of the amino acid constit-uents of the substances, i t was decided to employ the technique of 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 (37), and has become an immensely popular tool for several reasons: (a) i t is possible to detect the presence of every amino acid in a single experiment, (b) the technique is truly micro, the quantity of material required for analysis being a fraction of a milligram, (c) the procedure is 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 in the present work are based largely on a modification of those of Consden et al. by Williams and Kirby (38) and by Townsley ( 3 9 ) , and are as described below. It was first 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 in water to make a solution containing approximately 2 mgm. per ml. Two drops of each solution were placed in 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 for free amino acids). To the latter only were added 4 drops of 6 N hydrochloric acid. The spot plate was then placed in a vacuum dessicator over sodium hydroxide pellets until 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 of hydrochloric acid pre-treatment. The solvents required were then prepared. Phenol was dis-t i l l e d over zinc dust i n an all-glass apparatus i n order to remove impurities, particularly those giving the pink colour. While s t i l l l i q u i d , the phenol was thoroughly shaken with a small excess of water, thus producing a fine emulsion. The latter was allowed to cool to room temperature and was then centrifuged. Of the two clear layers which formed, the bottom one (phenol saturated with water) was carefully separated and kept i n the dark u n t i l used. Commercial 2,4, 6-collidine (light amber colour) was fractionally d i s t i l l e d under vacuum i n an all-glass apparatus, the fraction passing over at 55 to 60°C. at a pressure of 10 to 15 mm. of mercury being collected. This colourless collidine was shaken with d i s t i l l e d water at room tempera-ture u n t i l an emulsion formed. The latter was centrifuged, and of the two layers which formed, the upper one (collidine saturated with water) was kept for chromatography. A large sheet of Whatman f i l t e r paper no^I (approximately 18 by 22 inches) was selected and a pencil li n e drawn across i t s lesser dimension at least one inch from the edge. Care was takento avoid touching the paper except where necessary, with clean, dry fingers. Along the pencil line, at intervals of 3/4 of an inch, were placed small drops of the amino acid solutions, peptone and hydrolysati by means of a capillary tube. The wet spots, about 3 nun. i n diameter, were dried by warm a i r . 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 Spikes along the upper r i m o f the l a t t e r secured the t o p end o f the paper, w h i l e the end having t h e amino a c i d spots hung f r e e . The whole was placed u p r i g h t i n a pyrex p i e p l a t e , 6 inches i n diameter, r e s t i n g upon a l a r g e sheet o f p l a t e - g l a s s . The phenol s a t u r a t e d w i t h water, prepared as described above, was p i p e t t e d c a r e f u l l y i n t o the p i e p l a t e t o a depth o f about 5 mm, A sm a l l beaker of water was placed beside the p i e p l a t e and these, together w i t h the sheet and rack, were en-closed w i t h i n a l a r g e , i n v e r t e d g l a s s c y l i n d e r . The crack between the c y l i n d e r and sheet o f g l a s s was sealed w i t h p l a s t i c i n e . A f t e r 21+ hours the solvent f r o n t had r i s e n 27 cm. The c y l i n d e r was then removed, the wet sheet c a r e f u l l y detached from the rack, and suspended i n an e l e c t r i c a l l y heated oven maintained a t 100 t o 110° C, The sheet, having become dry a f t e r about 20 minutes, was removed from the oven and spray-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 hydrate) i n water-saturated n-butanol by means of an atomizer. J u s t enough s o l u -t i o n was sprayed on t o make the paper u n i f o r m l y damp, but w i t h no excess being allowed t o run over the s u r f a c e . The paper was then d r i e d a t 85 t o 90° C. f o r 10 minutes t o develop the c o l o u r s . The phenol f r o n t was marked and the coloured areas o u t l i n e d i n p e n c i l . I n the case o f coloured areas o f uniform i n t e n s i t y , a p e n c i l dot was placed a t the geometrical center; i n the case of areas o f non-uniform i n -tensity,,oh the other hand, a p e n c i l dot was placed a t the center o f greate s t i n t e n s i t y o f c o l o u r . The d i s t a n c e o f each p e n c i l dot from the base l i n e , d i v i d e d by the distance o f the phenol f r o n t from the base l i n e was determined. T h i s r a t i o was termed by Consden et a l . (37) the \"Rp value\", 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 acid. Another chromatogram was made i n the same way, and Rp values determined, A pair of two-dimensional chromatograms were then prepared as follows: Two sheets of 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 pencil dot. To one dot was applied a small drop of peptone hydrolysate, while to the other was applied a small drop containing a mixture of 19 amino acids prepared exactly as described previously, but this 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 of a few millimeters between the clipped edges. These cylinders were dried at 110°C. i n the oven for a few minutes and then treated with phenol as previously described, except that the cy-linders could stand alone, no glass support being needed. After 10 hours, the phenol fronts had risen nearly to the tops of the cylinders. The lat t e r were then dried at 100 to 110°G. for 20 minutes i n the oven. The clips were removed, the phenol front marked i n pencil, 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 distributed around the bottoms. The cylinders were placed upright i n pie dishes containing 2,4,6-colli-dine (saturated with water) to a depth of a few millimeters. Each cylinder and pie plate, together with a small beaker of water, was sealed under a glass tank as before. The collidine was thus allowed to ascend at right angles to the direction i n which the phenol had ascended. After 11 hours, the paper cylinders were removed, dried at 100 to 110° C. for 20 minutes and treated with ninhydrin as described previously. The coloured front (taken to be the collidine front) and coloured areas were outlined and marked as before, the R F values for collidine being measured at right angles to those for phenol. The results obtained are shown i n Tables VI and VII. The data of Table VI show a reasonable consistency.in Rp values found, although these are, on the average, approximately 0.04 higher than those of Williams and Kirby (38). This discrepancy i s probably due to the slight differences i n experimental conditions, such as quality 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 results for tyrosine, however, were not obtained only a long, faint streak appeared on the chromatogram. This may have been due to the insolubility 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 ninhy- ( drin colour reaction. 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) Rp Values (reported by 1st run 2nd run 3rd run Williams and Kirby) Serine X* 0.36 0.37 0.30 Glutamic acid 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 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 Hydroxy-proline -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 v om. om. 0.22 •* This indicates no spot appeared. Omitted, l . s . denotes the appearance of a long streak, f denotes that the spot was faint. TABLE VII RF VALUES FOUND FOR THE HYDROLYSATE OF ALPHA CASEIN PHOSPHOPEPTONE II BY THE ASCENDING METHOD OF FILTER PAPER PARTITION CHROMATOGRAPHY OF WILLIAMS AND KIRBY (38), USING PHENOL AND COLLIDINE AS SOLVENTS . Phenol R F Values Collidine Rp Values 2nd Run 3rd Run Two dimen-sional Two dimen-sional 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 faint. 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 third, presumably because there was insufficient 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 for glutamic acid and cystine (see table VI). On comparing the corresponding collidine Rp value (i.e. 0.10) with the collidine Rp values reported by Williams and Kirby for glutamic acid and .cystine, i t i s apparent that the value for the la t t e r corresponds more closely to the value found than does the former. The presence of cystine i n the hydrolysate i s thus inferred. The set of Rp values 0.32 (phenol) and 0.26 (collidine), given i n Table VII may now be considered. 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 collidine Rp value for glutamic acid i s , furthermore, reported (38) to be 0.27, a figure which i s very similar to the collidine Rp value of the unknown consti-tuent under consideration. That the latter i s glutamic acid 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 acid, serine i s the only amino acid having a phenol Rp value approximating 0.35. The collidine Rp value for this compound as reported i n the literature (38), agrees, moreover, with that obtained i n the case of the unknown, i.e . , 0.15. It i s reasonably certain from these data, therefore, that the peptone hydrolysate contained serine. The occurrence of the next set of two dimensional Rp values i n Table VII, i . e . 0.54 (phenol) and 0.28 (collidine), can reasonably be attributed to the presence of 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 res-pectively - which are comparable to the phenol Rp value - 0.54 - of the chromatogram spot now being considered. Of the collidine Rp values reported (38) for alanine and arginine, that of alanine corresponds f a i r l y closely with the collidine Rp value of the unknown spot. It i s to be concluded from this that alanine was a constituent of the peptone hydrolysate. Returning to the data of Table VII, we may now consider the set of Rp values 0.72 (phenol) and 0.39 (collidine), corresponding to another constituent of the peptone hydrolysate. The phenol Rp values found i n the neighbourhood of 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 collidine Rp value comparable with the collidine Rp value, i . e . 0.39, found for the unknown spot. (Evidence for the absence of tryptophane i s presented below i n section 4.) The single control determination of the phenol Rp value of valine (Table VI) differs by approximately 10$ from that reported by Williams and Kirby, and hence i t i s not considered to be a dependable criterion. The conclusion that valine was present i n the peptone hydrolysate i s , . f o r this reason, only tentative. The remaining spot of an intensity comparable to those already mentioned, was observed to have Rp values of 0.80 (phenol) and 0.50 (collidine); i t was therefore considered to be due to 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 available data, which ones were-present i n the peptone hydrolysate. •• , •• There remain to be discussed four faint spots. The upper one, having R values 0.88 (phenol) and 0.55 (collidine), i s thought F to be due to proline because of the correspondence of i t s phenol Rp value with that of the proline standard (see table VI), and of i t s two dimensional values with those of Williams and Kirby. The other faint 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 phos-phorylated serine. The l a t t e r possibility i s suggested by the work of Lipmann (40) who isolated serine phosphoric acid from among the pro-ducts of hydrolysis of casein by hot, .2.5 N hydrochloric acid for 10 to 12 hours. 4. Qualitative colour tests: The hydrolysate of alpha phosphopeptone II was tested by Ehrlich's reagent. An intense orange colour was produced, indicating the presence of tyrosine, histidine, or both. On reduction with zinc dust, followed by neutralization with ammonia, only a faint straw colour remained i n the solution. This was taken to indicate the presence of tyrosine, and the absence of histidine (Al). The same hydrolysate was tested with a solution of ammonium thiocyanate, whereupon an intense, blood-red colour formed, indicating the presence of iron. This impurity was thought to have come acciden-t a l l y from the stirring motor used during purification and fractionation of the casein. The presence of iron i n the hydrolysate was not con-sidered to invalidate the results obtained by chromatography because i t readily 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 finding was taken to indicate the absence of tryptophane, whereas the alpha casein gave a purple colour, as was expected, since alpha casein i s reported to contain trypto-phane (see Table I I ) . The alpha casein and i t s peptone hydrolysate II were then subjected to the Sakaguchi test (43), the former giving a bright red colour, changing to yellowish-pink after a few minutes, and the latte r giving a transient bright pink colour, changing to yellow. These re-sults were taken to indicate the presence of arginine i n the alpha casein, and i t s possible presence i n the hydrolysate. Considering the lack of satisfactory results with regard to tyrosine i n paper partition chromatography, i t was to be expected that the presence of this amino acid i n the hydrolysate of alpha phospho-peptone II might not be indicated by this method0 A specific test, as described above, was therefore resorted to. The presence of tyrosine lends support to the findings of Linderstrom-Lang (14) mentioned earlier; the same may be said for arginine. DISCUSSION The f i r s t problem which arose i n the present work, namely the possible identity of Warner's fractions with those of Cherbuliez and Jeannerat, has been solved with reasonable certainty. Since • Warner's alpha and beta caseins are reported to show large differences i n phosphorous content (0.99$ and 0.61$ respectively), their identity with or close similarity to the fractions of Cherbuliez and Jeannerat would have immediately become apparent upon determination of this element. Such, however, was by no means the case, as i s clearly shown by the results of Tables III and IV. An electrophoretic examination of the fractions obtained by the method of Cherbuliez and Jeannerat was not undertaken because the simpler and more direct approach of phos-phorous determination had fa i l e d to show any similarity between these fractions and those of Warner which were claimed to be electrophore-t i c a l l y homogeneous. In a l l probability 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 reason-able doubt, that an alpha casein similar to that described by Warner had indeed been prepared. The preliminary experimental hydrolysis of commercially pre-pared casein was merely an attempt to establish conditions for conven-iently 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 iden-t 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 in 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 is actually a mixture of two or more electrophore-tically 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 is 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 phos-phoric acid is resistant to hydrolysis by acid of higher concentration > \" -and temperature than that employed in 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 of l iberat ion of inorganic phosphorous under conditions similar to those employed i n the present work for the hydrolysis of alpha casein, was reported by Damodaran and Ramachandran (4) to be negl ig ible . We now arrive at the conclusion that the production of at least two phospho-peptones from alpha casein was probably not a result of the action of acid on the l a t t e r , but rather a result of the action of pepsin. Speculations concerning the structure of alpha casein i n an attempt to explain these findings are admittedly hazardous and lacking i n ex-perimental foundation. It seems not unreasonable, nevertheless, to suggest as a working hypothesis that phosphorous i s not distributed uniformly within the alpha casein molecule, but that at certain places i t i s more concentrated than at others. I f peptic hydrolysis occurred between these l oca l i t i e s of re la t ive ly high phosphorous content, several different phosphorous-containing products might resu l t . I t i s suggested that an examination of X-ray di f fract ion patterns of alpha casein before and after treatment with sodium hydroxide (which would remove organically bound phosphorous (2) ) might result i n the d i s -appearance of certain periodici t ies due to centers of phosphorous con-centration. Although highly speculative, the foregoing i s intended to outline a possible direction for further investigation. Examination of the alpha phosphopeptone II hydrolysate by f i l t e r paper part i t ion chromatography, demonstrated with reasonable certainty, the presence of serine, glutamic ac id , cystine, alanine, valine, proline and one or more of the leucines. In a l l cases the phenol R F values obtained with the controls were employed for purposes o f comparison, even when these d i f f e r e d somewhat from the values r e -por t e d i n the l i t e r a t u r e , as i n the case of glutamic a c i d and s e r i n e . I n the two dimensional chromatograms, where two phenol Rp values were s i m i l a r , the corresponding c o l l i d i n e Rp values d i s t i n g u i s h e d the s p o t s . I n such cases the c o l l i d i n e Rp values o f W i l l i a m s and K i r b y were suc-c e s s f u l l y used as a check. In a d d i t i o n t o t h e above-mentioned amino a c i d s , the h y d r o l y -sate was shown to 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 . No conclusion can be drawn, however, regarding the r e l a t i v e amounts of amino a c i d s nor t h e i r s p a t i a l arrangement i n the peptone. I n view o f i t s r e l a t i v e -l y l a r g e N/P r a t i o and the number o f amino a c i d s i t y i e l d s on h y d r o l y s i s the phosphopeptone examined appears t o be more eomplex than those l i s t e d i n Table I . An i n s t r u c t i v e comparison o f t h i s peptone w i t h the products of t r y p t i c h y d r o l y s i s of 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 the other hand, f a i r l y good agreement w i t h the r e s u l t s o f workers using pepsin (see p.3). I t must be re-emphasized, i n con-c l u s i o n , t h a t the present work i s the f i r s t , t o the author's knowledge, t o d e a l w i t h the products o f p e p t i c h y d r o l y s i s o f a s i n g l e c a s e i n c o n s t i t u e n t . The r e s u l t s p r e v i o u s l y reported by o t h e r workers i n t h i s f i e l d can thus be c o r r e l a t e d and c l a r i f i e d o n l y i n reference t o the present work and a necessary p a r a l l e l i n v e s t i g a t i o n o f beta c a s e i n . SUMMARY 1 Casein has been fractionated by two methods reported in 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 in the presence of sulphuric acid of pH 1.0 at 50-52°C. The residue from this hydrolysis was exhaustively hydrolysed under the same con-ditions, 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 parti-tion chromatography. The results indicate that the hydrolysate contained cystine, glutamic acid, serine, alanine, one or more of the leucines and possibly valine. In addition, proline may have been present. Three constituents, apparently present in traces, were not identified. 6 Qualitative colour tests indicated that the hydrolysate contained iron (presumably from the apparatus used i n fractionation), tyrosine and possibly arginine. BIBLIOGRAPHY 1 Posternak, S., U.S.Patent 1, 555, 517, Sept. 29, 1927. 2 Rimington, C , Biochem. J., 21, 204, 1179, 1187 (1927). 3 Levene and H i l l , J. B i o l . Chem.. 10. 711 (1933). 4 Damodaran and Ramachandran, Biochem. J., 35>122 (1941). 5 Svedberg, Carpenter and Carpenter, J. Am. Chem. Soc, 52, 241 & 701 (1930). 6 Carpenter, J. B i o l . Chem., 54, 1012 (1931). 7 Warner, R.C., J.Biol. Chem.. 66, 1725 (1944). 8 Lubavin (1871), from Raudnitz, Ergeb. Physiol., 2. 193 (1903). 9 Salkowski, Z. Physiol. Chem., 32, 245, (1901). 10 Dietrich, Biochem.Z.. 22, 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., 26, 1989 (1932). 13 Jones and Gersdorff, J. B i o l . Chem., 106, 707 (1934). 14 Linderstrom-Lang, Compt. Rend. Trav. Lab. Carlsberg, 17, No. 9 (1929). ' ' 15 Utkin, Biochem. Z., 283, 233 (1936). 16 Posternak and Pollaczec, Helv. Chem. Acta, 24, 1190 (1941). 17 Nicollet and Shinn, Abstracts. Chicago Meeting, Am. Chem. Soc. P20-B (1946). ' ~~ 18 Lowndes, Macara and Pliramer, Biochem. J., 35, 315 (1941). 19 Rimington, Biochem. 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(1949). 37 Consden, Gordon and Martin, Biochem. J., 38, 224 (1944). 38 Williams and Kirby^ S c i e n c e , 1 07, 481 (1948 ) . 39 Townsley, D., Bachelor's Thesis, Univ. of B.C. (1950). 40 Lipmann, Biochem. Z.. 262, 3, 9 (1934). 41 Totani, Biochem. J., % 385 (1915). 42 Lloyd, D.J., Chemistry of the Proteins, P. Blakiston and Co. Inc., (1938). 43 Sakaguchi, J. Biochem. (Japan), 5. 25 (1925). 44 Rimington and Kay, Biochem. J., 20, 777 (1926). 45 Fiske and Subbarow , J. B i o l . Chem. 66, 375 (1925). 46 Lipmann, Naturwissenschaften, 21, 236 (1933). "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0062318"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Chemistry"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Investigations into the constitution of casein"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/41429"@en .