THE PREPARATION. PROPERTIES AND METABOLIC 
 BEHAVIOR OF DEAMINIZED PROTEINS 
 
 MAX SHAW DUNN 
 
 A. B. Simpson College, 1916 
 M. S. University of Illinois, 1918 
 
 THESIS 
 
 Submitted in Partial Fulfillment of the Requirements for the 
 
 Degree of 
 
 DOCTOR OF PHILOSOPHY 
 
 IN CHEMISTRY 
 IN 
 
 THE GRADUATE SCHOOL 
 OF THE 
 
 UNIVERSITY OF ILLINOIS 
 1921 
 
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1 . 
 
 INTRODUCTION. 
 
 One of the important prohlems involved in the proof of 
 the structure of the protein molecule is that of the nature 
 of its basicity. It is believed that native proteins are 
 basic because of amino groups which, instead of being in 
 peptide linkage with carboxyl groups, exist free in the 
 molecule. In endeavoring to ascertain which of the various 
 amino acids contain the amino groups responsible for the 
 basicity of native proteins, Skraup (l) found that lysine 
 and tyrosine were lacking in proteins which had been deamin- 
 ized with nitrous acid while arginine and histidine were 
 present in diminished quantities. Therefore the assumption 
 that lysine and tyrosine and possibly arginine and histidine 
 contain amino groups free in the protein molecule seemed 
 tenable. Kossel and Cameron (2) have shown that arginine does 
 have an amino groun free in the protein molecule but that 
 this group is contained in the guanidine nucleus which does l 
 not react with nitrous acid. Van Slyke (3) and others (4) 
 have found that the nitrogen liberated from native proteins i 
 by the action of nitrous acid is approximately one-half of 
 the lysine nitrogen present. Because of these observations. 
 
 Van Slyke has put forth the theory that the free amino nitro- 
 gen of native proteins is due almost entirely to one of the 
 amino groups of lysine. Since it requires thirty minutes for 
 the deaminization of native proteins and for the cleavage of 
 

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 in 2016 
 
 https://archive.org/details/preparationpropeOOdunn 
 
 
the epsilon amino groun of lysine hy this reagent, the free 
 amino groups of native proteins are commonly thought to he 
 due, almost all if not entirely, to the terminal amino group 
 of lysine. However, it was the view of Kossol (5) that the 
 existence of a quantitative relation hetween the free amino 
 nitrogen and the lysine content of a protein was improbable . 
 Evidence in supnort of this view has recently been obtained 
 by Edlbacher (6) and by Felix (7). The former investigator 
 in studying the action of methylating agents upon native pro- 
 teins found that the lysine free proteins, clupeine and sal- 
 mlne, methylated as easily as other lysine containing proteins, 
 while formaldehyde reacted only with the proteins of the latter 
 character. These observations led to the conclusion that 
 there are free amino groups in the protein molecule other 
 than that of lysine. Herzig (8) has recently reported that 
 deaminized gelatin is methylated as easily as gelatin itself. 
 These observations would seem to establish the existence of 
 groups in the protein molecule, other than the free amino 
 group of lysine, which are capable of methylation. However, 
 it does not follow of necessity that the groups which have 
 been methylated are free amino in character. Additional ob- 
 jections to the theory proposed by Van Slyke have been raised 
 by Felix, who believes that the lysine determination by the 
 Van Slyke partition method gives high values for this amino 
 acid ov/ing to the precipi tation by phospho tungstic acid of 
 substances, such as phenyl alanine or proline, in addition to 
 the amino acids of the hexone bases. If, on the other hand, 
 the figures given for lysine by the Kossel method of direct 
 

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3. 
 
 
 isolation are accented, one half of the lysine nitrogen of the 
 proteins investigated will not evon approximate the free 
 amino nitrogen as determined by the nitrous acid method. This 
 author wishes to note further that the values for the free 
 amino nitrogen of certain proteins, particularly gelatin, as 
 determined by the nitrous acid method and by formol titration 
 are not in agreement. Furthermore, it was found by these in- 
 vestigators in the case of histones, sturin, gelatin and gly- 
 cinin that the free amino nitrogen as determined by the 
 nitrous acid method so exceeded one half of the lysine nitro- 
 gen of these nroteins that the existence of free amino groups 
 other than that due to lysine seemed probable. 
 
 The problem of the basicity of the protein molecule has 
 been attac-^ed in another way by studying the acid-combining 
 capacity of proteins. Robertson (9) believes that the ex- 
 periments of Blasel and Matula and those of Pauli and Hirsch- 
 feld are ” direct proof that the terminal groups are not 
 
 responsible for any apDreciable oortion of the acid combining- 
 
 capacity of proteins. These investigators found that 
 
 the combining-caoaci ty of deaminized gelatin for acids is but 
 slightly inferior to that of ordinary gelatin, indicating, 
 beyond any question, that the combining-capaci ty of gelatin 
 for acids, is in very large proportion, attributable to ele- 
 ments of the molecule other than the free -NH groups. The 
 inference is unavoidable that the elements of the molecule 
 which actually participate in the union with acids are, in 
 very large pronortion, the -COHN- groups within the body of 
 the protein molecule.” Bracewell (10) is of the opinion 
 
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4 . 
 
 that ” the amount of acid 'neutralized’ "by a considerable 
 number of proteins is determined by the free amino groups in 
 the protein molecule." By taking into account the possibility 
 of a free amino group in arginine, this investigator believes 
 that "the amount of acid absorbed by ordinary proteins is 
 determined mainly by their content of lysine and arginine." 
 
 In the oresent investigation it was considered to be of 
 interest and iraoortance to make a careful study of a typical 
 deaminized protein, i.e. deaminized casein, and to determine 
 to what extent the amounts of various amino acids, the dis- 
 tribution of nitrogen in the molecule, the metabolic behavior 
 and other properties had been altered by the processes of 
 deaminization. 
 
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5 . 
 
 EXPERIMENTAL . 
 
 A. The PreDaration of C asein and Deaminized Casein . 
 
 Although the original observations on the preparation of 
 casein from milk were made by Hoppe (11) in 1359, it remained 
 for Hammarsten (12), in 1877, to accurately describe the pre- 
 paration and nurification of this protein. Since that time 
 various modifications (13) of Hammarsten ' s method have been 
 proDOsed. 
 
 The procedure used in the present study is an adaptation 
 of the more modern methods for casein preparation. This 
 method is given in detail. Two gallons of skim milk are added 
 to 10 liters of distilled v/ater contained in a carboy of 
 about 50 liters capacity. Dilute acetic acid, made by adding 
 6 c.c. of glacial acetic acid to a liter of distilled water, 
 is very slowly added to this water-milk mixture, which is 
 mixed uniformly by stirring with a current of air. After the 
 addition of 5 to 6 liters of the acetic acid solution, the 
 casein flocks out completely and settles ranidly to the bottom 
 
 I 
 
 of the carboy. The yellowish, supernatant liquid is siphoned 
 off and the precipitated casein washed with 10 or more liters 
 of distilled water; the supernatant liquid is again siphoned 
 off and the washing process repeated. The casein remaining 
 from the final washing is dissolved by the addition of a 
 solution of dilute ammonium hydroxide, containing 6 c.c. of 
 concentrated ammonium hydroxide to a liter of distilled water. 
 About 7 liters of alkali will completely dissolve the casein 
 

 
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to give an onalescent solution, which is then filtered through 
 a thick layer of absorbent cotton. The whole process of pre- 
 cipitating with acid and dissolving in alkali is repeated 3 
 times. After the last precipi tation, the casein is washed 
 tvace with distilled v/ater, the sunernatant liquid decanted 
 and the purified product filtered on a Buchner funnel using 
 hardened filter naper and suction. The compact cake, which 
 is formed, is triturated in a mortar with 95 % alcohol and the 
 trituration with alcohol is repeated 3 times. After tritur- 
 ating 3 times with dry ether to completely dehydrate the 
 casein cake theproduct is dried for 3 hours in air and finally 
 for 30 minutes in the oven at 80° C. The resulting white 
 product may be powdered to a dust in a ball mill or by passing 
 it through an 80 mesh sieve. 
 
 Nitrous acid was first used as a deaminizing agent for 
 proteins in 1885 by Loew (14). This investigator found that 
 one-third of the nitrogen of peptones was liberated by the 
 action of nitrous acid. In 1896 Paal (15) used silver nitrite 
 and hydrochloric acid to deaminate gelatin peptones, while in 
 the same year Schiff (16) obtained a straw yellow compound by 
 the interaction of nitrous acid and egg albumin. Two years 
 later SchrStter (17) observed the formation of a similar sub- 
 stance from peptones. In 1908, Treves and Salomons (18) al- 
 lowed nitrous acid to react with egg albumin and obtained a 
 yellow product which they believed was diazo albumin. De- 
 aminized gelatin was obtained by Blasel and iiatula (19) in 
 
 1914. 
 
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7 . 
 
 SkrauD (20)and his pupils have prepared and studied the 
 deaminized products of the following proteins: casein (1); 
 
 gelatin (21); albumin (22); globulin (23); and edestin (24). 
 Deaminized albumin, gelatin, and casein (25) and deaminized 
 gliadin and vitellin (26) have been prepared and investigated 
 by Levites. 
 
 Levites and Skraup have been largely responsible for 
 perfecting the methods used in preparing deaminized protein 
 products. In the first (25) method of Levites, a paste made 
 from the nrotein and sodium nitrite was warmed on the water 
 bath with dilute acetic acid and the resulting olive green 
 product was dried in vacuo. In his second (25) procedure, 
 Levites produced an emulsion of the protein by agitating it 
 vigorously in a shaking machine with 10^ acetic acid. This 
 emulsion was warmed gently on the water bath with a 10^ solu- 
 tion of sodium nitrite and the resulting yellow product fil- 
 tered, washed with alcohol and ether and dried in vacuo. In 
 the method used by Skraup (1) an acid solution of the protein, 
 prepared by adding glacial acetic acid to a uniform suspension 
 of the protein in water, was warmed gently with sodium nitrite 
 on the water bath and the yellow product drained off on linen, 
 desiccated and dried in air. 
 
 In the present series of experiments deaminized casein 
 was prepared according to the methods outlined by Levites and 
 by Skraup. It was difficult to obtain a product of uniform 
 color and appearance while the yield of 70 percent reported 
 by Skraup was rarely exceeded. There are several objections 
 to the use of a shaking machine as employed by Levites. It 
 

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8 . 
 
 requires several hours to emulsify small amounts of protein 
 and the emulsified product invariably contains gelatinous 
 lumps which must he ground uo in a mortar to avoid contamin- 
 ation of the deaminized product with unchanged protein. All 
 of the methods used for the nraparation of deaminized pro- 
 teins employ heat up to 40° C. to effect complete deaminiza- 
 tion of the protein. It is possible that this apolication of 
 heat may cause a slight hydrolysis of the protein. 
 
 It is believed that the following procedure overcomes 
 the objections cited above. 100 grams of casein were added to 
 2 liters of distilled water contained in a 5-liter Pyrex 
 flask. After stirring vigorously for 30 minutes with an 
 electric stirrer a uniform suspension of the protein resulted. 
 To this suspension 140 c.c. of glacial acetic acid were added 
 dropwise, with continued stirring, during the course of 1.5 
 hours. At the expiration of 20 minutes a good emulsion was 
 formed while at the end of the period a solution was effected. 
 To this solution, 500 c.c. of a solution of sodium nitrite 
 containing 80 grams to a liter was added dropwise, with con- 
 tinued stirring, during the space of 1.5 hours. After 150 
 c.c. of this solution had been added a deep yellow precipitate 
 rose to the top of the liquid as a yellow layer which, after 
 standing for 18 hours, was filtered on a Buchner using suction 
 and a hardened filter paper. After triturating this substance 
 15 times with hot water to the disappearance of an acid re- 
 action to litmus, it was granular and light yellow in color 
 while the aqueous filtrate was similarly colored. The yellow 
 
'/ 
 
 
 i 
 
 h 
 
precipitate obtained by triturating four times with 95 % 
 alcohol was thoroughly desiccated by triturating three times 
 with dry ether, drying in air for 30 minutes and in the oven 
 at 80° C. for an equal length of time. Although the alcoholic 
 filtrate was highly colored the precipitate appeared to have 
 lost but little of its yellow color in the washing process. 
 
 The deaminized product was of a uniform color and 
 appearance and it was possible to secure practically a com- 
 plete transformation into the deaminized form. From three 
 100-gram samples of the original casein yields of 90, 95 and 
 97.5 grams of the oven dried product were obtained. These 
 deaminized products designated subsequently as deaminized 
 caseins A-64, A-66 and A-68 were very fine and pov/dery when 
 passed through an 80-mesh sieve. They were colored light 
 yellow when first prepared but surfaces exposed to the light 
 for a time became light brown. 
 
 With the second method of Levites, 17 grams of deaminized 
 casein (A - 18) were prepared from 15 grams of casein. How- 
 ever, instead of permitting a complete deaminization to 
 occur, the white precipitate which was formed upon the addi- ! 
 tion of the sodium nitrite solution was filtered on a Buchner ! 
 
 as soon as possible. This precipitate which was triturated 
 14 times with hot water, 3 times with 95^ alcohol, and twice 
 with dry ether, was only faintly colored yellow. 
 
10 . 
 
 B. The ProT)ertles of Deamlnl zed Proteins . 
 
 1. Color. Without exceotion, the deaminized protein 
 nroducts cited in the literature are yellow in color. Treves 
 and Salomons (18) believed that these substances were diazo 
 derivatives since they responded to the reactions given by 
 diazo compounds. It is known, however, that primary aliphatic 
 amines do not react with nitrous acid under ordinary condi- 
 tions to give stable diazo derivatives. On the other hand, 
 were this a reaction with the amide groups which are nresent 
 acids would be produced (27) and not diazo products. There- 
 fore, the assum-ntion of these authors seems untenable. 
 
 It is possible that deaminized proteins are colored be- 
 cause of the formation of nitroso compounds. There are numer- 
 ous possibilities for nitrosation in the protein molecule. 
 Histidine, tryptophane and proline each have one imino nitro- 
 gen, while there are two such nitrogens in the guanidine group 
 of arginine. Phenyl alanine would admit of nitrosation in 
 the para position of its benzene nucleus, while a nitroso 
 group might enter tyrosine in the position ortho to the hy- 
 droxy group in the benzene ring. It is unlikely that a nitro- 
 sation of the imide nitrogen making un the peptide linkage 
 has taken nlace because this imide is probably of insufficient 
 basicity, due to the neutralizing action of the adjacent car- 
 bonyl group, to react with nitrous acid. However it has re- 
 cently been sho\vn (23) that the nitroso derivative of methyl 
 phthalimidine is easily formed by treatment with nitrous acid 
 in water solution. Since the carbonyl imide linkage present 
 in methyl phthalimidine is the same as that found in peptides 
 
11 
 
 a possible nitrosation of the peptide linkage is suggested. 
 However, this cannot have taken place to any narked extent 
 because deaminized nroteins have been shown to contain less 
 nitrogen than the original substances. 
 
 Preliminary to carrying out some qualitative exneriments 
 upon several nure amino acids, it was found that the red solu- 
 tion formed by the action of nitrous acid upon phenol responds 
 to Liebermann’s nitroso reaction by giving a green solution 
 with concentrated sulnhuric acid. Tyrosine was found to give 
 a similar reaction after warming the solution gently while 
 indole reacts in much the same way although the resulting 
 color is of lesser intensity. Histidine and phenyl alanine 
 give no red color with sodium nitrite and glacial acetic acid 
 but upon the further addition of concentrated sulphuric acid 
 a green color is nroduced. It would appear from tnese tests 
 that the yellow to brown color of deaminized proteins may be 
 due in part to nitroso derivatives of tyrosine and possibly 
 tryptophane. Prom the fact that gelatine, a tyrosine free 
 protein, is renorted to give yellow deaminized derivatives, 
 it would seem that this color cannot be due alone to the | 
 
 formation of nitroso derivatives of tyrosine. However, it is 
 well known that samples of gelatin, considered to be tyrosine 
 free, still respond to Millon's test and Dakin (29) has re- 
 cently found traces of tyrosine in all specimens of gelatin 
 examined. 
 
 2. Solubility. Deaminized proteins are reported to be 
 insoluble in water and insoluble or only slightly soluble in 
 alkalies. Skraup noted the formation of a jelly-like substance 
 
 " — " -«i 
 

 r'lTipr'. ’ ’T- ■ 
 
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 rxld jj;f03 ffd 
 
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12 . 
 
 when deaminized proteins were brought into contact with strong 
 alkali. It was found in the present investigation that de- 
 aminized casein dissolved in .5^ sodium hydroxide after 
 standing for two days with the formation of a red solution 
 and a small amount of undissolved residuum. With 1.5!^ 
 sodium hydroxide solution, a red solution was formed in 24 
 hours, while with concentrated alkali an orange to brown 
 jelly was formed in a fev; minutes. 
 
 3. Color Reactions. Deaminized casein prepared ac- 
 cording to the method described above, gave positive tests 
 with the Hopkins-Cole , biuret and Millon's reagents. Millon's 
 test was unquestionably positive, although the color was less 
 intense than with casein, but the biuret reaction with de- 
 aminized casein was not characteristic ranging from a pink to 
 a reddish purnle color. Levites is the only investigator to 
 report a positive Millon's reaction with deaminized proteins, 
 while the biuret reaction was found to be positive by Levites 
 and by Treves and Saloraone. If it be true that deaminized 
 proteins give a positive biuret reaction, this would indicate 
 that the amide grouping which is responsible for the color 
 
 is not attacked or at least is only partially destroyed by 
 the action of nitrous acid. This grouping, according to 
 Schiff , is two CONH groups in union. 
 
 4. Composition. The elementary composition of native 
 proteins appears to be but little altered in the deaminiza- 
 tion process. Skraup (20) found a slight diminution in the 
 phosphorous content of deaminized casein and a constant in- 
 crease, with one excention, in the oxygen content of all of 
 

 Pwf ' ■ . • V 
 
 ' . T ^T'; 
 
 •J r’' •' C 'l ' ■ *. 7' un ' inir 
 . f«;.-'v'v^: ■.('»! ; X '■ ncrc'. 
 
 
13. 
 
 li 
 
 the deaminized -oroteins studied hut neither observation was 
 considered to be of particular signif icance . It is striking, 
 however, that the nitrogen content of deaminized proteins is 
 lower than that of the original substance. Schiff (16) re- 
 ports a reduction of 1 percent in the values for nitrogen 
 while the figures quoted by Skraup (20) range from .51 to 1.24 
 per cent lov/er in nitrogen than those of the original proteins. 
 In the case of edestin (24) for some unaccountable reason 
 the nitrogen of the deaminized product was found to be higher 
 than that of the original protein. 
 
 It will be noted from Table I that the nitrogen content 
 of the deaminized casein prepared in this research ranges for 
 the various sa’^nles from .22 to .68 per cent lower than the 
 figures given for the original casein. 
 
 TABLE I. 
 
 Nitrogen calc, on 
 
 Samnle 
 
 
 Ash 
 
 an ash free basis 
 
 
 
 
 
 io 
 
 Casein 1 
 
 
 
 .38 
 
 14.56 
 
 Deaminized 
 
 casein 
 
 A-18 
 
 .96 
 
 13.91 
 
 Deaminized 
 
 casein 
 
 A-64 
 
 1.21 
 
 14.34 
 
 Deamini zed 
 
 casein 
 
 A-66 
 
 .53 
 
 13.88 
 
 Deaminized 
 
 . casein 
 
 . A-63 
 
 .49 
 
 14.01 
 
 5. Free Amino Nitrogen. The first apnlication of the 
 reaction between nitrous acid and amino compounds was made by 
 Sachsse and Korraann (30) in 1874. The volume of nitrogen lib- 
 erated by the action of nitrous acid upon plants was considered 
 
14 . 
 
 to be a measure of the amide grouos present. In more recent 
 times Brown and Willar (31) have studied this reaction further 
 and have made certain improvements unon the apparatus used by 
 the original investigators. It remained, however, for Van 
 Slyke (32) in 1909 to perfect the method for purifying and 
 measuring the nitrogen evolved in this reaction. Because 
 of the fact that amides react very slowly or not at all with 
 nitrous acid under the conditions employed, it is believed 
 that the nitrogen liberated from proteins and from protein 
 derivatives comes entirely from the primary aliphatic amines 
 existing free in the molecule. 
 
 The determination of the free amino nitrogen of native 
 proteins is one of the many applications (33) which have been 
 made of the Van Slyke procedure. In the present study of 
 native proteins and their derivatives, the free amino nit- 
 rogen of casein, deaminized casein and gelatin was determined 
 according to the method outlined by Van Slyke (34) and using 
 the micro apparatus. To a suspension of 3 grams of the pro- 
 tein in distilled water was added a solution containing .375 
 grams of sodium carbonate. The proteins went completely in 
 solution usually within an hour. This protein solution was 
 transferred to a 100 c.c. volumetric flask, diluted to the 
 mark and 2 c.c. taken for amino nitrogen analysis. The 
 casein or gelatin solution formed in this manner is neutral 
 to litmus and Van Slyke (34) has shown that an inappreciable 
 hydrolysis takes place even after standing at room temperature 
 for 48 hours. In the deaminization of casein a foam inhibitor 
 was found to be indispensable . For this purpose, caprylic 
 
15 . 
 
 alcohol was found to he more effective than di phenyl ether, 
 although the blank resulting from the former substance was, 
 as has been recorted (35), considerably higher than that given 
 by di phenyl ether. Even without the caprylic alcohol, the 
 blank from the sodium nitrite was in general higher than that 
 reported by Van Slyke for good samples of this substance. 
 
 Casein was found to nrecipitate from solution immediately 
 upon contact with the acid solution in the deaminizing cham- 
 ber and to gradually change from a pure white to yellow. 
 Because of the fact that casein must undergo deamini zation 
 while in the solid state reliable results are to be obtained 
 only by maintaining constant conditions. Uniform results 
 were secured by shaking the deaminizing chamber for 30 minutes 
 at 300 vibrations ner minute. Gelatin is not precioi tated by 
 the acid solution of the deaminizing chamber and does not im- 
 part a color to the liquid during the process of deaminization . 
 
 Van Slyke (3) has renorted that the free amino nitrogen 
 of casein comnrises 5.51 ner cent of its total nitrogen con- 
 tent. Since there are various methods which are used for 
 the preparation of casein from cow's milk, it was considered 
 of importance to determine the free amincjbi trogen of samples 
 of casein prepared in a variety of ways on the assumption 
 that the reagents emnloyed in the purification of this protein 
 might possibly have altered its free amino groups. However, 
 the results which were obtained (see Table II) were not 
 widely divergent and it would seem that the reagents employed 
 in the prenaration of these samples of casein were without 
 appreciable influence unon the number of free amino groups 
 
Vi- irtif 
 
 f* ' I ‘ ^ \ *■ '.-' ^' , . S'y ‘ ■ VV 
 
 ;.t'-; '0iije£rOt»,y d ^ 
 
 I ' ■, ' '' . -Jp. -\ ■ ,'T-'' ‘■■‘♦Jr , 
 
 8 ‘' 
 
 i • * ' ’.'f:'/^Y% *_‘rt--A>*r ' ^ ^ .’1 I* ffu r W “'^Ur'JOsrV) a.- ^*rf. ■ " _ . ' ..^^■•'t' /^.J t , I 
 
 'r f 
 

 16. 
 
 of this protein. It should be noted, however, that the 
 average figure for the free amino nitrogen content of ten 
 samnles of casein was found to be 5.37 ner cent which is only 
 slightly higher than the 5.51 per cent reported by Van Slyke . 
 
 Casein A-18, which was only slightly yellow in color, 
 was prepared by treatment with nitrous acid but was removed 
 from the influence of this reagent as soon as nossible after 
 nrecipi tation . The color reactions given by this nroduct 
 were unquestionably nositive and quite comparable to the re- 
 actions given by casein itself. As is given in Table II, the 
 free amino nitrogen of this product was 3.10 per cent of the 
 total nitrogen, while that of completely deaminized casein 
 A-64 was 0.0. Since the value for casein A-18 lies midway 
 between that given by unchanged casein on one hand and com- 
 pletely deaminized casein on the other, it would seem that 
 at the time of nrecipi tation only partial deamini zation had 
 occurred. 
 
 The free amino nitrogen content of two samples of gelatin 
 was determined and found to be 5.29 and 5.41 per cent of the 
 total nitrogen figure. These values are in agreement with 
 the figure, 5.2 per cent, which was reported by Felix (7) 
 but differ from the nercent, 3.12, found by Van Slyke (3). 
 
 6. Total Amino Nitrogen. The conditions necessary for 
 the complete hydrolysis of proteins have been determined. 
 Comparable results have been secured by heating in an auto- 
 clave at 150 ° C. for 1.5 hours with 3.0 N hydrochloric acid 
 (37) and by boiling at 100*^ C. for 24 to 43 hours with 20^ 
 
1 
 
 17 . 
 
 TABLE II. 
 
 Casein, 
 
 Casein, 
 
 Casein, 
 
 Casein, 
 
 Casein, 
 
 If 
 
 II 
 
 ft 
 
 If 
 
 ft 
 
 The Free Amino Nitrogen Content 
 of Casein, Deaminized Casein and Gelatin. 
 
 Percent of total nitrogen 
 
 Samnles as free amino nitrogen 
 
 Kahlhaum 5.61 
 
 Kahlhaum nach Hammersten 5.99 
 
 after Van Slyke and Bosworth (13) 5.63 
 
 after Baker and Van Slyke (36) 5.52 
 
 Oshorne (# ) No. 1 6.13 
 
 ” "2 5.84 
 
 " ”3 6.24 
 
 ” ”4 6.22 
 
 " "5 6.04 
 
 " ”6 5.47 
 
 'Deaminized* casein, A-18 3.10 
 Deaminized casein, A-64 .00 
 Gelatin, renurified Gold Seal 5.29 
 Gelatin, Gold Seal 5.41 
 
 # Casein samples No. 1 to No. 6 were secured through 
 the kindness of Prof. T. B. Osborne of the Connecticut 
 Agriculture Experiment Station, New Haven, Connecticut. 
 
 In every case unusual procedures were used in the prepara- 
 tion of these samnles. 
 
-w. 
 
 
 
 
 
 k: 
 
 r 
 
 •( 
 
 V 
 
 ■V,., 
 
 &, 
 
 * ^ w 
 
 
 
 '■r:.r . 
 
 ( 
 
 f 
 
 i . 
 
 V 
 
 ('f' 
 
 y 
 
 I ' • < ■ 
 
 r • 
 
 ^ • . ' » 
 
 ► T 
 
 
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 ‘U, ti.'Ti 
 
 I.' 
 
 : 
 
 c 
 
 ; i' r> _ T;t- 
 
 o. ■ 
 
 r. 
 
 i . 
 
 > 
 
 . . ''■■ ’'■ 
 
 ■ji' ■ 
 
 
 
 / 
 
 Jr. 
 
 
 
 < . - ,;■ 
 
 
 
 \>3.i 
 
 
18 . 
 
 hydrochloric acid (r58). With either of these methods the 
 maximum amount of amino nitrogen is liberated from peptide 
 linkage but according to Van Slyke there is less tendency 
 towards deaminization of amino acids, particularly cystine 
 (39), at 100° C. than at 150 or 160° C. 
 
 Complete hydrolysis of 5-gram samoles of casein and deam- 
 inized casein was effected by autoclaving for 3 hours at 124° 
 
 C. with 200 c.c. of 3.0 N hydrochloric acid. Henriques and 
 Gjaldb8,k (37) found that under these conditions the results 
 of hydrolysis were anproximately the same as those obtained 
 by heating for 1.5 hours at the higher temperature. In both 
 cases some undissolved narticles remained after autoclaving 
 and the supernatant liquid of the deaminized casein was color- 
 ed a deeper brown than that obtained from casein. Each hydrol- 
 ysate was evaporated to dryness on the water bath, taken up 
 with water and diluted to the mark in a 500 c.c. volumetric 
 flask. Amino nitrogen analyses were made on 1 c.c. portions 
 of the hydrolysates by means of the Van Slyke micro apparatus. 
 
 The total amino nitrogen of casein was found to be 10.08 
 per cent of its total nitrogen content while 9.84 per cent of 
 the total nitrogen of deaminized casein was present in the 
 amino form. Since the difference observed between these per 
 cents isprobably within the range of experimental error, it 
 is believed that the total amino nitrogen of casein and de- 
 aminized casein is the same. 
 
 The Tyrosine Content of Deaminized Proteins . 
 
 Despite the fact that some investigators have obtained a 
 
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19 . 
 
 positive Millon’s reaction with deaminized proteins the 
 statement has been made that "Von den Monoaminos&uren ist zu 
 sagen, dass ihr Gehalt quantitativ wahrscheinlich unverS,ndert 
 hleibt, mit Ausnahme des Tyrosins, das bei alien Desaminido- 
 nroteinen fehlt," (26). This conclusion is based largely upon 
 the work of Skraun (l) who could not obtain crystalline 
 tyrosine from deaminized proteins although other amino acids 
 were isolated in amounts comnarable to those present in the 
 original protein. Since deaminized casein was found to give 
 a positive Millon's test in the present study, the^resence of 
 tyrosine would seem to be indicated. That this assumption 
 was correct was definitely proved by the following experiment. 
 Ten-gram samples of casein and deaminized casein were hydrolyz- 
 ed for 12 hours with 200 c.c. of 20 % sulphuric acid. Some of 
 the humin which was formed was removed from the hydrolysates 
 by filtering on fluted filter papers. The sulphuric acid 
 was neutralized with a suspension of nure barium hydroxide 
 and the resulting white precioitate of barium sulphate was 
 filtered on fluted filter pacers. After concentrating both 
 solutions upon the water bath until a scum of barium salts 
 began to form ucon the surface of the deaminized protein 
 solution the excess of barium was removed by precipitation 
 with dilute sulnhuric acid solution. Since a water clear 
 filtrate could not be obtained the barium sulphate was allowed 
 to settle out in tall cylinders and the clear supernatant 
 liquid, which was tinged slightly yellow in each case, was 
 siphoned off. Both filtrates were concentrated upon the 
 

 
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 y ' ( . 
 
 
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 Vi 
 
 
1 
 
 20 . 
 
 water bath until crystallization had begun. After standing 
 overnight the mother liquors were filtered on a Buchner 
 funnel and the crystals of tyrosine sucked dry. The impure 
 tyrosine from casein was tinged a grayish white while the 
 deaminized product was yellow but after recrystallization 
 from hot water, the tyrosine obtained from each source was 
 pure white. No effort was made to obtain a quantitative 
 yield of tyrosine from casein and deaminized casein. However, 
 the amount of this amino acid isolated from the latter protein 
 seemed only slightly less than that obtained from casein. 
 
 The amino acid isolated from casein and deaminized casein 
 was identified by its crystalline form. In addition the 
 color reactions with Millon's and Mftrner's reagents were 
 positive. 
 
 Prior to 1912, the quantitative estimation of the ty- 
 rosine in proteins was carried out by isolation of the pure 
 substance. Abderhalden (40) found 4.5 per cent of tyrosine 
 in casein and this is the figure which is usually quoted. 
 
 In 1912, the discovery that the blue color given by a solu- 
 tion of phospho tungstic and phosphomolybdic acids with phenols 
 was specific for the phenol group, led to the development of 
 Polin’ s (41) colorimetric method for the determination of 
 tyrosine in proteins. Although tyrosine is the only amino 
 acid in the protein molecule which has this groaning, Abder- 
 halden (42) found that this blue color was also given by 
 
 tryptophane, oxy tryptophane and oxy proline. However, Johns 
 and Jones (43) have shown that the blue color with tryptophane 
 
 was less intense than that given by an equivalent amount of 
 

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21 . 
 
 tyrosine while decomposition products of tryptophane gave no 
 color at all. Therefore, these authors concluded that re- 
 liable results for tyrosine in proteins can be obtained with 
 Folin's colorimetric method. Gortner (44) has recently 
 studied the action of Polin' s phenol reagent upon cure amino 
 acids. This investigator found that tryptophane, prolin, 
 cystin, histidine and valin gave the characteristic blue 
 color. The other amino acids which reacted with this reagent 
 were known to be impure. As a result of this study, the con- 
 clusion was reached that the colorimetric estimation of tyro- 
 sine in proteins is accurate only in the absence of trypto- 
 phane and other easily oxidizable substances. 
 
 The average figures for the gravimetric determination 
 of tyrosine in 27 different proteins was calculated by Folin 
 (41) to be 2.647 per cent while that obtained by the colori- 
 metric method was 5.065 per cent. According to Abderhalden 
 the values obtained by the colorimetric method are too high, 
 while Folin believes that the gravimetric figures are too low. 
 
 In all probability the true figure lies somewhere between 
 these extreme values. 
 
 In the nresent investigation, the tyrosine content of 
 casein and deaminized casein was determined according to the 
 colorimetric method of Folin and Denis (41). The results are 
 given in Table III. 
 
22 
 
 TABLE III. 
 
 Time of 
 Hydrolysis 
 
 Percent 
 Casein 1 
 
 of Tyrosine 
 Casein A-' 
 
 hours 
 
 
 
 12 
 
 5.56 
 
 3.83 
 
 16 
 
 5.98 
 
 4.03 
 
 19 
 
 5.78 
 
 3.57 
 
 Average 5.77 3.82 
 
 It will "Quoted that the average figure for the tyrosine 
 content of casein was found to be 5.77 per cent and that this 
 is .73 per cent lower than the value, 6.50 per cent, found by 
 Folin. Johns and Jones (43) believe that some tyrosine is 
 destroyed if acid hydrolysis is continued longer than 12 hours. 
 The data obtained in the nresent study do not support this 
 contention. It is evident, however, that some of the tyro- 
 sine or the substances reacting with the Folin reagent has 
 been destroyed during the process of deamini zation, since 
 the tyrosine content of deaminized casein was found to be 
 only about 66 per cent of that of casein. 
 
 D. The Distribution of Ni trogen in Gasein and Deaminized 
 
 Casein . Owing to the inadequacy of existing methods 
 for the quantitative isolation of the amino acids of the oro- 
 tein molecule coranarative determinations of the distribution 
 of nitrogen are of considerable value in the characterization 
 of proteins. 
 

 
 
 
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 jRiCf’jrb'r ©irL/y^bicr nlatfj 
 
 '■ ii . ’V, 1'- 'Y ' ' '„ 'V‘ 
 
 
2'3. 
 
 In 1899 Hausmann (45) determined the amide nitrogen, 
 the di amino nitrogen and the mono amino nitrogen of various 
 proteins. Many criticisms (46) of the methods used in esti- 
 mating the nitrogen nresent in these forms have "been made 
 and the value of the results has been questioned. The chief 
 difficulties seemed to lie in the accurate estimation of the 
 amide nitrogen and in the quantitative separation of the 
 hexone bases and cystine from the mono amino acids. Imnrove- 
 ments upon the original method of Hausmann and that developed 
 by Kossel and Kutscher (47) culminate in the well known Van 
 Slyke (48) procedure for the partition of protein nitrogen. 
 
 Although in general the Van Slyke procedure was followed 
 in the nresent investigation of the distribution of nitrogen 
 in casein and deaminized casein, certain modifications v/ere 
 incorporated from unpublished results of Grindley and Hamil- 
 ton. In each case 6 grams of casein or deaminized casein 
 were taken for dunlicate analysis and these samplos were 
 hydrolyzed in the usual manner. It was considered to be ad- 
 vantageous to distill off the excess hydrochloric acid in 
 vacuo before removing an aliquot portion for the estimation 
 of total nitrogen. By the use of a dual system for vacuum 
 distillation duplicate determinations were ’’ade under exactly 
 the same conditions of nressure. The apparatus for arginine, 
 as modified by Grindley and Hamilton permits the passage of 
 purified air through it during the entire six hours. Air 
 
 under pressure is passed through a purifying train of con- 
 centrated sulphuric acid and concentrated alkali to remove 
 carbon dioxide and ammonia and thence to the bottom of the 
 
;«■: t ■ • 
 
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 Kite' i,$L^ cjp^f - ^ f ’ ' ‘ ‘ 
 
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 ,F ■ e/k»’ 4 q x*^zV^.Pr»,. ‘^'K'' '^'*r .? f A 
 
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24 . 
 
 Kjeldattl flasks by means of a capillary glass tube which pass- 
 es through the stonoer in the Kjeldahl flask. It was found 
 that if a bubble of air per second is allowed to escape 
 from the Folin bulbs that the last traces of ammonia are 
 aerated into the standard acid and subsequent distillation 
 as recommended in the original nrocedure is unnecessary. 
 
 It has recently been shorn (49) that arginine loses 
 one-half of its nitrogen as amino nitrogen by the action of 
 nitrous acid at 20® C. for 2 or 3 hours. This observation 
 leads Sekine to believe that the formula for the calculation 
 of histidine nitrogen should be 3/2 (D- l/2 arginine nitrogen) 
 instead of 3/2 (D-3/4 arginine nitrogen). Although the de- 
 tailed renort of this investigation is not available, it 
 would seem that the action of nitrous acid upon arginine for 
 2 or 3 hours is not comnarable to the 30 minute reaction em- 
 ployed in the Van Slyke procedure. Therefore there appears 
 to be no justification for altering the formula for the cal- 
 culation of histidine nitrogen. In the present investigation 
 the Van Slyke form.ula was used. 
 
 The figures for the distribution of nitrogen in casein 
 and deaminized casein are given in Tables IV and V. On the 
 basis of these figures the percents of arginine, histidine, 
 lysine and cystine present in these proteins was calculated. 
 These results are given in Table VI. The incomplete recovery 
 of nitrogen in the first analysis was probably due to losses 
 in mono amino nitrogen. ■ The analysis for casein agrees 
 rather closely with the figures quoted in the literature (50) 
 
1 _ , •* • ' ?f :7 " ; ■ i ‘ ■*’ ■' \ ■ '■■ ' i. .., 
 
 <l • - 
 
 ’’iNM -"'‘••«d 4 (io«l 
 
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 'pr i 
 
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 rlr >€=^4 it . fer.poit^f; li. (t, 'll?- , i 
 
 ■ ^v ■:, '-i^ ' '■ ' ,^-,:/v ■ ■ V 'iS ^'. ■..-«-:%i- 
 
 ;•■- «% 3 ' 'iu i^^.l -^>19- ■ g^oi*! 
 
 'V ' ', '■' 1 ^,,'.. ; 'f :.(! ., ' . ' ' ** '■•^ * ", 5 ‘ ^ • *‘ ” *' 
 
 ‘m<t 
 
 vf^S 
 
 W'. 
 
 m ^ 
 
 ' ■ ^ '< «'■ . V'' ' ^ ■'*■ . ' ■' •■.r ■iJLfl«t?'. ' 
 
 ,__ _ , , - , ■ - , B-W'^ 
 
 ( Sift' Tvb iiti’^'J ;# e!l '8i’«:?^*'7t;|tvr3|r« 
 
 ■ -r^'O; .-^<5 irr jjiij:, - i jsi-14 t • *‘r •^flKfi 
 
 ,;... f... ,\: .^r/' ■ ,:/ ,:,„"-t'-''^ ■ \' 
 
 ' ff. ' ^'’ 'tia' I 
 
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25 
 
 TABLE IV. 
 
 The Distribution of 
 
 Nitrogen in 
 
 Casein 
 
 
 
 No. 1 
 Ni trogen 
 
 No. 2 
 Nitrogen 
 
 
 srams 
 
 per cent 
 
 srams 
 
 per cent 
 
 Amide 
 
 .0906 
 
 10.26 
 
 .0916 
 
 10.49 
 
 Humin 
 
 .0173 
 
 1.94 
 
 .0190 
 
 2.13 
 
 Arginine 
 
 .0720 
 
 8.15 
 
 .0673 
 
 7.42 
 
 Histidine 
 
 .0533 
 
 6.03 
 
 .0537 
 
 6.01 
 
 Lysine 
 
 .0637 
 
 7.21 
 
 .0812 
 
 9.09 
 
 Cystine 
 
 .0064 
 
 .73 
 
 .0043 
 
 .48 
 
 Mono amino 
 
 .4225 
 
 47.84 
 
 .5253 
 
 53.78 
 
 Non amino 
 
 .0788 
 
 8.92 
 
 .0530 
 
 5.93 
 
 Sum 
 
 .8046 
 
 91.08 
 
 .8954 
 
 100.33 
 
 Total nitrogen 
 by Kjeldahl 
 
 .8830 
 
 100.00 
 
 .8935 
 
 100.00 
 
 The figures given in determinations 1 and 2 renresent 
 in each case duplicate analyses. 
 

 > i 
 
 ___ - , , - . , . K' Tv 
 
 '^‘•: ' v.V;FiKfawv 
 
 '.•5' . ■' - ' icS' ^f''"'*- T ■^'^ ' '.'V'' ' A '.. .^- 
 
 IP''' ^ 'W» 
 
 "j.:.„'.i«2rf5ia itlass. -. 
 
 (<p ‘ - . ■' '‘wrvr. , ' ' .ifv '• ■ 
 
 - . - - , ; 
 
 ^■- i’> .>v ' -a' »r'- ’■■ 
 
 it’ '" ’,t ' ( ■• ' •^ ir 
 
 -^' '. I 
 
 ',*V 
 
 m 
 
 p. 
 
 ^J. 
 
 -— Ji 
 
 
 g^'A.0^0.- '■■ . ■ ^i^2^«A^Si||! 
 
 " ; 'SV,t^^;s . , kMiniA^ 
 
 t:ch '# ■ "^'- ■ 
 
 5'.'' . “■ ■' * ' .4 ■^•''^■■.* ^o^oox. f.ali^&.i 
 
 i’ 'v - .r>- — t ' 
 
 
 't ■> ' ^ ■' ^ ( j .■ I 
 
 ' r 
 
 -'V- Wj '• ' -r 
 
 . .: . ‘'"■’f# 1'. : 'fj^l 
 
 
 <t. <1 
 
 t# 1 
 
 bT ' * 1 
 
 ■y± 
 
 
 I ."^j 
 
 • •J'/ 
 
 I’W^ ' ■■■,. y"-- 't’ 
 
 Sr-^ '/kA/' ■/kl TJfcifcau ja.r-. , ,, ■■.>-> .-1, -m T 
 
 ■ 1 itJilfiari III I |M| I g*M Bii^ iiiti‘' ti I I'liii iiiiiir'i « III fall! i I'mi i i ■■' -“- ’ - - . . .. . 
 
 Lk)iL> .1 
 
 
 
 r'H 
 
 
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26 
 
 TABLE V . 
 
 The Distribution of Nitrogen 
 in Deaminized Casein. 45- 
 
 No. 1 No. 2 
 
 Nitrogen Nitrogen 
 
 
 grams 
 
 ner cent 
 
 grams 
 
 ner cent 
 
 Amide 
 
 .0921 
 
 11.11 
 
 .0921 
 
 11.09 
 
 Hum in 
 
 .0178 
 
 2.14 
 
 .0257 
 
 2.85 
 
 Arginine 
 
 .0608 
 
 7.55 
 
 .0588 
 
 7.09 
 
 Histidine 
 
 .0554 
 
 6.68 
 
 .0525 
 
 5.39 
 
 Lysine 
 
 -.0054 
 
 -.40 
 
 .0056 
 
 .67 
 
 Cystine 
 
 .0070 
 
 .84 
 
 .0022 
 
 .26 
 
 Mono amino 
 
 .4295 
 
 51.78 
 
 .5520 
 
 66.50 
 
 Non amino 
 
 .0859 
 
 10.12 
 
 .0486 
 
 5.85 
 
 Sum 
 
 .7465 
 
 90.02 
 
 .8155 
 
 98.20 
 
 Total nitrogen 
 hy Kjeldahl 
 
 .8290 
 
 100.00 
 
 .8500 
 
 100.00 
 
 45- The figures given in determinations 1 and 2 represent 
 in each case duplicate analyses. 
 

 
 
 
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27 
 
 
 TABLE VI 
 
 Deainini zed 
 
 Amino 
 
 
 Casein 
 
 1. 
 
 
 Casein A-64 
 
 Acid 
 
 1 
 
 2 
 
 Ave . 
 
 1 
 
 O 
 
 Ave 
 
 
 
 
 
 
 
 
 Arginine 
 
 3.72 
 
 3.44 
 
 3.58 
 
 3.14 
 
 3.03 
 
 3.08 
 
 Histidine 
 
 3.27 
 
 3 . 30 
 
 3.28 
 
 3.40 
 
 1.98 
 
 2.69 
 
 Lysine 
 
 5.53 
 
 7.05 
 
 6.29 
 
 -.29 
 
 .48 
 
 .09 
 
 Cystine 
 
 .85 
 
 .57 
 
 .71 
 
 .93 
 
 .28 
 
 .60 
 
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 : ! ’O. a;?.' y^ Ofl - t™. 
 
 «W,*. / -LJ • - SB*’ 
 
 •"'- (inW«iiC 
 
 
 
 ’p'-' -• smrmm 
 
 F- • ^ :■ 
 
 «.!' : Ibt'L' .f>V'';& ■■, - ' . .>;iSp|ltJ 
 
 
 •-<. M" W >4/ -i.!. . 
 
 •Ii-Ji 
 
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 ■'=i.,'v4 fr>V' -I 
 
 ? j-v-'i^rv', 
 
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 n v^^JT 
 
 ^•‘. ' './; ■'■ V** 
 
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 ;-.v'Y 4 
 
 L-:i43»3*T(.»"' 
 
 
 
28 . 
 
 and in general the figures obtained for casein and deaminized 
 casein agree closely. Skraup has renorted a diminution in 
 the per cent of arginine and histidine nitrogen in deaminized 
 casein and the entire absence of lysine in the deaminized 
 product. In the present research the average figure for two 
 determinations of arginine nitrogen in deaminized casein v/as 
 found to be 7.21 per cent. Since this value is only .56 per 
 cent lower than the average figure, 7.78 ner cent, for casein 
 it is believed that this difference is within excerimen tal 
 error and does not signify a destruction of this amino acid 
 in the deaminized product. This is what would be exnected if 
 nitrous acid is without action upon the guanidine group of 
 arginine which has been shovm to be free in the protein 
 m.olecule. If, on the other hand, it is true as reported 
 recently by Sekine (49) that the guanidine group of arginine 
 is slowly attacked within the space of a few hours by nitrous 
 acid at low temperatures some destruction of arginine would 
 be expected. In the first determination of the histidine 
 nitrogen of deaminized casein, 6.68 per cent was found, while 
 by the second analysis only 3.89 per cent was obtained. 
 
 Since the former value agrees approximately with the average 
 figure, 6.02 per cent, found for casein while the latter is 
 only about one-half of that amount, no definite conclusion 
 concerning the fate of histidine in the deaminized product 
 can be drawn. The average figure for lysine in casein was 
 found to be 8.15 per cent wrhile in the deaminized product 
 minus .40 per cent v/as obtained by the first analysis and 
 
 -- ■ — 
 
29 . 
 
 1 
 
 I 
 
 plus .67 per cent "by the second. The average figures for 
 these determinations is plus .13 per cent. It is considered 
 that probably no lysine is present since the amount found is 
 within the limits of accuracy of the method. It would seem, 
 therefore, that this observation is in harmony with the work 
 of Skraup (1), in which no lysine could be isolated from the 
 hydrolyzed products of deaminized proteins and with the cur- 
 rent theory concerning the nature of the free amino groups in 
 the nrotein molecule. According to this theory the epsilon 
 amino groun of lysine is free in the nrotein molecule and it 
 is this group which is attacked when deamini zation occurs. 
 
 It is further believed by Van Slyke and others that 
 
 the free amino nitrogen of native proteins is due almost 
 entirely to the epsilon amino group of lysine since the free 
 am.ino nitrogen content of these proteins is approximately 
 one-half of the nitrogen of the lysine which they have been 
 shown to contain. Those who oppose (6 and 7) this theory 
 have found that certain native proteins have a free amino 
 nitrogen content which is not one-half of the nitrogen of 
 the lysine present. However the belief that the free amino 
 nitrogen of native proteins is due at least in part to the 
 epsilon amino group of lysine renders likely the assumption 
 that deaminization of native proteins cleaves the terminal 
 amino group of lysine to form oc amino e hydroxy caproic 
 acid. Since this derivative of lysine would not be basic, 
 its subsequent reactions would be those common to the mono 
 amino acids, i.e., it would not be precipitated by phospho- 
 tungstic acid but would appear in the mono amino filtrate. 
 
'••jrics'r^..*^-..* .-v’jc~vV'J' ».;." 7 i 
 
 , h I. 
 
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 •.. ■ ' . ••’ ■■ ’ . ■ t. •: •' * .V .,ti 
 
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 ■ '•■ f * ■■•'.■. >*"'■'' I i‘r> ■'•.] yi'c^t t j ■% 
 
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 ^r-V.vX 
 
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 r, i '^:. ' : 1 3 “ J, .'. V •' 
 
 c*‘- •> 
 
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 31/ 
 
30. 
 
 The marked increase in mono amino nitrogen in the filtrate 
 hy the second analysis indicates that this or some other ly- 
 sine derivative has become associated with the mono amino 
 acid fraction. Attempts made by Skraup and others to isolate 
 this hydroxy derivative of lysine have failed. Skraup (22) 
 claims to have obtained the anhydride of oc amino S 
 hydroxy valerianic acid which he believes has been formed 
 from the hydroxy caproic acid derivative of lysine. 
 
 In the nresent investigation it seemed likely from di- 
 rect determinations of tyrosine in the original acid hydroly- 
 sates (see Table III) that some of this amino acid had been 
 destroyed in the process of deaminization. To obtain further 
 evidence bearing unon this point analyses of tyrosine in the 
 mono amino acid filtrates of casein and deaminized casein 
 were made. In the case of casein, '5.41 and 5.89 per cent 
 were found while 2.7 2 and 2.98 per cent of tyrosine 7<jas found 
 in the mono amino acid filtrate of deam.inized casein. It is 
 evident therefore, that a considerable part, about 50 per 
 cent of the tyrosine or the substance which gives a blue 
 color with the phenol reagent has been destroyed during the 
 process of deaminization. 
 

 u-r.T»,' . *. ‘I » ;.jr- iia^ 
 
 l-^T’ 
 
 k'^3^^1 ''**'^ ^' ' ' *'t' . 
 
 
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 ? '‘ V ',*,'»! ■; ,= -i \, 
 
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 W M; ;7 j;-' ■ V vfT 
 
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 w '4 '■ ■ }■''''■ ’• ■ ' -.j ‘ '■'‘'-I,' ^ ' 'f ' 
 
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 . I •, ,'i ■ •'■lY .. -:■ ' ’.■' - ^ , '.-w •' ■•'* . 
 
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 ;, • ■ sr.tl ^(f in ! 
 
 :: ;: • ■• : . . / “4. ,. ^ ' ,.. •• ‘-i:- . ki. ' '■ 
 
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 f ■, .:, ,■ ' ... W--^ * 
 
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 ^ '/ -..I'aa. *< ^.S Jt MT-. -*C. .i i 'tirmJlaB’ . 
 
 
 Si*t ‘ .' 
 
 , vr'if^M.yjjlSS 
 
 l^^.u •. .♦ ,;r ..f-'’-’ ^ li^ikltfeAis . I •... .i. / 'fi. /jk^i 
 
 
31. 
 
 — 
 
 V. Digestion in Vitro. 
 
 The observations renorted in the literature in regard 
 to the digestibility of deaminized proteins are conflicting. 
 Treves and Saloraone fl8) reported that deaminized proteins 
 were not digested by artificial gastric or pancreatic juices, 
 while Schiff (16) found them to be completely digested by 
 dog's gastric juice although the rate of digestion was much 
 slower than with the original proteins. Levites (26) found 
 digestion to be complete with dog's gastric juice except for 
 a small residue. 
 
 In a review of the methods used for studying digestion 
 in vitro the statement is made by Prankel (51) that "the 
 best index of the extent to which a protein has been disin- 
 tegrated is the ratio of the amino nitrogen at a given time 
 to the total amino nitrogen obtained after hydrolysis". Of 
 the various methods by which the amino nitrogen of nroteins 
 may be estimated Prankel chose the Van Slyke procedure as 
 "best suited to th^problem in hand." After examining eight 
 methods for the study of proteolytic action, Sherman and Neun 
 
 (52) concluded that "the quantitative determination of 
 
 the amino nitrogen of digestion products appears to be 
 
 more delicate as a means of detecting proteolysis than either 
 the biuret or the ninhydrin reaction and more delicate, ac- 
 curate, and generally applicable as a means for its measure- 
 ment than any of the other quantitative methods here studied." 
 These authors used the Van Slyke method for estimating amino 
 

 
 •: . ; Vi - 
 
 .j i-, 
 
 ilZ) 
 
 V,;j 'o£;; 
 
 
 “■ ..u n'c.t'f.: 
 
 
 m 
 
 ; 'I* Iv nl 
 
 
 c'*' - ‘io ' *■? 'Vo 
 
 K/: 
 
 
 
 : o.S ' 6 r<j Vj 
 
 
 ■ Y.f ;jq 1 }i'^v (tiiJ 
 
 ■ .t i .1* •-• 
 
 , VC, ^ s;{^IV ,fVt ' o e'f 
 
 • -. ‘’.Of:/!- 'dJ^ i -.iift 
 
 ' ’ '■ ■ ' ' \ ' • ' L'^/ ' " ■ ■'^.T' . ' r 
 
 hLD/,,[."^i$ r- ^ "^•. . T T'-' . i ’ 0 . r ^ »v •.'j'Jjj • ■ V-': fibc/.'J *ir 
 
 ^' r '••■•^ ’•V'r'f-'fo srJ '' ' Vz-r'i te-r/.r- '--cv '{OiL) 
 
 o3 l: I* *_r'i '-? .-.j’ vLv.i'^cr ' j ;:Aau;ji ‘j ;VtO> i.''i o:.’’:n>'- orfj 
 
 Vo -i.:v 
 
 ^ -■ .. .f ■ •■' 'V-'’ ■ ^ ‘ • :• ■ - 
 
 ■ tT’. r.s.' i^'Tor rip. J'-. o Or<r,,>/fNr{ r . ■ ; ,.-j 
 
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 • .‘c> 1 0;; J ,=?• £'^ j ff 3 lyo ■ j v n*' 
 
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 V ■ ^.•^.£Vrf 6 Up ' ;or' Vr ’. - r \t.Te«’ 
 
 ^ .'t ij, nc bCiVo •j''f 
 
 ii'f'V- 0 ^':iJj 'V -/i 5 
 
I 
 
 32. 
 
 nitrogen but assumed that results with the S^irenson method 
 would run parallel . 
 
 In the proteolytic studies of the present investigation, 
 the action o^epsin, trypsin and erepsin alone and in series 
 was studied. The samples of pepsin and trypsin used in these 
 experiments viere commercial preparations known to be active, 
 while erepsin was nrepared from the intestinal mucosa of a 
 dog according to Prankel's (51) modification of the method 
 originally outlined by Rice (53). The erepsin preparation 
 was considered to be trypsin free since it was without action 
 upon fibrin. The samples of casein and deaminized casein 
 were hydrolyzed according to the method of Henriques and 
 Gjaldb8.k (37) but the total amino nitrogen was determined 
 by the nitrous acid method. This figure, A^'/hen corrected 
 for free amino nitrogen, was considered to represent the 
 amino nitrogen in nentide linkage, i.e., the maximum amount 
 of amino nitrogen actually available for liberation by enzymes. 
 The liberation of amino nitrogen during digestion was follow- 
 ed by means of the Van Slyke micro apparatus. Prankel ran 
 controls ”with all reagents and ferments in the same quantity 
 except that no protein was added,” to correct for the amino 
 nitrogen present in the reagents. In the present study con- 
 trol experiments were carried out with the same amounts of 
 protein and reagents but using boiled enzyme solutions. 
 
 With the latter tecnique corrections are made not only for 
 the amino nitrogen present in the reagents and enzyme added 
 but for the free amino nitrogen content of the proteins and 
 
i - -i !. . - ;.. 
 
 ;i27' 
 
 B»;\’ Co fJu£re*T: vr'^W;lC 24 
 
 
 
 * *•; 
 
 
. 33 . 
 
 for that which may have been liberated by the hydrolytic 
 action of the reagents. It is believed, therefore, that the 
 corrected values for the free amino nitrogen are an accurate 
 measure of the amino groups actually liberated by the digest- 
 ive action of the enzymes employed. 
 
 The results given in Table VH were obtained by the si- 
 multaneous digestion of 5.0 gram samples of casein 1 and de- 
 aminized casein A-64. These proteins were suspended uniformly 
 in 250 c.c. of .2f^ hydrochloric acid and 20 c.c. of an aqueous 
 solution, containing .2 of a gram of pepsin, added. Controls 
 containing the protein, all reagents and boiled enzyme solu- 
 tion were run. After thoroughly mixing the contents of each 
 flask 5 c.c. of toluene were added as a preservative and in- 
 cubation at 38° C. begun. At the end of 3 hours incubation 
 the casein sample had gone entirely into solution, but the 
 deaminized casein and the controls had settled out. The 
 supernatant liquid of the controls was colorless but that of 
 the deaminized casein sample ?;as colored yellow, indicating 
 that a partial digestion had taken place in the latter case. 
 
 At the expiration of the 3 hour period, uniform samples from 
 each flask were taken for analysis of amino nitrogen in the 
 Van Slyke micro apparatus. To 5 c.c. portions from each 
 flask was added .5 c.c. of N sodium hydroxide to stop the 
 digestion. The resulting solution was diluted to 10 c.c. in 
 a volumetric flask and 2 c.c. taljen for analysis. At stated 
 intervals in the digestion of these proteins subsequent amino 
 nitrogen determinations were made. At the end of 110 hours 
 

 • 7 T♦*^4V,_ '•■. 
 
 ■ V-v.V.3C^^. ■.'.^. • '.’^V'' W-' •• 'iv • • 
 
 w- '/ n«:.^^r^.' sii'V!^' ’”«4> 
 
 |A ' ' '* *' ' <* J _ , ■ j, . '^■^''',tY,-f *'■ j^.'’'‘4*> '" 
 
 H' • < ’ ' ■ ■ ‘'■>*wi> . L K'o V, X (fCi; r 1^ v A'-ilij, . 
 
 ':r, 
 
 " ' «. 1 1 ^ I- 
 
 ’ V i^j! 
 
 s; k >v- 
 
 •'•1.-' ■ ' ... •,.' . ^ _ 'i,. ■ ' . '' 
 
 
 f4 ■ ' 
 
 - i. ■ ‘ ^»|- 
 
 ,;*■ 
 
 S' ,^^‘V 
 
 »v< V , ,\ 
 
 rf, ■ » 
 
 r f - ■ A. . '*-»i' * V tf ''’*■ 'S 
 
 •V ! '-rv: ^ ^ '«i. r ■ ". v.^-" 
 
 -r— i ,j ^ <L*» rf *. _4 xV<r.* A' . . ' . « • ._ ^ "w • i -: V •' -i_j . . . uV - «. flf -u 
 
i 
 
 34. 
 
 TABLE VII. 
 
 The Peptic, Tryptic and Ereptic Digestion of 
 Casein 1, and Deaminized Casein A-64. 
 
 The Percent of Total 
 Amino Nitrogen Liberated. 
 
 Enzyme 
 
 Hours 
 
 Casein 1. 
 
 Casein A 
 
 Pepsin 
 
 0.0 
 
 0.0 
 
 0.0 
 
 tf 
 
 14.5 
 
 9.6 
 
 2.6 
 
 ft 
 
 38.5 
 
 11.1 
 
 3.3 
 
 tf 
 
 86.5 
 
 11.7 
 
 3.3 
 
 tf 
 
 110.5 
 
 11.8 
 
 3.3 
 
 Trypsin 
 
 12.0 
 
 53.2 
 
 25.8 
 
 tt 
 
 36.0 
 
 68.3 
 
 30.9 
 
 tf 
 
 60.0 
 
 79.9 
 
 33.4 
 
 ft 
 
 132.0 
 
 78.7 
 
 33.5 
 
 Erepsin 
 
 17.5 
 
 91.7 
 
 56.2 
 
 tf 
 
 41.5 
 
 92.1 
 
 65.4 
 
 ft 
 
 65.5 
 
 95.8 
 
 65.8 
 
 ft 
 
 89.5 
 
 95.7 
 
 65.4 
 

160 200 240 2B0 320 ^0 
 
 The Peroent of Available Amino Nitrogen 
 
 rt h 
 
.4 
 
 t r 
 
 
36 . 
 
 digestion 200 c.c. aliquots from each peptic digest were 
 taken, 20 c.c. of 15^ sodium carbonate solution containing 
 .4 of a gram of trypsin added and incubation at 38*^ C. begun. 
 Boiled trypsin solution was added to the controls. After 
 several hours the deaminized casein sample went into solu- 
 tion giving a brovm but transparent liquid but there appeared 
 to be no change in the controls. At the end of 132 hours 
 digestion with trypsin 150 c.c. aliquot portions were removed 
 from each flask, 20 c.c. of erepsin solution added and incu- 
 bation continued for 89.5 hours. Boiled solutions of the 
 enzyme were added to the controls. For the determination of 
 amino nitrogen in the tryptic and ereptic solutions 5 c.c. 
 aliquot portions were used. After arresting digestion by 
 the addition of .5 c.c. of glacial acetic acid these samples 
 were diluted to 10 c.c. in a volumetric flask and 2 c.c. 
 used for the analysis of amino nitrogen. 
 
 Similar experiments were carried out with trypsin and 
 with erepsin to determine whether these enzymes could attack 
 deaminized casein without the preliminary action of pepsin. 
 
 2 gram samples of casein 1 and deaminized casein A-64 were 
 dissolved in 120 c.c. of sodium carbonate solution. 
 
 Casein 1 gave a solution of medium opalescence while casein 
 A-64 form.ed a deep red solution in which gelatinous particles 
 were suspended. 20 c.c. of a solution containing .2 of a 
 gram of trypsin or erepsin dissolved Inja. ,5% sodium carbonate 
 solution were added to each flask. 10 c.c. of an alcoholic 
 solution of thymol were added in each case as a preservative. 
 

 > , - . ; V’TTTffl? ^ w 
 
 T’ "S'- ,,-'''’W'r 
 
 . »?»*.!''#. . ■ »■ T Akiii-\ .- >1.^:. ^ L _i'. i. ^«; -i ' o -I ^-Tifi . 'i . r .'iji- ' ■* i»‘ 
 
 .£Ti 
 
 l » , y= 
 
 I' '-'j «*•«.’ ‘^,r*o b'i.fKr^_ io^}vUii Wt ^ ,^1 
 
 t |T 
 
 ■*': *. .LTC ■ : ; ^ _ * M • '■ 5 ’ . * JD ^ ^ J 
 
 i .:.iv4i<o-f‘ f)vOfi,ti%^ nrtf'i ' 
 
 ■" 'i ' ■* ,- - .' * . -'-w', V 1 '> /.' » <•■ '».ii.^* n. 
 
 • ‘ " ' ^‘ ' 
 
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 ^ 1 ;/ ’ iJTi ii.- V, ,'■ \iS 
 
 ii.S. .... ... _.. 
 
 
 
37 . 
 
 After incubating at 38° C. for 22.5 hours a 5 c.c. sample 
 from each flask was taken, .5 c.c. of glacial acetic acid 
 to arrest digestion and the resulting mixture diluted to 10 
 c.c. in a volumetric flask.* A clear but brown liquid in the 
 case of casein A-64 indicated some digestion had taken place 
 in each instance. The appearance of the proteins in the con- 
 trol flasks was not altered. 
 
 The results obtained from the enzymatic hydrolyses of 
 casein agreed closely with the observations of Frankel (51). 
 Pepsin liberated 11 per cent of the total amino nitrogen of 
 casein in 87 hours: trynsin superimposed upon the pepsin di- 
 gest set free 79 per cent in 60 hours, and by the further 
 action of erepsin 95 per cent of the total amino nitrogen was 
 liberated in 66 hours. These values are maximum for these 
 enzymes since the amino nitrogen was found to remain constant 
 over a period of 25 or more hours of additional digestion. 
 Frankel believes this to be due to the auto destruction of 
 the ferment rather than to the inhibiting action of the end 
 products as suggested bjr Abderhalden and Gigon (54). The 
 digestion of deaminized casein proceeded in every case at a 
 slower rate than that of casein and the total cleavage was 
 considerably less. Only 3 per cent of the total amino nitro- 
 gen of deaminized casein was liberated after 110 hours of 
 peptic digestion in contrast to 11 per cent with casein. 
 Tryptic digestion for 132 hours set free only 33 per cent of 
 the total amino nitrogen as compared with 73 per cent for 
 casein, while the further action of erepsin liberated only 
 
U.‘ ' ■' 
 
 
 • : I. 
 
 itV ,• » • f 
 
 n V ' I o ' 
 
 tiO.- J. , f 
 
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 *' . 
 
 s 
 
 
 
 
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 ' 
 
 
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 >.3^ 
 
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 : V '8. ! -fr 
 
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 'Iv ir v; 
 
 
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 r .-f '\ ^ • • 
 
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 0 ■■,. JLC 
 
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 ■ [’■ j Bf? . 
 
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 ■•LfO'f ’ V rl 
 
 ■:?r'^J>r-or n ', 
 
 
 ■ !<■' ' V 
 
 
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 ''9fK ^V. /■ 
 
 
 ' ' • 
 
 ■ ■ .' r.r 
 
 -* ,: 't-'K'” 
 
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 !■ A .N 
 
 \ 4 ^ i ^ ^ 
 
 ■ 
 
 ' "C 
 
 ■r: J ^.u.. 
 
 
 r o , '1 
 
 cd 
 
 
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 <, r- '.■ ,i '■- 
 
 
 i/n - 
 
 
 '■'I'' ’1 
 
 , f 
 
 ■>■'1 rv;,. 
 
 j .!• 
 
 
 ■ 1 ' r 
 
 i- 
 
 
 
 . _■ - 
 
 ::v:i.r r. . ■: 
 
 
 
 fl XO’j.r. 
 
 I 
 
 'rfi' 
 
 • T »% 
 
 . fJ'':' % 
 
 ►' n^'’;5 04i' ■' ' 
 
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 'is^ :'i 
 
65 per cent of the total value for amino nitrogen as con- 
 trasted with 95 per cent for casein. 
 
 It is possible that this difference in digestive action 
 hetv/een casein and deaminized casein may be due to the less- 
 ened solubility of the latter and its consequent less in- 
 timate contact with the enzymes. There is also the possibility 
 that reactions incidental to the process of deaminization 
 may have taken nlace to alter the peptide linkage in such a 
 way that its cleavage by enzymes became more difficult. 
 
 It is evident from the results reoorted in Table VIII 
 that trypsin will digest deaminized casein without the pre- 
 liminary action of pepsin. However, the rate of digestion 
 with trypsin is slower than that with the preliminary action 
 of pepsin and with trypsin alone a lessened cleavage is pro- 
 duced. The digestion of deaminized casein with trypsin is 
 less extensive and proceeds at a slower rate than that of 
 casein . 
 
 Since it is known that casein is attacked by erepsin 
 without the preliminary action of other enzymes it was of in- 
 terest to test the action of this enzyme towards deaminized 
 casein. It was found (see Table ’ IX.) that 11 per cent of the 
 total amino nitrogen of casein was liberated after 93 hours of 
 ereptic digestion but no amino nitrogen was obtained from de- 
 aminized casein. It would appear, therefore, that erepsin 
 does not attack deaminized casein directly or at least it 
 does so very slowly. 
 
;,<t -'‘Tf A.,-' 
 
 *<' 5 *Wr,P' 
 
 
 
 ^'\‘^'iV:.»..> .. 
 
 
 
 •;', -r^?3 •.'.. ■y:,^.-i:)isi.. .-/-Jito %.l’- ofti/jy' .Mtey 
 
 mxvis ' ■< s. . ' ■ 'I’v?*' ■-': . --v\ -i 
 
 ',*p 
 
 
 
 ‘ ‘ . *j. ’- |«^, - • ■ ^': : .‘.; ; *♦•; 
 
 L,., ■ ^ 
 
 '■'v *') :. *:’ 'r :.'i' >..*»»,’ . ' '^ ’ '<j -r - ' ** '*’ 
 
 W 
 
 j\. 
 
 i i < ~ -V ’ '■ ■ .1 
 
 V , "■ ■ *.’ ,.. • ’•■.,• 
 
 *■ ' •i'^ ..'■ • 
 
 * IV ■ " 
 
 ■ft 
 
 ; _ - '/V •V. ^V*^ .. h 
 
 fc Mi no !';»'$?’• -^/ivV i si^, iSi‘>’/v. 
 
 j* (‘f '*' ' * ' \ ' * ' ' ' ~'^" " ' "' '' 
 
 \s: . Jo ( ^^”5 -j^iX^^'Vtft^'is.)' V^Sv. 
 
 ■■ . AiTt&.V‘, to ' ■ * ■ — -.jr , , , . HIA 
 
 Bfei ‘%i.‘ • ;. . j*:' .r'r. 
 
 ■ viiM '■;-*5|^^i^fe.... __. .. ... 
 
 ' " 'P •'-' »J( ^ |iS»^ Ti r u‘ r (MiiTYiitffiii|-iina y j r » 1 1 ii'. y p r iit yj ii j i l i mimn aiin y i 
 
39 
 
 TABLE VIII. 
 
 The TryDtic Digestion of Casein 1. 
 and Deaminized Casein A-64. 
 
 The Percent of Total 
 Amino Nitrogen Liberated 
 
 Hours 
 
 Casein 1. 
 
 Casein A.-64 
 
 o 
 
 # 
 
 o 
 
 0.0 
 
 0.0 
 
 22.5 
 
 41.3 
 
 25.3 
 
 45.0 
 
 50.4 
 
 27.9 
 
 68.5 
 
 52.7 
 
 27.3 
 
 92.5 
 
 52.4 
 
 28 .7 
 
 
 TABLE IX. 
 
 
 The Ereptic Digestion of 
 and Deaminized Casein 
 
 Casein 1. 
 A-64. 
 
 Hours 
 
 The Percent of Total 
 Amino Nitrogen Liberated 
 Casein 1. Casein A-64 
 
 o 
 
 • 
 
 o 
 
 o 
 
 • 
 
 o 
 
 0.0 
 
 21.5 
 
 00 
 
 • 
 
 o 
 
 o 
 
 • 
 
 o 
 
 45.5 
 
 00 
 
 • 
 
 o 
 
 0.0 
 
 93.5 
 
 11.6 
 
 o 
 
 . 
 
 o 
 
 
II >i<|f|M>i i»iii'tt i » i, .5i . 'X.. .wi*.,. 
 
 
 I j ^ *ii ' * ' I' ■ • : t ^ ^ i 'i_ „. /''’/'•■ h-' V 
 
 S *V' , •'».'/ ■ ,, , '^>-s5' V ■’ .''t’ -jS" ■ '' ‘ r. 7'" 
 
 /'V '.-'■« 
 
 ■ 
 
 •3^^ ' ** '' *■-' • j2^'! ” ' 
 
 Ty. V ^ V rfftkp: :! •'< i ^ ■ , ■ kA|i4y| 
 
 • ;’.>vi •■• ‘i ■’ • ajJL’ '.v^ 
 
 i^: 
 
 - \>-5'' I ''■ ® y , I 
 
 #' -t 
 
 
 r -Iil^v'^' r 
 
 r;" -is J".?? . . % ■ ’ WjSJ i *v;»- 
 
 r ■'O' ^'• 
 
 i - 
 
 |f\, ' '* *>’ !■ fe ^ ' 4u • 
 
 '' . jil. ■'■'*- 't) 
 
 
 • 
 
 1 
 
 
 ' ,. .■ ■ 
 
 ‘ i > 
 
 
 , ^•■'' ■ .'6>0 ' 
 
 '■'‘- .''^ r. 
 
 -y^ 
 
 .1 V * . ^ • * \f9‘ 
 
 • 'i"'' ’■'• 
 
 ?v;' 
 
 >_ a{ 
 
 T ■ •. - 
 
 4 . . 
 
 
 .4r:; '■ 
 
 '■•i ' 'V . 
 
 . V >' '• •■'?r^'VV--.'fe‘. ‘ '■2*nR'...J Ly■.^ : .. ' v«V 
 
 } 1^ 
 
 '’• ■‘- iK • •} 
 
 ,; ':■ I’v 
 
 • a y, ^ 
 
 
 
 ;.V;". a'a'x ‘ilSA '‘'ar^ ®p 
 
 'V , •■ v-: ’;r V‘' 
 
 
 B .rV'K . ..'i. v.^u B., 
 
 I'b 
 
 I • \: 
 
40 . 
 
 Bn 
 
 F. The Behavior of Deaminized Casein in the Animal 0rg;anism . 
 
 Since in the nresent investigation digestion experiments 
 in vitro indicated that deaminized casein was digested al- 
 though at a slower rate than casein it was desirable to study 
 the behavior of deaminized casein in the animal organism. A 
 female dog, weighing about 9 kilos was maintained upon a uni- 
 form diet for 10 days to permit a constant level of nitrogen 
 excretion to be reached. At the expiration of this period 
 10 grams of deaminized casein A-64 was added to the standard 
 diet and the elimination of extra nitrogen in the urine de- 
 termined. 
 
 The standard diet as given in Table X was considered 
 to be calorifically adequate for a dog of the size used. 
 
 The beef heart, chosen in preference to other kinds of meat 
 because of its uniform composition, was dissected free from 
 fat, valves and tendons and ground to a pulp in a grinder. 
 
 The mass was kneaded to a uniform consistency and preserved 
 in glass jars at a low temperature. Since in feeding experi- 
 ments requiring the use of a synthetic diet it is important 
 to render the food as palatable as possible, the standard 
 diet used in the present research was prepared as follows: 
 
 Substance 
 
 Table X. 
 Amount (fi-ms) 
 
 Calories 
 
 Bone ash 
 
 10 
 
 
 Sucrose 
 
 35 
 
 140 
 
 Starch 
 
 25 
 
 100 
 
 Beef heart 
 
 50 
 
 120 
 
 Fat (lard) 
 
 25 
 
 225 
 
 Water 
 
 400 c . c . 
 
 — 
 
 Sum 
 
 585 
 
f t 
 
 
 1 
 
 
 
 OJ 
 
 . .1 V '■ •■ji’ ' 
 
 *r’.' 
 
 s 
 
 I 
 
 ■ \ 
 
 
 ■\' 
 
 ■ " .,■>.■ 
 • • - 
 
 ','1 
 
 r w. ' . C 
 
 iv> i ^ rn^^lv 'ni 
 
 
 - ;^V ’'' ■■.■; ■ ; '■:«»•.•.: J: u\rc ri^uoffi ' 
 
 *■ Z' "■ ■ •' '■ • • ^ .^'*' -' ■' r<'^ (. ■■ H • i 
 
41 
 
 Date 
 
 Weight 
 of Dos 
 
 TABLE XI. 
 
 Total (1) Total 
 
 Sulnhur Nitrosen 
 
 Urea and 
 Ammonia N. 
 
 % of total N 
 as urea N. 
 
 
 kg. 
 
 gms . 
 
 gms . 
 
 gms . 
 
 
 Jan. 7 
 
 — 
 
 — 
 
 
 
 
 " 8 
 
 — 
 
 — 
 
 — 
 
 — 
 
 — 
 
 " 9 
 
 — 
 
 — 
 
 — 
 
 -- 
 
 — 
 
 ” 10 
 
 9.90 
 
 — 
 
 — 
 
 — 
 
 — 
 
 " 11 
 
 9.90 
 
 
 1.355 
 
 .841 
 
 62 
 
 " 12 
 
 9.88 
 
 .068 
 
 1.342 
 
 .910 
 
 67 
 
 " 13 
 
 9.88 
 
 .069 
 
 1.251 
 
 .757 
 
 60 
 
 ft 14 
 
 9.86 
 
 .070 
 
 1.427 
 
 .872 
 
 61 
 
 
 9.82 
 
 .077 
 
 1.955 
 
 1.622 
 
 77 
 
 " 16 
 
 9.75 
 
 .064 
 
 1.363 
 
 .946 
 
 69 
 
 ” 17 
 
 9.75 
 
 .082 
 
 1.246 
 
 .960 
 
 77 
 
 " 18 
 
 9.75 
 
 .073 
 
 1.299 
 
 .968 
 
 74 
 
 " 19 
 
 9.73 
 
 
 
 
 
 •35- 10 grams of deaminized casein A-64 were fed in addition 
 
 to the standard diet. 
 
 (l). Thanks are due Miss Lucie E. Root for the sulphur de- 
 terminations reported in Table XI. 
 
’fMT.v ■■ , y ■' y 'Wiry s 
 
 
 
 
 
 i; :■ !>■ ■.(.,; •O'- v*>;-t, ‘’;if va 
 
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 k 'i-'si..' > , '■ iijj.1 4' 
 
 ulia I , S ■, . \ »lL'AI**.’.' 1 
 
 > -I^ifffastl,. ■ 4'u5iS3,tiit, • gi iS. . ft 5ii*s 
 
 ■“' » ' '•’ i»l ■•, . 1 ••'} I ^*s»BUi. '/,■' 
 
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 -,' 'ivT.>-'."- 
 
 tL' 
 
 ■y '- 
 
 
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 '■' ■ ■•' V ".f^^ 
 
 
 
 L-- 
 
 
 
 P- 
 
 <*I 'T '• 
 
 
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 • ^ .\! ‘ " •'1'' .1 ' .„•/ ‘ 
 
 ■lS 
 
 ; n&:.0 
 
 
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 l' ( f , 
 
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 ■'''• OS'. . t' ',i,«ir 
 
 ■ ^ ‘C ", s-(5.P* 
 
 ^ J *f' 1 *.' . w #*l tl «k'M ■ ( j 
 
 ■ '0 
 
 ■|) ’^,‘ •'<' '.^' '■’ ' 
 
 oe.e ■ ox ” 
 
 
 .' j,; - -ss-i. f^i-r 
 
 iy. -'ii i 
 
 A- 
 
 • X‘- , 
 
 \ * * . 1 ‘ • r. • ‘ . ■ fi •» r . '» : . 
 
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 r ; -t 
 
 •«tTV^' V«; 
 
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 I aK/'*'. ' ' -u- ‘ . V-. * ■"♦ 'i. 
 
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 3^:^' 
 
 
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 r-"- •' 1 - IM Iii1r>ini0««i*4ai11it. .lalfci I '^'iLr I.. I “ . .1 
 
 
 
42 . 
 
 10 grams of bone ash and 35 grams of sucrose were added to 
 350 c.c. of distilled water and the mixture heated to boiling. 
 
 To this mixture was added 25 grams of starch made into a 
 paste with 50 c.c. of distilled v/ater with constant stirring 
 to prevent burning of the starch. In a few minutes the mix- 
 ture thickened because of the swelling of the starch granules. 
 While the vessel containing this mixture was cooling, 50 grams 
 of beef heart and 25 grams of lard were added and stirring 
 continued until a uniform mixture was obtained. As a routine 
 procedure this diet was fed immediately after catheteriza- 
 tion of the dog at 9:00 A.M. 
 
 According to the figures given in Table XI, the total 
 nitrogen elimination for the experimental day after feeding 
 10 grams of deaminized casein represented 43 per cent of the 
 nitrogen added to the standard diet as deaminized casein. 
 
 This amount was .615 grams in excess of the average nitrogen 
 elimination for the fore and after periods. Since there 
 was a corresnonding or even greater increase in urea nitrogen 
 the assumntion that utilization of the deaminized casein in 
 the animal organism has occurred seems well founded. 
 
 j 
 
 Since the preliminary experiments seemed to indicate ! 
 
 i 
 
 utilization of deaminized casein in the animal organism it i 
 
 was desirable to determine whether an animal could be main- 
 tained in nitrogenous equilibrium upon deaminized casein. 
 
 The dog used in the preliminary experiment was put upon a 
 standard diet the nitrogen content of which was determined | 
 by Kjeldahl analysis. This diet given in Table XII ^as 
 
 f 
 

 
 4 V 
 
 ’ -wnr^'i r..i iiipiii 
 
 BX i'''- ” ^1. ^ a ^ 
 
 v;/- . , 
 
 fcv . ‘'^T^ 
 
 L' . ‘ , *■ V . • ’ 
 
 rtf. 
 
 
 jA »r ' L* - >?/■ 
 
 ,, ' ' ' I < 4'^.' ' . V ■ i ■ ' vl'' -’ ■ ■■‘!-'¥.'"'^ *■ ■''** ' ■ '• ■’ ■ ' •%:*' 
 
 f.;^ ■ ■ JM.V.ia-.' *' i.-.iL f >._. . ,?t* -,,nt' . Vi ■' fAif 
 
43 . 
 
 comparable to that 
 
 used in the 
 
 foregoing exoeriment although 
 
 it was made higher 
 
 in calorific 
 
 value because 
 
 the animal had 
 
 lost weight in the 
 
 first experiment. 
 
 
 
 TABLE XII 
 
 
 
 Substance 
 
 Amount 
 
 Calories 
 
 Ni trogen 
 
 
 gms . 
 
 
 gms . 
 
 Water 
 
 400 c.c. 
 
 — 
 
 — 
 
 Bone Ash 
 
 10 
 
 — 
 
 .0002 
 
 Cane sugar 
 
 40 
 
 160 
 
 .0160 
 
 Beef heart 
 
 25 
 
 60 
 
 .7400 
 
 Com starch 
 
 25 
 
 100 
 
 .0180 
 
 Lard 
 
 35 
 
 305 
 
 .0035 
 
 Casein II 
 
 25 
 
 
 
 or 
 
 
 100 
 
 3.3900 
 
 Casein A-64 
 
 24.19 
 
 
 
 
 Sum 
 
 725 
 
 4.1677 
 
 After 8 days on the standard diet, using the routine 
 procedure of the preliminary experiment, deaminized casein 
 was substituted for casein. Ordinarily the food was eagerly 
 and completely devoured but on the second day with deaminized 
 casein the dog ate with reluctance and after 2 or 3 hours time 
 the food which had been eaten was vomited up. This obser- 
 vation seems to indicate that deaminized casein becomes 
 toxic to the animal organism. The deaminized casein fed on 
 the first day was apparently not toxic or at least not 
 
44 . 
 
 sufficiently so to prevent its absorption and the elimination 
 of its nitrogen in the urine. No explanation for the ab- 
 normally high elimination of nitrogen on this experimental 
 day has been found. 
 
 The original intention was to continue the experiment 
 with deaminized casein for a longer time but the toxicity of 
 this product rendered further experimentation inadvisable, 
 if not im.possible. It would seem, however, from this isolat- 
 ed experiment that deaminized casein cannot replace casein 
 in the diet because of its toxicity but whether the deaminized 
 product will maintain an animal in nitrogenous equilibrium 
 is still to be determined. 
 

 • •- ' y '<■ 1 ■;.> ® V- ®T*W 
 
 p 
 
 i^- ^ '' ’ .ff ^ 
 
 *:;■■ -f T-VS ’ .ct . ' ■ - '•■ ■' 
 
 I - ' '. ^. £ •’•'jT *1 .. • ito AJ '" , «T ^ JriiacJ 
 
 '^'- Lll 
 
 '-yi 
 
 '•j "’’fv-r' 
 
 f yy ' ■'. ^ 
 
 ■ A' ^.AS^ M I i- r . 
 
 □ I*' rt' 
 
 
 
 
 
 ■^- J 
 
 ■ • " I J V,.*J -A 
 
 ■’ r . 'V, * '5,'j4^-; 
 
 
 
 &’liSj 
 
 ■/•■«< •ihiC'.v w.’ 
 
 'i\' ' I'i 
 
 Jw'' . A" 
 
 ^ 
 
45 
 
 TABLE XIII 
 
 Date 
 
 
 Weight of Dog 
 
 Nitrogen of food 
 
 Nitrogen in 
 
 
 
 kg. 
 
 gms. 
 
 gras . 
 
 Jan. 
 
 24 
 
 9.75 
 
 4.4177 
 
 
 M 
 
 25 
 
 9.71 
 
 4.1677 
 
 — 
 
 tf 
 
 26 
 
 9.73 
 
 If 
 
 2.005 
 
 »f 
 
 27 
 
 9.77 
 
 It 
 
 2.240 
 
 If 
 
 28 
 
 9.86 
 
 ff 
 
 2.490 
 
 If 
 
 29 
 
 9.84 
 
 If 
 
 2.640 
 
 ff 
 
 30 
 
 9.86 
 
 If 
 
 2.660 
 
 ff 
 
 31 
 
 9.88 
 
 It 
 
 2.939 
 
 Feb . 
 
 1 
 
 9.88 
 
 It 
 
 3.868 
 
 If 
 
 2 
 
 9.86 
 
 ff 
 
 
 ■}{■ Deaminized casein A-68 was substituted for casein in 
 the diet. 
 
■V'''*^t,f''My’.^i*?w»^swF^ % ^.•'7-.7P. fi 
 
 
 
 m 
 
 i ' I • '''„''> ,H: 
 
 "V* 
 
 ^v:‘S^ 5 Vr:K'V ’'® 
 
 );■ ■■' , ' .. -- V , ' . f ' jc^^-(.r _ Ja’ >. ■' -ife.:/ ' r'.'rt'i 
 
 Si ^ > I 
 
 
 
 ■• 1 - " -KMt? -T HI' 
 
 I'vt'.rsp;' 
 
 ■'* -■■ 
 
 ■ '. / „ ^ ■“'■ 
 
 f ^'Y 
 
 i 
 
 ' . ■■v>..i« ™ )> ;: vrwi Saiil yi :’*Jp , 
 
 ho ^*. i 
 
 p: . ‘ “^-'V-:- 'V 'i" ■ 4 '. ■'.•!/ .»■ ^it.- 'f V- '""'Vft. ' -■‘‘ 
 
 Sf^- ■ ■ ■ ^ •-•■- ■ -M- . -. ■ 
 
 «l 
 
 /■ I 
 
 
 Vf 
 
 
 S' "f. 
 
 ■ • 9 ” ■■ ■ :■ . .'’ ''fc'tv , " ■ - li^ia 
 
 rtSiSMW^ s.:- '■, ■!<’»* \....~ ...'•# 1 ^ aM 
 
 A' 's " ,, ■ ■. , ^ V: ■ v^'-'»'.' 'r : ‘V^PHfJ sn*Pi niflHp 
 
 ^ ^ -f i, " ’ ■ • '.V ■ 
 
 r" ' I'J'- ' ' f ■' ' f 
 
 ^ “■ .P: ... .' .. . i? . ' „ S.p^-Va ^ 
 
 js;i . ' 
 
 
 
 !., . .1 1 ' 
 
 •a ‘'M':4! & .... . in ^ mSI 
 
46 . 
 
 SUMMARY . 
 
 1. The free amino nitrogen of a number of sam.ples of 
 casein, prepared in various ways, has been determined and 
 found to be constant. 
 
 2. Deaminized casein has been orepared by the action 
 of nitrous acid unon casein. 
 
 3. The distribution of nitrogen in casein and deaminized 
 casein has been determined by the Van Slyke partition method. 
 In harmony with the current theory, as to the nature of the 
 free amino groups of. the protein molecule, deaminized casein 
 was found to contain no lysine. No other notable differences 
 between casein and deaminized casein were found. 
 
 A. 
 
 4. Tyrosine was found to be partially destroyed by the 
 deaminization of casein but not completely so as maintained 
 by Skraup. 
 
 5. Casein and deaminized casein were found to be di- 
 gested in Vitro by nensin and trypsin. Erepsin was found to 
 digest casein readily but to attack deaminized casein only 
 after the preliminary action of pepsin or trypsin. In every 
 case the digestion of deaminized casein proceeded at a slower 
 rate than the digestion o:^casein. 
 
 The nitrogen of deaminized casein, administered per os, 
 was excreted as urea. Deaminized casein, administered per 
 os, appeared to be somewhat toxic to the organism. 
 
I"' I ’ 
 
 .m 
 
 ^^ ' “ >., , ■ p' t '; ,.; ^ ■■ '. •^■'^ ■ ' ■ ’*• 7< . ,i 1 ' *1 
 
 ’•V' v^’’ 
 
 < 
 
 
 ■ ' \ . 'j ■. ■': ^*v ■• i\A 
 
 :■ ,mv«(js :. ^ 4 ,-'t' 
 
 ' , : vT'^j 
 
 o' 
 
 >-‘’(.i.i«i'ma,T “vp: jiscncar p tfuvr&Ui o.-iii(ii' ao-t'f/ejW- v ’’ JT? 
 
 ' , " - ■ S f; ’ ' - ’*' ■ '^, ,^\ '• tSJB 
 
 6 iV ^ •. PS'Vj^yJ' 
 
 * ■' / " ■ ’' ' " "^y -‘H ^ 
 
 jp. ,• ;/i';\ 'i ■ '•(■/.* ./ ■, >Kj^< ^ ii 
 
 'K- •<. i** .•• i •■ ^ '■ ■ ‘i \ - , »■ r^'-ar ’.■>"?''•***'. ^ ^ . TA»^ ■'' ^ If 
 
 ■\'.\ ‘ . ,- ■.* •■ ■, ■ •-■ -;SS ' ’> r ' 1 
 
 J».. • . '*■" V ■ . f :N:?' ^ rv'r^' ■ ,;.\V\>-h.\' 
 
 b’-T)' i*fl 4 ‘ ' 
 
 t* :? ■'. i hH'^gny>rfir, .^p' i;qi:/.^.<>?T':^^ ^.’ 
 
 0“ 1 ;• ' , ■■‘'•'.''■r ; - ‘‘-' : ,, . , ,r., 'i '«-•<■•!•■''■ '^>y: 
 
 ^ -'^' ' ' 'i^ I ' . .1 * ■ '* *' > 
 
 ^■; viitt & - ^;jr ;.ft f ‘ .^.. r flJHtei*' 
 
 lar j ■ ^ " p. ' ' -, jIa *1 ' 
 
 .. ’ ■. 7*^ * »« IMMlf IT 
 
 iS’ 
 
 i 
 
 ■pv ..Jjrjijpp' -^Tr i'/^q6*ii?r . vii$co<r ort;f'M ftd- ty^sS^f^- 
 
 ■"■ , ' ’ *■ , '•■ • * '^'St - .' \ 
 
 ?^\T£C'Vf- 0i • >f v»cf iP frpil:C«; 
 
 '*'. 'f A ■ ' ' ■^ . I r ■*<i*'',Vt'i ’«. 'v ■A'*’ ' 
 
 . 'ipvpXji « 3^ Xjej^itjQpp'xg ^ 
 
 ;• . ■' .\/ ‘ ,v 
 
 t-. ■ '^ ■ .. - ■ ''i‘-*‘‘'-‘'" -."x. • *''' ^ " r ‘' ' ' ' V"'*^ . 
 
 [jT s$7C^fi*^<jT(pJtp'*3ai^ 
 
 ^ ^ • • . . . • >.. ‘ ' "'''’i ‘"^-rv 
 
 ' .. »1PQ J^^i'^‘{^^IQ artJ 0±/ C’lSdOd^' ■liJfflff'/ittAR ftprf.. (V.?’- ^A^.iwrrrtfe . »A < 
 
 V* 
 
I 
 
 47 . 
 
 BIBLIOGRAPHY. 
 
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48 
 
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ACKN0\7LEDGMENT 
 
 I wish to acknowledge my indebt- 
 edness to Dr. H. B. Lewis, at whose 
 suggestion this research was undertaken, 
 for his interest and helpful advice. 
 
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VITA. 
 
 Educational Career - 
 
 A. B. Simpson College 1916. 
 
 M. S. University of Illinois 1918. 
 
 Assistant in Chemistry, University of Illinois, 
 1917-1918; 1919-1920. 
 
 Fellow in Chemistry, University of Illinois, 
 1920-1921. 
 
 Publications - 
 
 Studies in Uric Acid Metabolism. 2. Proteins 
 
 and Amino Acids as Factors in the Stimula- 
 tion of Endogenous Uric Acid Metabolism. 
 Howard E. Lewis, Max S. Dunn and Edward A. 
 Doisy. Journal of Biological Chemistry, 
 
 Volume 36, 9 (1918).