SOME of the earliest methods of amino

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1 The Amino Acid Content of Fresh and Stored Shell Eggs. II. Arginine, Histidine, Lysine, Methionine, Cystine, Tyrosine, Tryptophan, Phenylalanine, and Proline* ROBERT JOHN EVANS, J. A. DAVIDSON, SELMA L. BANDEMER AND HELEN A. BUTTS Departments of Agricultural Chemistry and Poultry Husbandry, Michigan State College, East Lansing, Michigan SOME of the earliest methods of amino acid determination were for cystine, tyrosine, tryptophan, and the three basic amino acids, arginine, histidine, and lysine, so consequently many data are available for the content of these amino acids in egg proteins. Furthermore, values are also available for methionine and phenylalanine, because of their classification as essential in the diet and the existence of suitable methods of determination. However, few data on the proline content of egg proteins can be found in the literature. Although no study has been reported of changes occurring during storage in the arginine, histidine, lysine, methionine, cystine, tryosine, tryptophan, phenylalanine, and proline content of egg proteins, changes in the content of some of these in eggs during incubation have been studied (Sendju 1925, 1927; Cho 1926; Plimmer and Lowndes 1927; Calvery 1929, 1930). It is the purpose of this paper to present data on the percentages of the above mentioned amino acids in the proteins of egg * Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No (n.s.) (Received for publication February 28, 1949) albumen, egg yolk, and the whole egg contents from fresh and stored shell eggs and to determine if changes in the total content of these amino acids in shell eggs occurred during storage. EXPERIMENTAL Descriptions of the eggs used, methods and time of storage, preparation of samples for analysis, and method of acid hydrolysis of the proteins have been given previously (Evans, Davidson, and Butts 1949a, Evans, Butts, Davidson, and Bandemer 1949b). Amino acids were determined by microbiological assay. Lysine, methionine, cystine, and proline were assayed with Leuconostoc mesenteroides P-60, phenylalanine and tryptophan with Lactobacillus arabinosus 17-5, histidine and tyrosine with Streptococcus faecalis R, and arginine with Lactobacillus casei. The media of Sauberlich and Baumann (1946) were used for arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, and proline assays, and that of Lyman, et al. (1946) was used for methionine and cystine. Enzymatic digest of the egg protein samples were prepared by hydrolyzing in vitro with pepsin, trypsin, and erepsin (Evans 1946), and 697

2 698 EVANS, DAVIDSON, BANDEMER AND BUTTS TABLE 1. The arginine content of egg proteins Literature values: Block and Boiling (1943), 7.0; Block and Mitchell (1946), 5.8,8.2,6.4; Calvarv and Titus (1934),, 7.5; Edwards, et al. (1946)', 9.7; Hess, et al. (1948), 5.6, 7.0, ; Horn, et al. (1948b), 9.5; Munks, et al. (1945), 5.7,169,6.8,168, 6.4, 344. these digests used for the determination of tryptophan. Cystine was also determined chemically by separating as the cuprous mercaptide (Csonka, et al. 1944) and then determining the cystine colorimetrically (Evans 1944). Results. The results obtained are presented in Tables 1-9. The amino acid contents of the egg proteins have been calculated to a basis of 16 percent nitrogen in the protein. The amino acid content of the whole egg is on the basis of milligrams of amino acid per egg. Values from the literature for the amino acid content of egg proteins are included for comparison. DISCUSSION protein contained a higher TABLE 2. The histidine content of egg proteins Literature values: Block and Boiling (1943), 2.4; Block and Mitchell (1946), 2.2,,; Calverv and Titus (1934), 1.4, 1.3; Edwards, et al. (1946)", 3.6; Hess, et al. (1948), 1.2,1.5,1.5; Horn, et al. (1948a), 2.3; Munks, et al. (1945), 1.4, 42, 1.5, 37, 2.0, 108. percentage of methionine, cystine, and phenylalanine and a lower percentage of arginine, histidine, lysine, and proline than yolk protein; the percentages of tyrosine and tryptophan were similar in yolk and albumen proteins. The data presented in Tables 1-9 are for the most part in good agreement with TABLE 3. The lysine content of egg proteins Literature values: Block and Boiling C1943), 6.0; Block and Mitchell (1946),, ; Calvery and Titus (1934), 5.3, 5.2; Edwards, et al. (194o), 7.8; Hess, et al. (1948), 4.8, 5.8, 5.1; Horn, et al. (1947), 7.9; Munks, et al. (1945), 4.9,145, 5.7,141, 5.2, 280. TABLE 4. The methionine content of egg proteins Literature values: Beach and Teague (1942), ; Block and Boiling (1943), 3; Block and Mitchell (1946), 4.4, 3.0, ; Edwards, et al. (1946), 3.9; Hess, et al. (1948),, 3.6,3.9; Horn, et al. (1946a), 3.1; Horn, et al. (1946b), ; Munks, et al. (1945), 6.6,196, 3.0,, 5.2, 280. published values where values are available for the amino acid content of the albumen, yolk, or whole egg proteins. No appreciable change in the concentration of histidine, lysine, methoinine, cystine, tryptophan, phenylalanine, and proline in egg proteins occurred during storage. protein from stored eggs

3 AMINO ACID CONTENT OF EGGS 699 contained less arginine and tyrosine than that from fresh eggs. The fewest milligrams per egg albumen of most of the amino acids were found in the 9-month old eggs, but the fewest milligrams per whole egg were in the 26-month TABLE 5. The cystine content of egg proteins Literature values: Block and Boiling (1943), ; Block and Mitchell (1946), 2.3,, 2.4; Calvery and Titus (1934),, 1.6; Hess, et al. (1948), 2.5,, 2.3; McFarlane, el al. (1930),,1.7; Munks, et al. (1945),, 56, 2.2, 54, 2.2,. TABLE 6. The tyrosine content of egg proteins Literature values: Abderhalden and Kempe (1907), ; Block and Boiling (1943), 5.0; Block and Mitchell (1946), 4.8, 5.3, ; Calvery and Titus (1934),, ; Grace (1947), ; Gunness, et al. (1946), ; McFarlane, et al. (1930), 5.0, 5.1; Munks, et al. (1945),, 133, 4.7, 116, 4.8, 258. old ones. The one exception was methionine, which occurred in smaller amounts in the 9-month old eggs. The loss from the 26-month old eggs was from the yolk rather than from the albumen. Only serine, threonine (Evans, et al. 1949b), arginine, and tyrosine were found in smaller amounts in the 18- than in the 9-month old eggs. In contrast with the low level of tyrosine and arginine in the TABLE 7. The tryptophan content of egg proteins OO OOOO Ov Literature values: Block and Boiling (1943), 1.6; Block and Mitchell (1946) 1.6,1.6,1.5; Celvery and Titus (1934), 1.5, 1.3; Edwards, et al. (1946), 1.6; Fiirth and Lieben (1921), 2.0,, ; Hess, et al. (1948), 1.4, 1.2, 1.4; McFarlane, et al. (1930), 1.5, 1.5; Munks, et al. (1945), 1.4, 42, 1.4, 34, 1.4, 75. TABLE %. The phenylalanine content of egg proteins Literature values: Block and Boiling (1943), 5.6; Block and Mitchell (1946), 5.5, 5.7, 6.3; Edwards et al. (1946), 6.1; Grau (1947), 7.6; Hess, et al. (1948),, 3.7, ; Horn, et al. (1948), 7.3; Motomura (1929), 1.6; Munks, et al. (1945), 6.2,184, 4.4, 108, 5.8, 312. TABLE 9. The proline content of egg proteins Literature value: Motomura (1929), protein of eggs which had been stored for, the proline level was high compared with fresh eggs and with those stored for other lengths of time. This is reflected in a comparatively large number

4 700 EVANS, DAVIDSON, BANDEMER AND BUTTS of milligrams of proline in both the albumen and the yolk of the 18-month old eggs. There was more phenylalanine in the 18- than in the 9-month old eggs, although it is very similar structurally to tyrosine differing only in the absence of the hydroxyl group. There was no evidence that tyrosine was converted to phenylalanine. s of the 23-month old eggs contained more of each of the amino acids except methionine than yolks of any other stored eggs. There was less methionine in the yolks of 23- than of 26-month old eggs. The - percent methionine in egg albumen protein (Table 4) agrees well with literature values except for that of Munk etal. (1945) who estimated methionine by difference, subtracting the cystine sulfur, determined by precipitation as the cuprous mercaptide, from the total organic sulfur. Values presented in Table 4 for the methionine content of yolk protein are lower than most reported in the literature. Good agreement was obtained between chemical and microbiological methods for the determination of cystine. The average values determined colorimetrically and microbiologically respectively were for albumen protein 2.44 and 2.45, yolk protein 1.76 and 1., and whole egg protein 5 and 4 percent. Except for the tryptophan data reported by Fiirth and Lieben (1921), the value of percent tryptophan in whole egg protein presented in Table 7 is the highest coming to the authors' attention. Other investigators obtained values of 1.4 to. 1.6 percent. The use of enzymatic digestion in vitro for hydrolyzing the proteins previous to making the tryptophan determinations rather than the usual alkaline hydrolysis, may have been, partially at least, responsible for the high tryptophan values obtained in the present investigation. Nutritionally it is doubtful if any of the eggs except those that were stored for need be given consideration, because shell eggs are not usually kept in storage for a longer time. The greatest losses of essential amino acids from the 9-month old eggs were of methionine and tryptophan, each of which decreased by 18 percent, and the least were of arginine and lysine with 12 percent decreases. Losses of most of the essential amino acids were not much greater from the 26- than from the 9-month old eggs, except that arginine and threonine losses from the 26-month old eggs were nearly twice those from the 9-month old ones. 20 percent of the arginine and 30 percent of the threonine (Evans etal. 1949b) were lost. Of the essential amino acids only threonine occurred in lower concentration in the protein of 9-month old eggs than in that of fresh ones, and the decrease was so small as to be unimportant nutritionally (4 percent of the threonine). The differences of amino acid concentration in yolk and albumen proteins show the importance of eating the whole egg in order to supply a well balanced protein. Such is especially true for the two sulfurcontaining amino acids, cystine and methionine, because egg albumen protein contains 1.6 times as high a concentration as egg yolk protein. Mitchell and Block (1946) consider whole eggs to be the most ideal protein source nutritionally and have used the amino acid composition of whole egg protein as a basis of comparison for other proteins and protein concentrates. According to their standards the nutritive value of egg yolk protein would be limited by the low concentration of cystine plus methionine, because it contains only percent compared to 5.7 percent in whole egg protein. When one considers that

5 AMINO ACID CONTENT OF EGGS 701 egg albumen protein contains 6.6 percent of cystine plus methionine, the necessity of eating the egg albumen along with the yolk is indicated if the most economical utilization of the egg protein is to be obtained. Otherwise more protein must be consumed to meet the body's need for cystine and methionine. SUMMARY Arginine, histidine, lysine, methionine, cystine, tyrosine, tryptophan, phenylalanine, and proline contents of fresh eggs and of eggs stored for 9, 18, 23, and 26 months were determined by microbiological assay. Cystine was also determined colorimetrically, and good agreements were obtained between the two methods. Higher values for tryptophan were obtained by employing enzymatic hydrolysis in vitro than had previously been reported with alkaline hydrolysis. Arginine and tyrosine were the only ones of these amino acids present in albumen protein of stored eggs in a lower concentration than in albumen protein of fresh eggs. Egg albumen protein contained 1.6 times as high a concentration of cystine plus methionine as yolk protein. The concentrations of the basic amino acids and proline in egg yolk protein were higher than in the albumen protein. s of eggs stored for had fewer milligrams of each of the amino acids than did those from fresh eggs or eggs stored longer. s from 26-month old eggs contained fewer milligrams of each of the amino acids, except methionine than the other eggs, but the yolks of the 23-month old eggs had the least methionine. Of these amino acids, only tyrosine and arginine were present in larger amounts in 9- than in 18-month old eggs. REFERENCES Abderhalden, E., and M. Kempe, Comparative investigations concerning the content of tyrosine, glycocoll, and glutamic acid in fertilized hens' eggs at various periods of development. Z. physiol. Chem. 53: Beach, E. F., and D. M. Teague, A gravimetric method for the determination of methionine. J. Biol. Chem. 142: Block, R. J., and D. Boiling, The amino acid yield from various animal and plant protein after hydrolysis of the fat free tissue. Arch. Biochem. 3: Block, R. J., and H. H. Mitchell, The correlation of the amino-acid composition of proteins with their nutritive value. Nut. Abst. and Rev. 16: Calvery, H. O., Some chemical investigations of embryonic metabolism. IV. An investigation of the basic amino acids of the hen's egg during development. J. Biol. Chem. 83: Calvery, H. O., Some chemical investigations of embryonic metabolism. V. The tyrosine, tryptophan, cystine, cysteine, and uric acid content of the developing hen's egg. J. Biol. Chem. 87: Calvery, H. O., and H. W. Titus, The composition of the proteins of eggs from hens on different diets. J. Biol. Chem. 105: Cho, B., The quantitative changes in cystine, tryptophan, and tyrosine in the proteins of hen egg during incubation. Chem. Abst. 22: 2967; Bui. sci Fakultato Terkultura 2: (1), 6 pp. Csonka, F. A., H. Liechtenstein, and C. A. Denton, Cystine determination in proteins and foods. J. Biol. Chem. 156: Edwards, L. E., R. R. Sealock, W. W. O'Donnell, G. R. Bartlett, M. B. Barclay, R. Tully, R. H. Tybout, J. Box, and J. R. Murlin, Biological value of proteins in relation to the essential amino acids which they contain. IV. The analysis of fifteen protein foods for the ten essentials. J. Nutrition 32: Evans, R. J., Use of the spectrophotometer in the determination of cystine by Sullivan's reaction. J. Biol. Chem. 156: Evans, R. J., Hydrolysis of soybean oil meal proteins by some proteolytic enzymes. Arch. Biochem. 11: Evans, R. J., J. A. Davidson, and H. A. Butts,. 1949a. Changes in egg proteins occurring during cold storage of shell eggs. Poultry Sci. 28: Evans, R. J., H. A. Butts, J. A. Davidson, and S. L. Bandemer, 1949b. The amino acid content of fresh and stored shell eggs. I. Leucine, isoleucine, valine, glycine, serine, threonine, aspartic acid, and glutamic acid. Poultry Sci. 28:

6 702 EVANS, DAVIDSON, BANDEMER AND BUTTS Ftirth, O., and F. Lieben, Colorimetric studies of tryptophane. VI. On the tryptophane content of some foodstuffs and the tryptophane requirement of grown men. Biochem. Z. 122: Grau, C. R., The phenylalanine and tyrosine contents of chicks and eggs. J. Biol. Chem. 158: Gunness, M., I. M. Dwyer, and J. L. Stokes, Microbiological methods for the determination of amino acids. III. Extension of the uniform assay method for the ten essential amino acids to include tyrosine. J. Biol. Chem. 163: Hess, W. C, E. H. Kramke, J. C. Fritz, and H. W. Howard, A comparison of the nutritive value of egg proteins and their amino acid content. Proc. Soc. Exptl. Biol. Med. 67: Horn, M. J., D. B. Jones, and A. E. Blum, 1946a. Colorimetric determination of methionine in proteins and foods. J. Biol. Chem. 166: Horn, M. J., D. B. Jones, and A. E. Blum, 1946b. Microbiological determination of methionine in proteins and foods. J. Biol. Chem. 166: Horn, M. J., D. B. Jones, and A. E. Blum, Microbiological determination of phenylalanine in proteins and foods. J. Biol. Chem. 176: Lyman, C. M., 0. Moseley, A. Wood, and F. Hale, Note on the use of hydrogen peroxidetreated peptone in media for the microbiological determination of amino acids. Arch. Biochem. 10: McFarlane, W. D., H. L. Fulmer, and T. H. Jukes, Studies in embryonic mortality in the chick. I. The effect of diet upon the nitrogen, amino nitrogen, tyrosine, tryptophan, cystine, and iron content of the proteins and on the total copper of the hen's egg. Biochem. J. 24: Mitchell, H. H., and R. J. Block, Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. Biol. Chem. 163: Motomura, J., Hydrolysis of the protein of hen's eggs. Chem. Abst. 23: 2732; Bull. Sci. Fakultato Terkuturo, Kjusu Imp. Univ. 3: (reprint). Munks, B., A. Robinson, E. F. Beach, and H. H. Williams, Amino acids in the production of chicken egg and muscle. Poultry Sci. 24: Plimmer, R. H. A., and J. Lowndes, Changes in the amino-acids in the proteins of the hen's egg during development. Biochem. J. 21: Sauberlich, H. E., and C. A. Baumann, The effect of dietary protein upon amino acid excretion by rats and mice. J. Biol. Chem. 166: , Sendju, Y., The behavior of the vitally important amino acids during the incubation of the hen egg. Chem. Abst. 20: 2362; J. Biochem. (Japan) 5: Sendju, Y., The behavior of vitally important amino acids during the incubation of the hen's egg. II. Cystine and cysteine. Chem. Abst. 21: 2309; J. Biochem. (Japan) 7: