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An improved method of analysis for glycine using Streptococcus faecalis
An Improved Method of Analysis for Glycine Using Streptococcus faecalis Thomas A. McCoy, M. K. Patterson, Jr. and Simon H. Wender From the Laboratory Research Division of The Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma, and the Chemistry Department of the University
of Oklahoma,
Norman,
Oklahoma
Received November 6, 1952 INTRODUCTION
While glycine has been reported essential for several lactic acid bacteria, the only organisms which have been generally used to date to analyze for this amino acid are Lewonostoc mescnteroides P-60 (l-3) and Leuconostoc citrovorum (3). This report describes the successful use of Streptococcus faecalis A.T.C.C.’ 6057 in the determination of glycine in protein hydrolyzates. EXPERIMENTAL The stock culture was maintained on a medium contaning O.SoJopeptone, 1% glucose, 1% yeast extract, and 1.5% agar. The organism was transferred as a stab weekly, incubated at 37” for 24 hr., and stored in the refrigerator at 4’ until used. The maintenance of this organism was carried out with extreme care, since the condition of the culture was shown to affect analytical results. The assay techniques employed were similar to the ordinary microbiological procedures with the following exceptions. The inoculum used was an S-hr. culture. All incubation periods were carried out in a special 55.gal. constant-temperature water bath (American Instrument Company). The growth response of the organism was measured by the turbidity of the suspension at the end of 16 hr. of growth. The turbidities were measured in a Fisher electrophotometer using a filter of 650 rnp and the attachment for micro cells. Several basal media have been developed for this organism (4), and the one used in the present study was basal medium III (see Table I). The tubes were sterilized at 15 lb. pressure for 8 min. and were chilled in an ice bath before and during inoculation. The method of inoculation used was the “automatic pipet” method. This consisted of autoclaving the double-strength medium and the test solutions separately. The washed cell suspension was introduced into the doublestrength medium and allowed to circulate through a Filamatic Pipettor (National 1 American Type Culture Collection, Washington 6, D. C. 435
486
MCCOY,
PATTERSON
AND
WENDER
Instrument Company) for 5 min. at a speed of 4.5. During this time the inoculum and the medium were stirred with a magnetic stirrer. The inoculated doublestrength medium was introduced aseptically into each test solution. At the end of the incubation period, the tubes were again chilled in an ice bath to inhibit growth. Just previous to measuring the turbidity of the tubes, the solutions were warmed to prevent the condensation of water vapor on the surface TABLE Composition
I
of the Basal Medium III” nrb.
Alanine Arginine . HCI Aspartic acid Cystine Glutamic acid. HCI Histidine . HCl Hydroxyproline Isoleucind Leucine * Lysine *HCl Methionine* Norleucine* Phenylalanine” Proline Serine* Threonine” Tryptophan Tyrosine Valine* Uracil Adenine sulfate Guanine*HCl.2HzO
25 1ooo 50 166 200 20 50 30 60 30 20 50 36 50 30 50 10 50 40 10 10 10
NH&l Na citrate.2HzO Na acetatem3HtO KHzPO, K,HPO, MgS0,.7HzOC NaClc FeS0d.7HtOc MnSOd.4HtOc Na citrate.2HrOC Glucose Thiamine. HCI Riboflavin Ca pantothenate p-Aminobenzoic acid Pyridoxamine Niacin Folic acid Biotin
2 g. 10 g. 5 g. 160 mg. 160 mg. 800 mg. 40 mg. 40 mg. 120 mg. 30 mg.
6 g. 0.5 0.25 0.5 0.1 1.0 0.5
2
mg. mg. mg. mg. mg. mg. Pg.
4 Pg.
a All amounts are based on the L-isomer in a solution of 1 1. final concentration. * Source of amino acid was racemic mixture. c Modified salt C. of the optical cell, and the bacterial cells were suspended in the medium with a magnetic stirrer. For the purposes of this paper, the growth response will be referred to in terms of the optical density of the suspension multiplied by ten to the second power (O.D. X lo*), and the concentration of glycine will be referred to on the basis of amount per milliliter of final solution. The protein hydrolyzates were prepared in the following manner: Ten milligrams of protein was hydrolyzed with 1 ml. of 6 N HCl for 24 hr. At the end of this time, the excess HCI was removed by distillation. The solutions were filtered,
GLYCINE ANALYSIS METHOD neutralized with NH,OH, and made up to a volume of 5 ml. Appropriate of the hydrolyzates were made from these stock hydrolyzates.
487 dilutions
RESULTS
S. jaecalis is more sensitive to glycine than some of the other lactic acid bacteria employed in glycine analysis. The range of the glycine curve was found to be from 0 to 6 pg. as compared with Leuc. mesenteroides P-60 whose range is from 3.5 to 14 pg. (2) and Leuc. citrovmum whose range is from 0 to 12.5 pg. (3). Further, S. fuecaZi.sdoes not exhibit an induction period at lower concentrations of glycine as was noted with Leuc. mesenteroides P-60 (2), or the high blanks using Leuc. citrovorum (3). The results of analyses of lysozyme, casein, silk fibroin, gelatin, fibrin, pepsin, and wheat gluten, together with recovery data, are listed in Tables II and III. The percentages of glycine in the proteins are reported on the basis of the moisture-free and ash-free protein. Glycine in Lysozyme Isoelectric lysozyme was prepared by the method of Alderton and Fevold (5). The average mean value of glycine in lysozyme was found to be 6.15 y0 (Table II). This value is slightly higher than those values previously reported in the literature. For example, Lewis et al. (6) using Lew. mesentercides P-60 reported 5.7 Y0 glycine in lysozyme. Fromageot et al. (7) using the Fisher paper-partition method (8) obtained a value of 5.3 %, and a value of 5.8% was reported using the colorimetrie procedure of Alexander et al. (9). Krueger (10) has recently pointed out, however, that the chromotropic acid method of Alexander et al. (9) yielded low values. In view of these factors, it seems that the glycine content of lysozyme is in the general range of 6.1%. Glycine in Remaining Proteins The glycine content of the other purified proteins (listed in Table II) compare favorably with previous reports in the literature. Some of the other analytical methods referred to in Table II include the solubility product method (ll), paper partition (7,12), isotope dilution (13), other chemical methods (14, 15), as well as microbiological assays. The ranges for glycine content together with average deviations are self-explanatory and will not be discussed here. Since S. foe&is seemed so sensitive to minute amounts of glycine, it was deemed advisable to see if this method could be used in micro
488
MCCOY,
PA’M’ERSON
AND
WENDER
analysis. Three milligrams of casein was hydrolyzed and analyzed for glycine content. There were six replicate tubes at three levels on the curve, and the average glycine content was found to be 2.02 f .03. These results compared favorably with previous results using larger test volumes. In view of this fact, it appears that the glycine content of proteins can be satisfactorily determined when limited amounts of the TABLE Glycine
II
Content oj Proteins
All values are reported on an oven-dried ash-free basis and are the average of 15 tubes at five-sample levels. The sources of proteins were as follows: lysozyme prepared from egg white by the method of Alderton and Fevold (5) ; casein from milk by the method of Cohn and Hendry (18) ; silk fibroin was kindly supplied by Dr. M. S. Dunn, University of California at Los Angeles; gelatin was precipitated in 0.04 N sodium acetate and acetic acid buffer at pH 4.7 using 950Joethanol as a precipitating agent followed by dialysis; fibrin was Catalog No. F-3, Fisher Scientific Company; pepsin was porcine origin, Lot No. 80802, Armour and Company; wheat gluten was obtained from Nutritional Biochemicals Corporation. -Per cent nitrogen
Protein
i
Per cent glycine Literature
values
Lysozyme Casein Silk fibroin Gelatin Fibrin Pepsin Gluten (wheat)
-
18.1 15.7 18.7 17.6 16.3 14.1 15.4
6.096.26 1.98-2.12 42.944.3 24.4-27.6
’ 6.15 ’ 2.06 ~ 43.6 1 26.2
f f f f
.07 .05 .50 .70
5.3-5.8 (7) 2.1 (15) 41.5-44.9 (2, 11, 12) 26.6 (14) 5.1-5.8 (12, 13) 6.4 (16) 3.5 (17)
protein are available for analysis. This is one of the greatest assets of this analytical method. Recovery of Glycine The glycine recovery data are listed in Table III. The glycine recovered averaged about 100% with an average deviation of approximately ~3 %. In no case was there any direct evidence indicating additional stimulation or inhibition at the different levels of glycine on the curve. As one check for the validity of the assay. two amino acid test mixtures
GLYCINE
ANALYSIS
489
METHOD
were prepared and analyzed. The concentration of glycine in the amino acid mixture No. 1 was 0.271%. This ext,remely low amount of glycine was used since Leuc. mesenteroides P-60 yielded recoveries of 112 y0 and 108% when the glycine content of Shankman’s et al. amino acid mixture was 0.292% (2). The average recoveries from the amino acid mixture No. 1 was 100.5 y0 with an average mean deviation of f0.5 %. TABLE Glycine
Recovery
Protein
Lysozyme Casein Silk fibroin Gelatin Fibrin Pepsin Gluten Amino acid mixture No. la Amino acid mixture No. 2b
III
Data from Protein
Hydrolyzates
Per cent glycine added to hydmlyzate (to be recovered)
3.0 1.01
23.4 13.0 2.6 3.0 1.7 .271 5.17
99.0-102.0 96.0-102.4 96.8-114.0 96.7-106.0 93.9-104.7 94.0-103.0 101.6-102.5 100.0-101.0 98.0-99.6
100.6 99.7 102.5 100.3 100.7 98.4 102.0 190.5 98.9
f * f f f f f f f
1.0 1.8 4.6 2.3 3.4 3.1 .18 0.5 0.5
a Composition of the amino acid mixture No. 1 (in grams) was as follows: arginine, 14.83; histidine, 7.24; lysine, 26.21; tyroeine, 23.10; tryptophan, 4.14; phenylalanine, 17.24; cystine, 1.38; methionine, 11.72; threonine, 13.10; serine, 26.55; leucine, 33.45; isoleucine, 21.72; valine, 22.41; giutamic acid, 80.35; aspartic acid, 21.03; glycine, 1.00; alanine, 18.96; proline, 24.14. b Composition of the amino acid mixture No. 2 (in grams) was as follows: arginine, 2.230; histidine, 0.175; lysine, l.ooO; tyrosine, 0.632; tryptophan, 1.860; phenylalanine, 0.573; cystine, 1.193; methionine, 0.368; threonine, 0.965; serine, 1.175; leucine, 1.210; isoleucine, 0.912; valine, 0.842; glutamic acid, 0.754; aspartic acid, 3.193; glycine, 1.009; aianine, 1.017; proline, 0.246.
The concentration of glycine in the amino acid mixture No. 2 was 5.17 %. The average recovery of glycine from this mixture was 98.9 % with an average mean deviation of f0.5 %. These results speak well for the validity of the analytical method employed. DISCUSSION
From these experimental data, it is apparent that S. ja.ecalis can be used very satisfactorily for the routine analysis of micro amounts of glycine in protein hydrolyzates. The authors have been unable to find any other generally applicable method of analysis for small amounts of
490
MCCOY, PATTERSON
AND WENDER
glycine that is as sensitive as and still retains the degree of accuracy of the present reported method. The results of analyses of purified proteins agree favorably with values of previous workers. The recoveries of glycine from protein hydrolyzates (ranging in glycine content from 2 % in casein to 43 % in silk fibroin) averaged around 100 ‘j& which further supports the accuracy of the assay. It is significant, however, that recovery of glycine at the lowest sample level has consistently the largest deviation from the average mean. For example, the 114 $&recovery in silk fibroin, the 106 % recovery in gelatin, the 93 y0 recovery in fibrin, and the 94 y0 recovery in pepsin were values obtained at the lowest sample level. Since the amount of glycine to be recovered in all of these cases was in the magnitude of 0.50 pg., it seems that the over-all accuracy of the method is limited at this level of glycine. The accuracy, however, was greatly improved at the recovery level of 1 pg. of glycine. SUMMARY
Streptococcus faecalis A.T.C.C. 6057 can be used satisfactorily to determine glycine in a final medium solution concentration of from 0 to 6 pg./ml/tube. This analytical procedure is more sensitive and is as accurate as previous methods of glycine determinations. Advantages of the method include (a) the small amount of protein required for assay, and (b) the accuracy of the assay. The glycine content of seven purified proteins was reported. REFERENCES 1. BRAND, E., SAIDEL, L. J., GOLDWATER, W. H., KASSEL, B., AND RYAN, F. J., J. Am. Chem. Sot. 67, 1524 (1945). 2. SHANKMAN, S., CAMIEN, M. N., AND DUNN, M. S., J. Biol. Chem. 166, 51 (1947). 3. STEELE, B. F., SAUBERLICH, H. E., REYNOLDS, M. S., AND BAUMANN, C. A., J. Biol. Chem. 177, 533 (1949). 4. MCCOY, T. A., AND WENDER, S. H., Presented at the 7th Southwest Regional Meeting of the American Chemical Society, Austin, Texas, 1951. 5. ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164.1 (1946). 6. LEWIS, J. C., SNELL, N. S., HIRSCHMANN, D. J., AND FRAENKEL-CONRAT, H., J. Biol. Chem. 186, 23 (1959). 7. FROMAQEOT, C., AND DE GARILHE, M. P., Biochim. et Biophys. Acta 4. 509 (1959). 8. FISHER, R. B., PARSONS, D. S., AND MORRISON, G. A., Nature 161, 764 (1948). 9. ALEXANDER, B., LANDWEHR, G., AND SELIGMAN, A. M., J. Biol. Chem. 166, 51 (1945).
GLYCINE
10. 11. 12. 13. 14.
ANALYSIS
METHOD
491
KRUEGER, R., Helv. Chim. Acta 32, 238 (1949). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 136, 113 (1943). TRISTRAM, G. R., Advances in Protein Chem. 6, 101 (1949). RITTENBERQ, D., AND FOSTER, G. L., J. Biol. Chem. 133, 737 (1940). BLOCK, R. J., AND BOLLINQ, D., The Determination of the Amino Acids, p. iii. Minneapolis, 1948. 15. BLOCK, R. J., AND MITCHELL, H. H., Nutrition Abstr. & Revs. 16,249 (1946-47). 16. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., Crystalline Enzymes, p. 26. New York, 1948. 17. PENCE, J. W., MECHAM, D. K., ELDER, A. H., LEWIS, J. C., SNELL, N. S., AND OLCOTT, H. S., Cereal Chem. 27, 335 (1950). 18. COHN, E. J., AND HENDRY, J. L., in BLATT, A. H., Organic Syntheses, Collective Vol. II, p. 120. Wiley, New York, 1947.
of Oklahoma,
Norman,
Oklahoma
Received November 6, 1952 INTRODUCTION
While glycine has been reported essential for several lactic acid bacteria, the only organisms which have been generally used to date to analyze for this amino acid are Lewonostoc mescnteroides P-60 (l-3) and Leuconostoc citrovorum (3). This report describes the successful use of Streptococcus faecalis A.T.C.C.’ 6057 in the determination of glycine in protein hydrolyzates. EXPERIMENTAL The stock culture was maintained on a medium contaning O.SoJopeptone, 1% glucose, 1% yeast extract, and 1.5% agar. The organism was transferred as a stab weekly, incubated at 37” for 24 hr., and stored in the refrigerator at 4’ until used. The maintenance of this organism was carried out with extreme care, since the condition of the culture was shown to affect analytical results. The assay techniques employed were similar to the ordinary microbiological procedures with the following exceptions. The inoculum used was an S-hr. culture. All incubation periods were carried out in a special 55.gal. constant-temperature water bath (American Instrument Company). The growth response of the organism was measured by the turbidity of the suspension at the end of 16 hr. of growth. The turbidities were measured in a Fisher electrophotometer using a filter of 650 rnp and the attachment for micro cells. Several basal media have been developed for this organism (4), and the one used in the present study was basal medium III (see Table I). The tubes were sterilized at 15 lb. pressure for 8 min. and were chilled in an ice bath before and during inoculation. The method of inoculation used was the “automatic pipet” method. This consisted of autoclaving the double-strength medium and the test solutions separately. The washed cell suspension was introduced into the doublestrength medium and allowed to circulate through a Filamatic Pipettor (National 1 American Type Culture Collection, Washington 6, D. C. 435
486
MCCOY,
PATTERSON
AND
WENDER
Instrument Company) for 5 min. at a speed of 4.5. During this time the inoculum and the medium were stirred with a magnetic stirrer. The inoculated doublestrength medium was introduced aseptically into each test solution. At the end of the incubation period, the tubes were again chilled in an ice bath to inhibit growth. Just previous to measuring the turbidity of the tubes, the solutions were warmed to prevent the condensation of water vapor on the surface TABLE Composition
I
of the Basal Medium III” nrb.
Alanine Arginine . HCI Aspartic acid Cystine Glutamic acid. HCI Histidine . HCl Hydroxyproline Isoleucind Leucine * Lysine *HCl Methionine* Norleucine* Phenylalanine” Proline Serine* Threonine” Tryptophan Tyrosine Valine* Uracil Adenine sulfate Guanine*HCl.2HzO
25 1ooo 50 166 200 20 50 30 60 30 20 50 36 50 30 50 10 50 40 10 10 10
NH&l Na citrate.2HzO Na acetatem3HtO KHzPO, K,HPO, MgS0,.7HzOC NaClc FeS0d.7HtOc MnSOd.4HtOc Na citrate.2HrOC Glucose Thiamine. HCI Riboflavin Ca pantothenate p-Aminobenzoic acid Pyridoxamine Niacin Folic acid Biotin
2 g. 10 g. 5 g. 160 mg. 160 mg. 800 mg. 40 mg. 40 mg. 120 mg. 30 mg.
6 g. 0.5 0.25 0.5 0.1 1.0 0.5
2
mg. mg. mg. mg. mg. mg. Pg.
4 Pg.
a All amounts are based on the L-isomer in a solution of 1 1. final concentration. * Source of amino acid was racemic mixture. c Modified salt C. of the optical cell, and the bacterial cells were suspended in the medium with a magnetic stirrer. For the purposes of this paper, the growth response will be referred to in terms of the optical density of the suspension multiplied by ten to the second power (O.D. X lo*), and the concentration of glycine will be referred to on the basis of amount per milliliter of final solution. The protein hydrolyzates were prepared in the following manner: Ten milligrams of protein was hydrolyzed with 1 ml. of 6 N HCl for 24 hr. At the end of this time, the excess HCI was removed by distillation. The solutions were filtered,
GLYCINE ANALYSIS METHOD neutralized with NH,OH, and made up to a volume of 5 ml. Appropriate of the hydrolyzates were made from these stock hydrolyzates.
487 dilutions
RESULTS
S. jaecalis is more sensitive to glycine than some of the other lactic acid bacteria employed in glycine analysis. The range of the glycine curve was found to be from 0 to 6 pg. as compared with Leuc. mesenteroides P-60 whose range is from 3.5 to 14 pg. (2) and Leuc. citrovmum whose range is from 0 to 12.5 pg. (3). Further, S. fuecaZi.sdoes not exhibit an induction period at lower concentrations of glycine as was noted with Leuc. mesenteroides P-60 (2), or the high blanks using Leuc. citrovorum (3). The results of analyses of lysozyme, casein, silk fibroin, gelatin, fibrin, pepsin, and wheat gluten, together with recovery data, are listed in Tables II and III. The percentages of glycine in the proteins are reported on the basis of the moisture-free and ash-free protein. Glycine in Lysozyme Isoelectric lysozyme was prepared by the method of Alderton and Fevold (5). The average mean value of glycine in lysozyme was found to be 6.15 y0 (Table II). This value is slightly higher than those values previously reported in the literature. For example, Lewis et al. (6) using Lew. mesentercides P-60 reported 5.7 Y0 glycine in lysozyme. Fromageot et al. (7) using the Fisher paper-partition method (8) obtained a value of 5.3 %, and a value of 5.8% was reported using the colorimetrie procedure of Alexander et al. (9). Krueger (10) has recently pointed out, however, that the chromotropic acid method of Alexander et al. (9) yielded low values. In view of these factors, it seems that the glycine content of lysozyme is in the general range of 6.1%. Glycine in Remaining Proteins The glycine content of the other purified proteins (listed in Table II) compare favorably with previous reports in the literature. Some of the other analytical methods referred to in Table II include the solubility product method (ll), paper partition (7,12), isotope dilution (13), other chemical methods (14, 15), as well as microbiological assays. The ranges for glycine content together with average deviations are self-explanatory and will not be discussed here. Since S. foe&is seemed so sensitive to minute amounts of glycine, it was deemed advisable to see if this method could be used in micro
488
MCCOY,
PA’M’ERSON
AND
WENDER
analysis. Three milligrams of casein was hydrolyzed and analyzed for glycine content. There were six replicate tubes at three levels on the curve, and the average glycine content was found to be 2.02 f .03. These results compared favorably with previous results using larger test volumes. In view of this fact, it appears that the glycine content of proteins can be satisfactorily determined when limited amounts of the TABLE Glycine
II
Content oj Proteins
All values are reported on an oven-dried ash-free basis and are the average of 15 tubes at five-sample levels. The sources of proteins were as follows: lysozyme prepared from egg white by the method of Alderton and Fevold (5) ; casein from milk by the method of Cohn and Hendry (18) ; silk fibroin was kindly supplied by Dr. M. S. Dunn, University of California at Los Angeles; gelatin was precipitated in 0.04 N sodium acetate and acetic acid buffer at pH 4.7 using 950Joethanol as a precipitating agent followed by dialysis; fibrin was Catalog No. F-3, Fisher Scientific Company; pepsin was porcine origin, Lot No. 80802, Armour and Company; wheat gluten was obtained from Nutritional Biochemicals Corporation. -Per cent nitrogen
Protein
i
Per cent glycine Literature
values
Lysozyme Casein Silk fibroin Gelatin Fibrin Pepsin Gluten (wheat)
-
18.1 15.7 18.7 17.6 16.3 14.1 15.4
6.096.26 1.98-2.12 42.944.3 24.4-27.6
’ 6.15 ’ 2.06 ~ 43.6 1 26.2
f f f f
.07 .05 .50 .70
5.3-5.8 (7) 2.1 (15) 41.5-44.9 (2, 11, 12) 26.6 (14) 5.1-5.8 (12, 13) 6.4 (16) 3.5 (17)
protein are available for analysis. This is one of the greatest assets of this analytical method. Recovery of Glycine The glycine recovery data are listed in Table III. The glycine recovered averaged about 100% with an average deviation of approximately ~3 %. In no case was there any direct evidence indicating additional stimulation or inhibition at the different levels of glycine on the curve. As one check for the validity of the assay. two amino acid test mixtures
GLYCINE
ANALYSIS
489
METHOD
were prepared and analyzed. The concentration of glycine in the amino acid mixture No. 1 was 0.271%. This ext,remely low amount of glycine was used since Leuc. mesenteroides P-60 yielded recoveries of 112 y0 and 108% when the glycine content of Shankman’s et al. amino acid mixture was 0.292% (2). The average recoveries from the amino acid mixture No. 1 was 100.5 y0 with an average mean deviation of f0.5 %. TABLE Glycine
Recovery
Protein
Lysozyme Casein Silk fibroin Gelatin Fibrin Pepsin Gluten Amino acid mixture No. la Amino acid mixture No. 2b
III
Data from Protein
Hydrolyzates
Per cent glycine added to hydmlyzate (to be recovered)
3.0 1.01
23.4 13.0 2.6 3.0 1.7 .271 5.17
99.0-102.0 96.0-102.4 96.8-114.0 96.7-106.0 93.9-104.7 94.0-103.0 101.6-102.5 100.0-101.0 98.0-99.6
100.6 99.7 102.5 100.3 100.7 98.4 102.0 190.5 98.9
f * f f f f f f f
1.0 1.8 4.6 2.3 3.4 3.1 .18 0.5 0.5
a Composition of the amino acid mixture No. 1 (in grams) was as follows: arginine, 14.83; histidine, 7.24; lysine, 26.21; tyroeine, 23.10; tryptophan, 4.14; phenylalanine, 17.24; cystine, 1.38; methionine, 11.72; threonine, 13.10; serine, 26.55; leucine, 33.45; isoleucine, 21.72; valine, 22.41; giutamic acid, 80.35; aspartic acid, 21.03; glycine, 1.00; alanine, 18.96; proline, 24.14. b Composition of the amino acid mixture No. 2 (in grams) was as follows: arginine, 2.230; histidine, 0.175; lysine, l.ooO; tyrosine, 0.632; tryptophan, 1.860; phenylalanine, 0.573; cystine, 1.193; methionine, 0.368; threonine, 0.965; serine, 1.175; leucine, 1.210; isoleucine, 0.912; valine, 0.842; glutamic acid, 0.754; aspartic acid, 3.193; glycine, 1.009; aianine, 1.017; proline, 0.246.
The concentration of glycine in the amino acid mixture No. 2 was 5.17 %. The average recovery of glycine from this mixture was 98.9 % with an average mean deviation of f0.5 %. These results speak well for the validity of the analytical method employed. DISCUSSION
From these experimental data, it is apparent that S. ja.ecalis can be used very satisfactorily for the routine analysis of micro amounts of glycine in protein hydrolyzates. The authors have been unable to find any other generally applicable method of analysis for small amounts of
490
MCCOY, PATTERSON
AND WENDER
glycine that is as sensitive as and still retains the degree of accuracy of the present reported method. The results of analyses of purified proteins agree favorably with values of previous workers. The recoveries of glycine from protein hydrolyzates (ranging in glycine content from 2 % in casein to 43 % in silk fibroin) averaged around 100 ‘j& which further supports the accuracy of the assay. It is significant, however, that recovery of glycine at the lowest sample level has consistently the largest deviation from the average mean. For example, the 114 $&recovery in silk fibroin, the 106 % recovery in gelatin, the 93 y0 recovery in fibrin, and the 94 y0 recovery in pepsin were values obtained at the lowest sample level. Since the amount of glycine to be recovered in all of these cases was in the magnitude of 0.50 pg., it seems that the over-all accuracy of the method is limited at this level of glycine. The accuracy, however, was greatly improved at the recovery level of 1 pg. of glycine. SUMMARY
Streptococcus faecalis A.T.C.C. 6057 can be used satisfactorily to determine glycine in a final medium solution concentration of from 0 to 6 pg./ml/tube. This analytical procedure is more sensitive and is as accurate as previous methods of glycine determinations. Advantages of the method include (a) the small amount of protein required for assay, and (b) the accuracy of the assay. The glycine content of seven purified proteins was reported. REFERENCES 1. BRAND, E., SAIDEL, L. J., GOLDWATER, W. H., KASSEL, B., AND RYAN, F. J., J. Am. Chem. Sot. 67, 1524 (1945). 2. SHANKMAN, S., CAMIEN, M. N., AND DUNN, M. S., J. Biol. Chem. 166, 51 (1947). 3. STEELE, B. F., SAUBERLICH, H. E., REYNOLDS, M. S., AND BAUMANN, C. A., J. Biol. Chem. 177, 533 (1949). 4. MCCOY, T. A., AND WENDER, S. H., Presented at the 7th Southwest Regional Meeting of the American Chemical Society, Austin, Texas, 1951. 5. ALDERTON, G., AND FEVOLD, H. L., J. Biol. Chem. 164.1 (1946). 6. LEWIS, J. C., SNELL, N. S., HIRSCHMANN, D. J., AND FRAENKEL-CONRAT, H., J. Biol. Chem. 186, 23 (1959). 7. FROMAQEOT, C., AND DE GARILHE, M. P., Biochim. et Biophys. Acta 4. 509 (1959). 8. FISHER, R. B., PARSONS, D. S., AND MORRISON, G. A., Nature 161, 764 (1948). 9. ALEXANDER, B., LANDWEHR, G., AND SELIGMAN, A. M., J. Biol. Chem. 166, 51 (1945).
GLYCINE
10. 11. 12. 13. 14.
ANALYSIS
METHOD
491
KRUEGER, R., Helv. Chim. Acta 32, 238 (1949). MOORE, S., AND STEIN, W. H., J. Biol. Chem. 136, 113 (1943). TRISTRAM, G. R., Advances in Protein Chem. 6, 101 (1949). RITTENBERQ, D., AND FOSTER, G. L., J. Biol. Chem. 133, 737 (1940). BLOCK, R. J., AND BOLLINQ, D., The Determination of the Amino Acids, p. iii. Minneapolis, 1948. 15. BLOCK, R. J., AND MITCHELL, H. H., Nutrition Abstr. & Revs. 16,249 (1946-47). 16. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., Crystalline Enzymes, p. 26. New York, 1948. 17. PENCE, J. W., MECHAM, D. K., ELDER, A. H., LEWIS, J. C., SNELL, N. S., AND OLCOTT, H. S., Cereal Chem. 27, 335 (1950). 18. COHN, E. J., AND HENDRY, J. L., in BLATT, A. H., Organic Syntheses, Collective Vol. II, p. 120. Wiley, New York, 1947.