MICROBIOLOGICAL METHODS FOR THE DETERMINATION OF AMINO ACIDS II. A UNIFORM ASSAY FOR THE TEK ESSEXTIAL AMINO ACIDS BY JACOB I,. STOIiES, MARION GUNNESS, IRLA M. DWYER, AND MURIEL C. CASWELL (From the Research Laboratories, Merck and Company, Inc., Rahway, New Jersey) (Received for publication, May 8, 1945) Microbiological methods for the determination of amino acids, becauseof their specificity, accuracy, sensitivity, and ability to yield many replicate results within a short time, promise to become of increasing importance in investigations of the chemistry and biochemistry of proteins and amino acids. Although such methods are of relatively recent origin, a considerable and rapidly increasing number are already available for the assay of many of the amino acids in purified proteins and natural products. Since a pre- vious review of the literature (l), a method for lysine (2) and additional methods for tryptophane (3) and glutamic acid (4,5) have been published. Also a fungus method is available for the assay of leucine (6). With the exception of leucine (6-8), isoleucine (7), valine (7, 8), and tryptophane (3,9), microbiological methods have either not been developed or existing methods have not been shown to be applicable to the determina- tion of the essential amino acids in meats, grains, milk, and other natural materials. It is in the analysis of such substances rather than purified proteins that microbiological methods will find their most extensive appli- cation. Also the analysis of a protein for those amino acids which can be measured by established microbiological methods involves use of a variety of microorganisms, media, and details of procedure. This is confusing and troublesome even to those who have considerable experience with micro- biological assay methods. Experience with the latter suggested that a large degree of standardization and, therefore, simplification of amino acid methods was possible and highly desirable. The present paper describes a basic method for the assay of the ten essential amino acids, namely histidine, arginine, lysine, leucine, isoleucine, valine, methionine, threonine, trypto- phane, and phenylalanine, which is applicable to foodstuffs and other nat- ural products as well as to purified proteins and synthetic amino acid mix- tures. A complete analysis can be made with 1.5 gm. or less of sample. Nine of the amino acids are determined with Streptococcus faecalis and phenylalanine with Lactobacillus delbriickii LD5.l Although two organisms 1 These organisms can be obtained from the American Type Culture Collection, Georgetow’n University School of Medicine, Washington, D. C., where Streptococcus jaecalis (also known as Streptococcus Zactis R) is listed as No. 9790 and Lactobacillus delbrtickii LD5 as No. 9595. 35 by guest on August 18, 2020 http://www.jbc.org/ Downloaded from
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MICROBIOLOGICAL METHODS FOR THE DETERMINATION OF AMINO ACIDS
II. A UNIFORM ASSAY FOR THE TEK ESSEXTIAL AMINO ACIDS
BY JACOB I,. STOIiES, MARION GUNNESS, IRLA M. DWYER, AND
MURIEL C. CASWELL
(From the Research Laboratories, Merck and Company, Inc., Rahway, New Jersey)
(Received for publication, May 8, 1945)
Microbiological methods for the determination of amino acids, because of their specificity, accuracy, sensitivity, and ability to yield many replicate results within a short time, promise to become of increasing importance in investigations of the chemistry and biochemistry of proteins and amino acids. Although such methods are of relatively recent origin, a considerable and rapidly increasing number are already available for the assay of many of the amino acids in purified proteins and natural products. Since a pre- vious review of the literature (l), a method for lysine (2) and additional methods for tryptophane (3) and glutamic acid (4,5) have been published. Also a fungus method is available for the assay of leucine (6).
With the exception of leucine (6-8), isoleucine (7), valine (7, 8), and tryptophane (3,9), microbiological methods have either not been developed or existing methods have not been shown to be applicable to the determina- tion of the essential amino acids in meats, grains, milk, and other natural materials. It is in the analysis of such substances rather than purified proteins that microbiological methods will find their most extensive appli- cation. Also the analysis of a protein for those amino acids which can be measured by established microbiological methods involves use of a variety of microorganisms, media, and details of procedure. This is confusing and troublesome even to those who have considerable experience with micro- biological assay methods. Experience with the latter suggested that a large degree of standardization and, therefore, simplification of amino acid methods was possible and highly desirable. The present paper describes a basic method for the assay of the ten essential amino acids, namely histidine, arginine, lysine, leucine, isoleucine, valine, methionine, threonine, trypto- phane, and phenylalanine, which is applicable to foodstuffs and other nat- ural products as well as to purified proteins and synthetic amino acid mix- tures. A complete analysis can be made with 1.5 gm. or less of sample. Nine of the amino acids are determined with Streptococcus faecalis and phenylalanine with Lactobacillus delbriickii LD5.l Although two organisms
1 These organisms can be obtained from the American Type Culture Collection, Georgetow’n University School of Medicine, Washington, D. C., where Streptococcus jaecalis (also known as Streptococcus Zactis R) is listed as No. 9790 and Lactobacillus delbrtickii LD5 as No. 9595.
are used, only one standard medium and one procedure are employed. The response of the two organisms to the amino acids is measured by titrating, with standard alkali, the lactic acid produced during growth.
Procedure
The described procedure applies to both Streptococcus faecalis and Lacto- bacillus delbriickii except for two indicated minor details and is similar in most respects to that already outlined (1).
Sa1t.s B MgSO1.‘iH?O. NaCl...................... FeSOc.7HzO. MnSOd.4H20.
Adjust to pH 6.8 Add distilled Hz0 to.. . .
- 3 gm. 5 mg. 5 cg 5 (‘
100 y 100 “ 100 ‘( 100 “ 200 (‘
20 “ 0.1 “ 1.0 1‘s
250 mg. 250 I[
loo “ 5 “ 5 “ 5 “
250 cc.
* The amino acid being assayed is omitted from the medium. t Sold by Merck and Company, Inc. $ Obtainable from Dr. R. J. Williams, University of Texas, Austin, Texas. 5 Equivalent to 1.0 y of material of “potency 40,000.”
Inoculum-The cells are prepared according to previous directions (1) except that Streptococcus faecalis is suspended in 100 cc. of water after being centrifuged and washed. Although one experiment indicated that Lacto- btiUus delbriickii also could be diluted to the same degree, most of the assay media were inoculated with cells suspended in 20 cc. of water.
Assay Medium-The composition of the basic assay medium is shown in Table I. The amino acid which is being determined is omitted from the
medium. Stock solutions of the amino acids, salts, and vitamins are pre- pared as indicated previously (1) with the following changes and additions. Owing to difficulty in obtaining I(-)-tryptophane, the dl form is used. Because of its lesser solubility, stock solutions are prepared with 0.2 N HCI to contain 20 mg. of dl-tryptophane per cc. A stock solution containing 1 mg. per cc. of adenine, guanine, and uracil is prepared by dissolving 870 mg. of adenine sulfate, 620 mg. of guanine hydrochloride, and 500 mg. of uracil in 200 cc. of water containing 10 cc. of concentrated HCl and adjusting the volume to 500 cc. Individual aqueous solutions containing per cc. 100 y of thiamine hydrochloride and 10 y of p-aminobenzoic acid are also re- quired. A solution of pyridoxamine dihydrochloride is prepared in a con- centration of 100 y of pyridoxamine per cc., in place of the more dilute solution previously described, to avoid excessive dilution of the basal medium. It is stable for at least a month when preserved with toluene and stored in the refrigerator.
Preparation of Samples for Assay-Fresh, moist substances such as vege- tables or meats are sliced and dried at 100” sufficiently to permit grinding into a homogeneous mass. It is not necessary to free the samples of fatty materials or any other constituent prior to hydrolysis or assay. 1 gm. of dried impure protein is autoclaved with 10 cc. of 10 per cent HC12 in sealed ampuls of 20 cc. capacity for 10 hours at 15 pounds pressure and prepared for assay as out,lined earlier (1). This quantity should be sufficient for determining all of the essential amino acids except tryptophane which re- quires a separate alkaline hydrolysis. With purified proteins and other matserials available only in small quantities, the amount hydrolyzed may be reduced considerably, the degree depending upon the amino acid content of the sample. This can be calculated, approximately, from the sensitivity of each assay as indicated by the standard curves (Fig. 1). Use of smaller samples is possible also if only some of the essential amino acids are to be determined. A further reduction should be possible by decreasing, pro- portionately, the scale of the assay from 10 cc. total volume per assay tube to 2.5 cc. or less (10, 11).
For the determination of tryptophane, 0.5 gm. of sample is hydrolyzed with 10 cc. of 5 N NaOH in 50 cc. Pyrex Erlenmeyer flasks plugged with non-absorbent cotton. Hydrolysis in sealed ampuls should also be satis- factory and perhaps preferable, since appreciable amounts of water some- times enter the flasks during autoclaving. In the case of purified proteins, 25 mg. are hydrolyzed with 2 cc. of alkali in sealed ampuls. The quantity of sample may be reduced as indicated above for acid hydrolysis. Diges- tion is carried out at 15 pounds steam pressure for 10 hours as for acid hy-
2 Concentrated HCl (about 38 per cent) is diluted with water to 10 per cent, by volume, of HCl .
drolysis so that both types of digestion may be made simultaneously in one autoclave. Frequently a copious precipitate appears on neutralization of the alkaline digest which may contain considerable quantities of silica dissolved from the flask. The precipitate is best removed by centrifuga-
MIcRoGR(MS OFL%WNE
FIG. 1. Typical standard curves. The quantities of each amino acid are those of the 2 or naturally occurring isomer.
tion, since filtration through paper may be extremely slow. It is washed once with water which is combined then with the remainder of the precipi- tate-free hydrolysate.
Activity of Optical Isomers and Preparation of Standards-With the ex- ception of histidine of which only the 1 isomer was available, the synthetic
dl racemate of each amino acid, under our conditions, was exactly one- half as active as the I isomer, indicating that the d or unnatural isomer is inactive (12). Data for threonine, which is typical of all the amino acids, are given in Table II. Identical standard curves were obtained with the 1 and dl forms when twice as much of the latter as compared to the 1 isomer was used. Therefore, either form can be used as the standard. It is im- portant, obviously, that amino acids of known purity be employed. It may be necessary to purify commerical dl-leucine (13) and dl-isoleucine.
It is convenient to prepare solutions of the amino acids, to be used as standards, in a concentration of 100 y per cc. of the I isomer. These may be stored under toluene in the refrigerator. Dilutions are made from these primary solutions so that the quantities needed for construction of the stand- ard curves (Fig. l), which are in terms of the 1 isomer, can be pipetted
TABLE II
Activity of l’hreonine Enantiomorphs for Streptococcus jaecalis
conveniently. These secondary dilutions, also, may be preserved. The dilutions must be made so that the maximum volume of standard added to the assay tubes does not exceed 5 cc.
Assay Procedure-This is identical with that already outlined (1) except for the following minor changes applicable only to assays with Streptococcus faecalis; there are no changes for the assay of phenylalanine with Lacto- bacillus delbriickii. Streptococcus famalis assays are titrated with approxi- mately 0.05 N NaOH after 40 hours incubation at which time maximum acid production has occurred. Some experiments have indicated that it may be possible to reduce the incubation period to 16 hours for most routine work and that as an alternative to titration the turbidity of the cultures may be measured with a photoelectric cell. However, the latter can be used only if the sample does not impart any appreciable amount of color or turbidity to the assay medium.
Recording and Calculation of Results-An example of a convenient method is the assay of beef liver for threonine. 1 gm. of dried liver was digested
with 10 cc. of HCl and the hydrolysate neutralized, filtered, and adjusted to 100 cc. ; a further 1: 12.5 dilution was made prior to assay (Table III).
Alkaline hydrolysis used to liberate tryptophane from proteins results in complete racemization of this amino acid. Since the d form is inactive for Stre@ococcus juecalis, assay values must be multiplied by a factor of 2 to arrive at the final correct tryptophane content of the protein.
Diluted sample added to assay tube
NaOH required to neutralize assay tube
after incubation
Amino acid equivalent of Amino add content titration figure. as read calculated for 1 cc. from standard curve diluted sam le at
I ‘xl 3 Average...................................................., ____
dilution
20.2 X -12.5 X 100 = 25.25 mg. of threonine per gm., or 2.53 per cent.
DISCUSSION
Streptococcus jaecalis was selected for use in the assays because, in con- trast to the commonly employed Lactobacillus casei and Lactobacillus arab- inosus, its amino acid requirements are not influenced by pyridoxamine or pyridoxal; those members of the vitamin Bc group can substitute for lysine and threonine in the nutrition of the lactobacilli (14). The latter are un- suitable, therefore, for the assay of lysine and threonine, since pyridoxa- mine and pyridoxal or closely related compounds are commonly present in natural products (15) or are formed readily in the assay medium during sterilization (14, 16).
Although initial, short time experiments appeared to indicate that Streptococcus jaecalis required all ten essential amino acids for full growth, subsequent experience disclosed that the almost imperceptible development evident in 16 to 20 hours, in the absence of phenylalanine, rapidly increased to maximum growth and acid production in 40 hours. Inability to obtain cultures from thirty-five single colonies which had an absolute requirement for phenylalanine suggests that Streptococcus jaecalis is able to synthesize phenylalanine, although too slowly to permit a normal rate of growth. This situation is analogous to that of the rat with respect to arginine (17).
Lactobacillus arabinosus also developed without phenylalanine. This was not unexpected, since its need for phenylalanine is dependent, critically, on the composition of the growth medium (18). However, Lactobacillus del- briickii LD5, Lcwtobacillus casei, and Leuconostoc mesenteroides P-60 failed to grow unless phenylalanine was in the medium and they responded in ap- proximately linear fashion to increments of the amino acid. The former was chosen for development of the phenylalanine assay, because previous experience with it in amino acid assays was available (1).
The basal medium (Table I.) resembles, closely, those proposed for other microbial amino acid assay methods. Although the number of ingredients is formidable, most of them are essential for growth. The amino acids and growth factors are in excess both in number and quantity. Thus proline, hydroxyproline, norleucine, and glycine are not essential for the growth of either assay organism and the same applies to thiamine andp-aminobenzoic acid and probably also to guanine and uracil. However, such a medium seemed preferable to a “minimum” medium which might be unduly sensi- tive to non-specific stimulatory substances or inhibitory substances that might be introduced with the sample. Doubling of the glucose and sodium acetate increases acid formation only slightly and has no effect on the assay values.
In the development of the method for lysine, standard curves were ob- tained frequently which “dipped” in the center in contrast to normal linear curves. The medium in use at the time was one which had been and still was entirely satisfactory for histidine and threonine assays developed earlier. Amino acid values from such irregular curves did not agree closely when calculated from the different assay levels and were high compared to those from assays with normal standard curves. The dip was eliminated by doubling the concentration of adenine, guanine, uracil, and vitamin sup- plements, except folic acid. Increase of the purine and pyrimidine bases was the most important factor in eliminating the dip, followed by biotin and nicotinic acid. However, the higher concentration of all three was neces- sary for linear curves. The modified basal medium proved to be satisfac- tory for the assay of histidine and threonine and also for the remainder of the amino acid assay methods developed subsequently.
The proposed assay method fulfils, for each of the amino acids, the con- ventional but adequate criteria of reliability: (a) Essentially the same amino acid values are obtained for a particular protein irrespective of the amount of sample assayed, thus indicating that the method is stable to non-specific stimulatory or inhibitory substances which may be introduced with samples (Table IV). Thii holds for such complex materials as tankage, blood meal, and yeast and also for carrots and potatoes which contain only small amounts of amino acids and must be added, therefore, in relatively large
amounts to the assay medium. (b) Th e results are readily reproducible, so that the same values are obtained for a given protein in repeated assays with fresh hydrolysates, different batches of medium, and different opera-
TABLE IV
Amino Acid Content of Proteins at Different Assay Levels
The results are calculated for partially dried material.
tors (Table V). (c) Recoveries of known amounts of amino acids added to proteins prior to hydrolysis are quantitative, generally within f2 per cent (Table VI). (d) Compounds related chemically or physiologically to the amino acids, other than a few very closely related exceptions, are inactive
(Table VII). Thus ornithme and citrulline which can individually replace arginine in the nutrition of certain Neurospora mutants (19) and the chick (20) are unable to do so for Streptococcus faecalis. Likewise, the combina- tion of choline plus homocystine which can substitute for methionine in the growth of the rat (21) and the chick (22) is inactive for S. faecalis. Also, indole and anthranilic acid which can replace tryptophane for Lactobacillus arabinows (9, 23) and L. casei (23) are inactive for S. faecalis. This last observation simplifies the present tryptophane method, since samples do not need to be extracted with ether and toluene to remove indole and anthranilic
TABLE VI
Recovery of Amino Acids Added to Proteins Prior to Hydrolysis
The results are calculated in mg. per gm. of partially dried material.
acid as is the case with one of the L. arabinosus methods (9). (e) In general, the microbiological values for purified and impure proteins are in reasonably good agreement with those obtained by the more recent, improved chemi- cal methods. The degree of agreement for lysine is characteristic of all of the essential amino acids with the exception of methionine (Table VIII) for which lower microbiological values were obtained, consistently. In view of the remarkable specificity of the microbial response to methionine (Table VII) and also to the other amino acids, it seems possible that the higher chemical values may include substances other than methionine. For fur- ther comparisons of the microbiological values for the purified proteins and
natural products listed in Tables IX and X, with chemical data, reference can be made to the recent extensive compilations by Block and Bolling (36). If desired, the amino acid values in Table X can be recalculated to esqn-ess
TABLE VII Activity of Compounds Related Chemically or Physiologically to the Essential Amino
* Less than 0.25 per cent (weight basis). t Weight basis.
Active
Arginine flavinate
l(-)-Leucine - 3 - naphthalene sul- fonate
a-Bromoisocaproic acid
Fonllyl-l(f)-valine
dl-Tryptophane methyl ester
a-Bromo-@-phenyl- propionic acid
Per cent activ- ityt
36.5
35.5
19.5
2-5
50
67
percentages of dry weight of sample by multiplying each value by the factor (per cent nitrogen)/l6.
Investigation of the effect of 5 to 30 hours of acid hydrolysis on liberation of each of the essential amino acids, except tryptophane, from representative protein materials indicates that autoclaving for 5 hours is sufficient to give
maximum amino acid values. The data in Table XI are typical. For some of the proteins, there is evidence of slight destruction or racemization of some of the amino acids after 30 hours. The results are in accord with
similar microbiological data of other investigators (7,8, 10) and emphasize, unequivocally, in contrast to conflicting chemical data (36), the marked stability of the amino acids to acid hydrolysis. Since periods of hydrolysis considerably in excess of those required for maximum liberation of amino acids from proteins cause no apparent. destruction, the data support the view that the amino acid values of protein hydrolysates are the same as those of the intact proteins.
TABLE X
Amino Acid Content of Natural Products
Substance
Rye, seed. Wheat, seed.. Flour, patent. Soy bean flour, de.
* The results are not calculated to 16 per cent nitrogen because of difficulty in obtaining concordant nitrogen values.
For tryptophane assays with Luctobucillus arabinosus, Ba(OH)z (9) or enzymatic digestion of proteins (3) has been recommended. Although ir- regular results have been reported with NaOH digestion (3), it has proved satisfactory under our conditions. To insure complete liberation and race- mization of tryptophane, it is necessary to hydrolyze proteins for at least 10 hours (Table XI) and to limit the sample to an amount which does not con- tain more than 10 to 15 mg. of tryptophane. There is little danger of exceeding that quantity if not more than 0.5 gm. of impure or 100 mg. of purified protein is used.
The described method is eminently suitable for obtaining many analyses within a short time. One experienced operator can assay six proteins for all essential amino acids in little more than a week. The techniques re- quired are relatively simple although elementary knowledge of bacteriologi- cal technique is essential in maintaining purity of stock cultures and pre- venting contamination of assays.
TABLE XI Effect of Time of Hydrolysis on Liberation of Amino Acids from Proteans
The results are calculated in per cent, on a dry basis.
The authors are greatly indebted to the following individuals for generous supplies of invaluable compounds: Dr. W. H. Stein and the late Dr. Max Bergmann, Rockefeller Institute, for egg albumin, silk fibroin, p-lactoglob- ulin and I(-)-serine; Dr. W. C. Rose and Dr. Madelyn Womack, Univer- sity of Illinois, for Z( -)-phenylalanine, d(+)-phenylalanine, I( -)- methionine, d(+)-methionine, and dl-argiriine; Dr. M. S. Dunn, University of California, for dl-citrulline; Dr. C. M. Lyman, Texas Agricultural Ex- periment Station, for I(+)-isoleucine; Dr. W. M. Stanley, Rockefeller Institute, for tobacco mosaic virus; Dr. V. du Vigneaud, Cornell University Medical College, for dl-homocystine and I-cystathionine; Dr. W. W. Moyer, A. E. Staley Manufacturing Company, for Z(-)-phenylalanine and Z(+)- isoleucine; Dr. C. P. Berg, State University of Iowa, for I(-)-methionine;
and Dr. E. E. Howe, Dr. M. Tishler, and Dr. S. A. Harris, Merck Labora- tories, for many of the compounds listed in Table VII.
SUMMARY
An accurate, specific, and sensitive microbiological method is described for the determination of the ten essential amino acids, namely histidine, arginine, lysine, leucine, isoleucine, valine, methionine, threonine, trypto- phane, and phenylalanine, in foodstuffs and other natural products as well as in purified proteins and synthetic amino acid mixtures. A complete amino acid analysis can be made with 1.5 gm. or less of sample. With only one medium and procedure, nine of the amino acids are determined with Streptococcus faecalis and phenylalanine with Lactobaeillus delbriiclcii LD5. The response of the two organisms to the amino acids is measured by titrating, with standard alkali, the lactic acid formed during growth. The method yields many replicate results within a short time and lends itself readily to routine use. The quantities of essential amino acids in casein, gelatin, egg albumin, p-lactoglobulin, silk fibroin, tobacco mosaic virus, rye, wheat, patent flour, soy bean flour, whole milk, peas, carrots, potatoes, beef liver, brewers’ yeast, blood meal, tankage, alfalfa meal, and linseed meal are presented.
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