[77] Microbiological determination of amino acids

[77]

MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

477

provides the basis for adopting this method to study rate and degree of protein hydrolysis. Application of the method to the study of protein hydrolysis is described in the original paper, and use of the method in following proteolysis of cottonseed allergen has been described. 15 Test solutions of 500 ~M (twice the concentration of amino acid usually employed) in glucose, galactose, sucrose, mannitol, inositol, and ammonium chloride did not interfere with the test. Noninterference by ammonia is an advantage of the method. The necessity of avoiding traces of copper or amino acids in reagents, distilled water, or on glassware is obvious from consideration of the sensitivity of the method. Particular care must be taken to exclude contaminating copper from blank solutions. The precision of the method determined from duplicate analyses of eight 300 ~M alanine solutions is: standard deviation of the mean _ 1.1%, and standard deviation of a single pair of duplicates _+3.2 %. is j. R. Spies, D. C. Chambers, E. J. Coulson, H. S. Bernton, and H. Stevens, J. Allergy 24, 483 (1953).

[77] M i c r o b i o l o g i c a l

Determination

of A m i n o A c i d s

B y ESMOND E. SNELL

Introduction The use of microorganisms for quantitative estimation of amino acids was a natural outgrowth of their earlier successful use in the determination of vitamins. The general principles on which such methods are based, an account of their development, and details of many individual assay methods, have been treated in several review articles. 1-s The methods depend on employment of microorganisms for which individual amino acids are essential nutrients. In a basal medium lacking a single essential amino acid but complete in other respects, the growth of such an organism is, within limits, a function of the amount of the amino acid added. The concentration of amino acid in a given sample can thus be determined by comparing the growth response (measured in 1E. E. Snell, Physiol. Revs. 28, 255 (1948); Advances in Protein Chem. 2, 85 (1945); Ann. N. Y. Acad. Sci. 47, 161 (1946). s E. E. Snell, in "Vitamin Methods" (P. GySrgy,ed.), Vol. 1, p. 327, AcademicPress, New York, 1950. 3B. S. Sehweigertand E. E. Snell, Nutrition Abstr. & Revs. 16, 497 (1947). 4 E. C. Barton-Wright, "The MicrobiologicalAssay of the Vitamin B-Complex and Amino .acids," Pitman, London, 1952. i M. S. Dunn, Physiol. Revs. 29, 219 (1949) ; Food Technol. 1, 269 (1947).

478

PROTEINS AND DERIVATIVES

[77]

any convenient fashion) to aliquots of the sample with that permitted by known amounts of the pure amino acid. A great many microorganisms that require one or more amino acids are known, and, in theory, assay methods based on any one of these could be developed. In practice, the lactic acid bacteria show many advantages and have been utilized widely with great success. Although different authors have employed different organisms of this group and slightly different basal media for determination of various amino acids (and even of a single amino acid), all the methods are fundamentally similar. Once the technique is learned for a single amino acid with a single assay organism, its application to any amino acid in any of several variations with respect to test organism or basal medium offers no difficulty. In proposing variations in test media and test organisms for an amino acid, individual authors have not, in general, established by comparative tests the superiority of one organism or test medium over another. Rather, they have been content to develop methods that give apparently accurate and reproducible results and are conveniently operable in their own laboratory. It is probable that each of the methods of recent origin are of comparable accuracy, and there is little basis for choice among them. Some of the methods, however, can be applied to any of several amino acids without change in methodology save for the necessary change in amino acid composition of the basal medium. Some of them, too, have been used successfully in several different laboratories. These are the methods selected for presentation here.

Preparation of Samples for Assay Many investigations of the utility of peptides as sources of amino acids for microorganisms have been made. 5,6-13 Depending on the amino acid being determined, the assay medium, and the test organism, individual peptides may exhibit activity lower than, equal to, or higher than that expected from their amino acid content. In general, organisms that utilize a given peptide for growth are able to hydrolyze it; 6,9-13 the presence or absence of peptidases for such peptides suffices to explain activities equal to or less than that expected from the composition of the H. Kihara and E. E. Snell, J. Biol. Chem. 197, 791 (1952). H. Kihara, O. Klatt, and E. E. Snell, J. Biol. Chem. 197, 801 (1952). A. I Virtanen and V. Nurmikko, Acta Chem. Scand. 5~ 97, 681 (1951). 9 j. M. Prescott V. J. Peters, and E. E. Snell, J. Biol. Chem. 202, 533 (1953). ~0 M. S. Dunn and L. E McClure, J. Biol. Chem. 184, 223 (1949). ~ M. Klungsoyr, R. J. Sirny, and C. A. Elvehjem, J. Biol. Chem. 189, 557 (1951). ~2 V. J. Peters and E. E. Snell, J ~acieriol. 6~/, 69 (1954). ~ D. Stone and H. D. Hoberman, J. Biol. Chem. 202, 203 (1953).

[77]

MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

479

peptide. Tentative explanations for instances in which peptides are more active than their component amino acids also have been advanced; here, too, the organisms are generally able to hydrolyze the peptides in question.8,7,9,~ These results make it clear that as a general rule proteins must be hydrolyzed completely to their component amino acids before application of microbiological methods can be expected to yield valid results. In some instances, enzymatic digests or partial acid hydrolyzates have been assayed with results that check those obtained after complete acid hydrolysis. 11 Only where such a check has been obtained on the specific proteinaceous material being investigated should partial hydrolyzates be employed for assay. The variable activity of different peptides for individual assay organisms also emphasizes that the increase in amino acid content observed in a sample after acid hydrolysis is not a reliable measure of the " b o u n d " amino acid content of the sample. Procedure. The hydrolytic procedure necessary to release amino acids completely (for purposes of microbiological assay) from a protein-containing sample can be readily determined by sampling the hydrolyzate at intervals, assaying the several samples, and selecting a time interval at which further hydrolytic treatment fails to alter the values obtained. Studies of this nature (summarized elsewhere TM) show that the time required for this purpose varies somewhat with the sample and with the test organism. For routine application, it is best to adopt a procedure known to effect release of the amino acids from the most resistant samples to an extent sufficient for complete utilization by the most demanding assay organisms. A convenient procedure I~,~5for acid hydrolysis consists in placing the finely divided, weighed sample together with a 40-fold excess of 3 N hydrochloric acid in a test tube. The tube is sealed, then heated at 15 lb. of steam pressure (121 °) in the autoclave for 5 to 10 hours. The tube is opened, and the sample neutralized before assay. Interfering materials present in the humin of such hydrolyzates of some foodstuffs can be removed by adjustment to pH 4.0 and filtration through a fritted glass filter before final adjustment to neutrality. 16 Since tryptophan is destroyed by acid hydrolysis, it must be liberated by alkaline or enzymatic hydrolysis. Autoclaving (121 °) of 0.5 g. of sample with 10 ml. of 5 N NaOH in a sealed Pyrex tube for 5 to 15 hours 14L. M. Henderson and E. E. Snell, J. Biol. Chem. 172, 15 (1948). 15j. L. Stokes, M. Gunness, I. M. Dwyer, and M. C. ~aswell, J. Biol. Chem. 160, 35 (1945). ~6M. J. Horn, A. E. Blum, C. E. F. Gersdorff, and H. W. Warren, J. Biol. Chem. 203, 907 (1953).

480

PROTEINS AND DERIVATIVES

[77]

has been recommended 1.,15.17 for this purpose; hydrolysis with Ba(OH)2 should not be used. 17 This procedure appears to racemize the peptidebound L-tryptophan of proteins completely (it does not racemize free L-tryptophan), and, since D-tryptophan is inactive for assay organisms so far utilized, one must multiply the assay result by 2 if a standard of L-tryptophan is employed. In carbohydrate-containing foods, acid hydrolysis also destroys much tyrosine; 18 hydrolysis by autoclaving the sample with 5 N NaOH for 5 hours in the autoclave at 15 lb. of pressure (121°) is recommended. 18 This effects complete racemization of the tyrosine. As mentioned earlier, generally applicable enzymatic digestion procedures have not been developed. In a recommended procedure for tryptophan analysis, a suspension of the finely divided sample (200 rag.) is layered with toluene and shaken with a mixture of pancreatin (20 mg.) and hog intestinal mucosa (4 mg.) for 1 to 5 days. 14,~9 Tryptophan analyses of such digests agreed well with those obtained by alkaline hydrolysis. A m i n o A c i d S t a n d a r d s . The development of highly sensitive microbiological methods, and later of paper chromatographic methods, for analysis of amino acids revealed significant and previously unsuspected contamination of certain amino acids (both natural and synthetic) with other amino acids. Awareness of this situation has led to marked improvement in the quality of products now available from chemical supply houses. Despite this improvement, the analyst should assure himself of the chemical and optical purity of the amino acids used in establishing standard dose-response curves. Criteria of purity and methods for recrystallization of the amino acids have been summarized by Dunn and Rockland. 2° The optical purity of most of the amino acids can be checked conveniently by means of D- and L-amino acid oxidases as described by Greenstein et al. 2~ Special care should be taken with isoleucine, since the synthetic product may contain significant amounts of alloisoleucine, and the product isolated from proteins by older procedures is also frequently impure. Convenient procedures for purification of this amino acid are now available. 22 Many of the older microbiological values for the iso17W. A. Krehl, J. de la Huerga, and C. A. Elvehjem, J. Biol. Chem. 164, 551 (1946). 18 M. Gunness, I. M. Dwyer, and J. L. Stokes, J. Biol. Chem. 163, 159 (1946). 19I. T. Greenhut, B. S. Schweigert,and C. A. Elvehjem, J. Biol. Chem. 165, 325 (1946). :0 M. S. Dunn and L. B. Rockland, Advances in Protein Chem. 3, 295 (1947). 21j. p. Greenstein, S. M. Birnbaum, and M. C. Otey, J. Biol. Chem. 204, 307 (1953) ; A. Meister, L. Levintow, R. B. Kingsley, and J. P. Greenstein, J. Biol. Chem. 192~ :. 535 (1951). 2~j. p. Greenstein, S. M. Birnbaum, and L. Levintow, in Biochem. Preparations $, 84 (1953).

[77]

MICROBIOLOGICALDETERMINATION OF AMINO ACIDS

481

leucine content'of proteins are high because of an impure standard. In general, only the L-amino acids should be taken as standards unless it has been established that the D-isomers have no activity under the test conditions. Methods Excellent procedures studied in connection with one or more individual amino acids are available in the literature but cannot be considered here. A particularly valuable compilation of individual procedures that permit determination of eighteen amino acids is that of Barton-Wright ;4 Dunn and co-workers 23 have summarized the several individual procedures adopted in their laboratory for twelve amino acids. The procedures to be presented below are the general ones of Henderson and Snell, TM Baumann and co-workers, 24 and Stokes et al. 15,~8 Since the methodology in all cases is similar, detailed description of one method will suffice to illustrate the procedure and certain general principles involved. Summaries of the various methods then will be presented; where clarification is desired, details will become clear from examination of the one procedure presented in full. A s s a y Organisms

The assay organisms utilized with methods to be presented later are listed in Table I, together with the amino acids for assay of which they are most commonly employed. I t will be observed that several different organisms can be employed for determination of certain of the amino acids. Agreement of assay results obtained with different test organisms is an excellent indication of the reliability of the values obtained for a given amino acid. 1-3 Stock cultures of these organisms are conveniently carried as stab cultures in any of several media. 2 One of the simplest of these contains 1% yeast extract, 1.0 % glucose, and 1.5 % agar. TM In recent years there has been a tendency to employ more complex media for the stock cultures and to increase the frequency of transfer from monthly to biweekly or even weekly. Barton-WrighO recommends the medium of N ym on and Gortner, 25 which contains 1 g. of glucose, 1 g. of tryptone (Difco), 0.2 g. of K~HP04, 0.3 g. of CaC03, 0.5 ml. each of inorganic salts A and B, 26 23M. S. Dunn, M. N. Camien, R. B. Malin, E. A. Murphy, and P. J. Reiner, Univ. Calif. (Berkeley) Publs. Physiol. 8, 293 (1949). 2~B. F. Steele, H. E. Sauberlich, M. S. Reynolds, and C. A. Baumann, J. Biol. Chem. 177, 533 (1949). 25M. C. Nymon and W. A. Gortner, J. Biol. Chem. 168, 277 (1946). ~6Salts A contain 25 g. of KH2PO4and 25 g. of K2HPO4in 250 m]. of water; salts B contain 10 g. of MgSO4.7HaO, 0.5 g. of FeSO,.TH20, 0.5 g. of NaC1, and 0.5 g. of MnSO4.4H20 in 250 ml. of water.

482

PROTEINS AND DERIVATIVES

[77]

10 ml. of liver extract, 27 and 1.5 g. of agar per 100 ml. Henderson and Snell 14 employ their basal assay medium (Table I I ) modified b y using glucose and sodium citrate at one-haft the specified level, and adding per 100 ml. of single-strength medium 0.5 g. of tryptone, 0.5 g. of yeast extract, and 2 g. of agar. However, limited studies of D u n n and coworkers ~s indicate t h a t the medium chosen for carrying stock cultt~res TABLE I COMMONLY USED CULTURES FOR DETERMINATION OF AMINO ACIDS

Name and strain Lactobacillus arabinosus 17-5b Lactobacillus delbriickii 5c Leuconostoc citrovorum d Leuconostoc mesenteroides P-60 • Streptococcus faecalis R !

ATCC~

For determination of

8014 Glutamic acid, leucine, isoleucine, valine, phenylalanine, tryptophan 9595 Phenylalanine, tyrosine, serine 8081 Alanine; could also be used for most other amino acids 8042 All amino acids except alanine 8043 Glutamie acid, histidine, lysine, arginine, leucine, isoleucine, valine, methionine, threonine, tryptophan

a American Type Culture Collection, 2029 M Street, N. W., Washington 6, D. C. b A strain of Lactobacillus plantarum. c Possibly identical with Lactobacillus casei or Lactobacillus helveticus [M. S. Dunn, S. Shankman, M. N. Camien, and H. Block, J. Biol. Chem. 168, 1 (1947)]. Recent studies indicate that this organism belongs to the genus Pediococcus rather than Leuconostoc [E. M. Jensen and H. W. Seeley, J. Bacteriol. 67, 484 (1954)]. • Studies of C. S. McCleskey [J. Bacteriol. 64, 140 (1952)] indicate that this culture is misclassified and is probably a strain of Streptococcus equinus. s Designated in some early literature as Streptococcus lactis R or RglA. is not a critical factor in success of these methods. The procedure had little effect on the subsequent quantitative response of Lactobacillus arabinosus to several essential amino acids. Stab cultures prepared in the medium of choice are incubated at 37 ° until good growth appears in the line of the stab (usually 24 hours; and no longer t h a n 48 hours), then refrigerated at 4 to 12 ° until the next transfer. Fresh transfers are prepared at intervals of 1 to 4 weeks. Streptococcus faecalis grows satisfactorily either in stab cultures or on agar 27Approximately 450 g. of fresh liver is ground and suspended in 2 1. of water. The suspension is heated for 1 hour on the steam bath, filtered through cheese cloth, the filtrate adjusted to pH 7.0, again heated for 15 minutes, filtered through paper, and stored in a brown bottle under toluene in the cold. 2s M. S. Dunn, S. Shankman, M. N. Camien, and H. Block, J. Biol. Chem. 168, 1 (1947).

[77]

483

MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS TABLE II COMPOSITION OF Assxr MEDIA FOR AMINO ACIDS (Milligrams per 100 ml. of double-strength medium) Medium

Constituents

(1) Henderson and Snell*

Glucose 4000 Sodium citrate 4000 Sodium acetate 200 Inorganic salts NH~C1 600 K~HPO4 1000 KH~PO4 MgSO4.7H20 160 MnSO4-4H20 32 FeSO4.7H20 8 NaC1 8 Purine and pyrimidine bases Adenine sulfate 2 Guanine hydrochloride 2 Xanthine 2 Uracil 2 Vitamins p-Aminobenzoic acid 0.04 Biotin 0. 002 Calcium pantothenate 0.2 Folic acid 0.002 Folinie acid __d Nicotinic acid 0.2 Pyridoxal-HC1 0.04 Pyridoxamine.2 HC1 Pyridoxine.HC1 Riboflavin 0.2 Thiamine 0.2 Amino aeidsf.g DL-Alanine 200 L-Arginine.HCl 40 L-Asparagine L-Aspartic acid 100 L-Cysteine-HC1 L-Cystine 10 L-Glutamie acid 200 Glycine 10 L-Histidine.HCl 10

(2) Steele, Sauberlich, Reynolds, and (3) Stokes, Gunness, Baumann b Dwyer, and Caswell° 5OOO

2OO0

40OO

1200

600 120 120 40 4 2 2

100 100 40 2 2 2

q

2 2 2

2

0.02 0.0002

0.008 0.00004

0.1 0.002

0.04 0.0004

2

_ _ e

0.2 0.06 0.06 0.2 0.1 0.1 40 48.5

80 20 10 60 20 12.4

0.04 0.08 0.04 0.04 40 40 2O 4O 2O 4O 4O

484

P R O T E I N S AND

DERIVATIVES

[77]

TABLE II (Continued) Medium

Constituents Amino acids (Continued) DL-Isoleucine DL-Leucine L-Lysine.HC1 DL-Methionine D~Phenylalanine L-Proline DL-Serine DL-Threonine DL-Tryptophan L-Tyrosine DL-Valine DL-Norleucine

(1) Henderson and Snell a

20 20 40 20 20 10 20 20 20 10 20 --

(2) Steele, Sauberlich, Reynolds, and (3) Stokes, Gunness, Baumann b Dwyer, and Caswell ~

50 50 50 20 20 20 10 40 8 20 50 --

40 40 20 40 40 40 40 40 80 40 40 40

a j . Biol. Chem. 172, 15 (1948). b j . Biol. Chem. 177, 533 (1949). J. Biol. Chem. 160, 35 (1945).

d Must be added (ca. 10 ~ per 100 ml.) if the medium is used with Leueonostoc eitrovorum.

• When used with L. citrovorum, 0.08 ml. of injectable liver concentrate (reticulogen, 20 U.S.P.) is added to supply this subsequently identified growth factor. This can now be replaced by the synthetic growth factor at a convenient level (e.g., 10 ~, per 100 ml.). The amino acid to be determined is omitted from the basal medium. a L- or DL-Amino acids may be used interchangeably; ~-amino acids are employed at one-half the concentration specified for DL-amino acids, DL-amino acids at twice that specified for L-amino acids. slants; a c c o r d i n g t o B a r t o n - W r i g h t , 4 b e t t e r results are o b t a i n e d in some assays if t h e o r g a n i s m is carried in t h e l a t t e r fashion. Method

of Henderson

a n d S n e l l I~

B a s a l M e d i u m . T h e c o m p o s i t i o n of t h e basal m e d i u m is given in T a b l e I I . Glucose, s o d i u m citrate, a n d s o d i u m a c e t a t e are a d d e d in solid f o r m ; inorganic salts, purine a n d p y r i m i d i n e bases, vitamins, a n d a m i n o acids are c o n v e n i e n t l y c o m b i n e d f r o m p r e v i o u s l y p r e p a r e d s t o c k solutions. W h e r e m a n y r o u t i n e assays for a single a m i n o acid are being m a d e , it is c o n v e n i e n t t o p r e p a r e a m i x t u r e of t h e d r y a m i n o acids lacking t h e one t o be d e t e r m i n e d . This m e d i u m was developed as a single a s s a y m e d i u m suitable for

[77]

MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

485

culture of any of the common assay organisms listed in Table I except Leuconostoc citrovorum. It becomes suitable for the latter organism on addition of folinic acid (leucovorin, 10 ~ per 100 ml.), which this organism requires and which was discovered subsequent to the development of this medium. 29 The medium is suitable for assay of any of the amino acids required by such organisms. For this purpose, the single amino acid to be determined is omitted from the medium. The citrate of this medium serves only as a buffer; the above-cited members of the genus Lactobacillus (but not S. faecalis) grow equally heavily and somewhat more rapidly if the sodium citrate is omitted and the sodium acetate is increased to 4 g. per 100 ml. When this change is made, the concentration of manganese sulfate should be reduced to 8 rag. per 100 ml. Precipitation of inorganic salts is minimized by adding them after the other ingredients have been dissolved and the solution made almost to volume. For assay of most amino acids, the pH is adjusted to approximately 6.8; for assay of glutamic acid and proline, the initial pH of the medium is 6.0.14 Inoeulum Cultures. For growing inocula, basal medium 1 (Table II) is modified by lowering the glucose and sodium citrate to one-haft the level specified and adding 0.5 g. of tryptone and 0.5 g. of yeast extract per 50 ml. of the double-strength medium. The volume is adjusted to 100 ml., and separate aliquots of 5 ml. are transferred to individual tubes. These are plugged with cotton, sterilized in the autoclave at 15 lb. of pressure for 20 minutes, and held in the cold room until required. A transfer from the appropriate stock culture is made to this inoculum medium, and the subculture is incubated at 37 ° for 10 to 15 hours. The cells are centrifuged and resuspended in a volume of sterile 0.9 % sodium chloride solution five to twenty-five times as great as that of the medium in which they were grown. 3° One drop of the resulting suspension is used to inoculate each assay tube containing 2 ml. of medium. If volumes other than 2 ml. are employed, the amount of inoculum suspension is altered correspondingly. Procedure. Assays may be carried out in any convenient volume from 0.2 ml. 31 to 10 ml. ; the concentration (amount per unit volume) of sample and standard required to permit growth remains constant whatever the volume. The range over which growth is a function of concentration is illustrated for L-leucine in Fig. 1 and summarized for the various amino 29 It. E. Sauberlich a n d C. A. B a u m a n n , J. Biol. Chem. 176, 165 (1948). a0 Suspensions of Streptococcus faecalis were diluted approximately 25-fold, those of the remaining organisms from 5- to 10-fold, depending on the density of growth. T h e a m o u n t of inoculum is not a critical factor in determining success of the assay. sl L. M. Henderson, W. L. Brickson, a n d E. E. Snell, J. Biol. Chem. 172~ 31 (1948). Special small-size test tubes are used for assays conducted on the micro scale.

486

PROTEINS AND DERIVATIVES

[77]

acids in Table III. With few exceptions it varies only slightly from one organism to another or from one assay medium to another. A sufficiently large total volume for convenient manipulation but one that is saving of materials is 2 ml., and the following description pertains to assays conducted on this scale. Samples and standard are dispensed to 18 × 150-mm. Pyrex culture tubes in volumes of 0.2 to 1.0 ml. The standard amino acid should be added at several different concentrations within the range shown in Table I I I (as illustrated for leucine in Fig. 1) ; I0

I

I

l

I

i

l

I

I

I

I

I

I

I I0

I

I 20

I

I 30

I

I 40

I

I 50

i

o

z

g2 d ..i 0 0

r

L- LEUCINE

PER

60

2 ML.

Fro. 1. A typical standard curve showing the response of Lactobacillus arabinosus

to leuclne." the sample also is added at several different concentrations estimated to supply the amino acid being determined in similar amounts. The volume in each tube is brought to 1 ml. by addition of water, and 1 ml. of the double-strength basal medium (from which the amino acid to be determined has been omitted) is added. Each tube is plugged with cotton or covered by aluminum caps or by a special cover made to fit the rack for the tubes and lined with a heavy padding of cotton, then sterilized by autoclaving at 10 to 12 lb. of pressure for 10 minutes. 3~ The tubes are cooled and then inoculated with 1 drop of the inoculum suspension prepared as described in the preceding section. They are then incubated for 60 to 72 hours at 37 °, after which acid production in each tube is measured a2Excessive autoclaving produces extensive browning of the medium (especially where the initial pH is 6.8 or higher) and should be avoided. Even shorter sterilization times can be employed successfullyif care is taken to avoid contamination and the assays are conducted in a dust-free environment.

[77]

487

MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

b y titration with 0.04 N alkali. 38 Since the medium is heavily buffered, use of indicators gives a less satisfactory end point t h a n does electrometric titration ~4 to approximately p H 7.3. An inexpensive assembly for the latter purpose has been described. I4,31 A s t a n d a r d curve is constructed b y plotting acid production against a m o u n t of the s t a n d a r d amino acid added. Such a standard curve, showing the response of L. arabinosus to increasing a m o u n t s of L-leucine, is TABLE III CONCENTRATIONS OF VARIOUS AMINO ACIDS SUITABLE FOR ESTABLISHING A STANDARD CURVE

Amino acid a Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine

Assay range? 7/ml. 0-25 0-20 0-20 0-5 0-25 0-15 0-5 0-15 0-15

Amino acid • Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Assay range, b 7/ml. 0-20 0-5 0-5 0-10 0-10 0-10 0-2 0-10 0-15

a L-Isomer in all cases. In addition to setting up several points on the standard curve within this range, it is well to have one higher concentration (e.g., two to four times as great as the highest listed here) to establish a point of maximum growth. b The values are taken from many papers on assay of individual amino acids and from the following summary papers: E. E. Snell, Ann. N. Y. Acad. Sci. 47, 161 (1946) ; M. S. Dunn, S. Shankman, M. N. Camien, W. Frankl, and L. B. Rockland, J. Biol. Chem. 166, 703 (1944); B. F. Steele, H. E. Sauberlich, M. S. Reynolds, and C. A. Baumann, J. Biol. Chem. 177, 533 (1949). shown in Fig. 1. Acid production in response to k n o w n a m o u n t s of sample is interpolated onto this curve to determine directly the a m o u n t of amino acid present in the sample. The amino acid c o n t e n t per unit weight of sample as calculated from several increasing concentrations should agree closely (e.g., within ___5%), and the values are averaged for the final result.

Assay Organisms for Individual A m i n o Acids and Specificity toward Optical Isomers. I n the original s t u d y of this method, ~4 three organisms tt For special purposes, turbidimetric estimation of growth after 18 to 36 hours of incubation is useful and gives results that agree closely with those obtained aeidimetrically. Two-milliliter cultures may be diluted to 8 ml. with water prior to such estimation if desired.

488

PaOTEINS AND DV.RIV.~T~WS

[77]

were found most useful. Lactobacillus arabinosus was employed for determination of giutamic acid, leucine, phenylalanine, valine, and tryptophan; Streptococcus faecalis for arginine, histidine, methionine, and threonine; and Leuconostoc mesenteroides P-60 for aspartic acid, glycine, 34 histidine, lysine, proline, tyrosine, and isoleucine. In some instances the choice is arbitrary; for example, both S. faecalis and Leuco. mesenteroides give excellent results in tryptophan determination and, unlike L. arabinosus, are unable to utilize either indole or anthranilic acid in place of the intact amino acid. 36 Use of the medium for determination of alanine, serine, and cystine was not investigated; Leuconostoc citrovorum 8081 requires each of these amino acids 24,3e,37 and might be applied to their determination in the basal medium supplemented with folinic acid, as noted earlier. Leuconostoc mesenteroides has been applied successfully to the determination of cystine in a somewhat different medium; 38 it appears probable that the same technique would permit utilization of this organism and the present medium for determination of this amino acid. Certain a-keto acids and a smaller number of a-hydroxy acids can replace the corresponding a-amino acids in these test organisms. 89 In most instances, these related compounds are much less active than the amino acid itself, and their stability is such that they would not commonly occur in samples for assay. To the extent that they do occur, they must be considered as possible interfering materials. A check of the apparent amino acid content before and after extraction of the acidhydrolyzed sample with ethyl acetate would in most cases reveal their presence. For all the test organisms listed above, the D-isomers of the essential amino acids are essentially inactive; that is, the DL-isomers of the amino acids are 50% as effective on the weight basis as are the L-isomers. 3 Exceptions to this general rule are D-alanine, which has 100% of the activity of L-alanine for Leuco. citrovorum 8081 ;24 D-aspartic acid, which has 100% of the activity of L-aspartic acid for Leuco. mesenteroides under the assay conditions recommended here ;24.4oD-methionine, which has low activity ( ~ 10% that of L-methionine) for Leuco. mesenteroides P-6024 and S. faecalis R TM but none for Leuco. citrovorum; 24 D-tyrosine, which has low 34Samples of DL-alanine available in 1947 were contaminated with glycine. It was necessary, therefore, to recrystallize this amino acid before satisfactory determinations of glycine could be obtained. ~5E. E. SneU, Arch. Biochem. 2, 389 (1943). a6H. E. Sauberlich and C. A. Baumann, J. Biol. Chem. 177, 545 (1949). 37E. M. Jensen and H. W. Seeley, J. Bacteriol. 67, 484 (1954). 38M. N. Camien and M. S. Dunn, J. Biol. Chem. 183, 561 (1950). 39j. T. Holden, R. B. Wildman, and E. E. Snell, J. Biol. Chem. 191, 559 (1951). 40 M. N. Camien and M. S. Dunn, Proc. Soc. Exptl. Biol. Med. 75, 74 (1950).

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MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

489

activity for both Leuco. mesenteroides and Leuco. citrovorum 24 but is inactive for L. delbri~ckii 5; 18 and D-glutamic acid, which has variable low activity (usually less than 5 % that of L-glutamic acid) for L. arabinosus in the presence of L-glutamic acid. s In such instances, only the L-amino acids should be employed as standards. Atypical Standard Curves and Their Significance. In the above and similar assay media an atypical sigmoidal standard curve is obtained with L. arabinosus in response to glutamic acid. 1,41-43 Frequently, such standard curves can be used without difficulty, since the organism responds to standard and sample alike (cf. Lewis and Olcott41). Where this is not true, it can be readily recognized by a "drift" in assay values calculated from different concentrations of the sample. In general, it is more satisfactory to modify the procedure in such instances to obtain a regular standard curve. With glutamic acid, for example, the tendency toward sigmoidal curves is overcome successfully in any of several ways: (1) addition of small amounts of glutamine insufficient to replace glutamic acid completely but sufficient to initiate growth, 48 or addition of small amounts of an enzymatic casein digest sufficient for the same purpose ;42 (2) use of an inoculum approximately five times as large as for other amino acids and initial adjustment of the basal medium to pH 6.0; T M or (3) use of asparagine (at a concentration of 20 mg. per 100 ml. of doublestrength medium) in place of aspartic acid in the medium. 4~ Of these alternatives, the second is simplest and suffices to permit excellent assays for glutamic acid to be obtained. The reasons for this behavior are, however, instructive and warrant further examination. The lag in the response of L. arabinosus to low concentrations of glutamic acid has been shown 4~ to be due to. the inhibition of utilization of low levels of glutamic acid by the high level of aspartie acid (which functions as a structurally related antimetabolite of glutamic acid) in the medium. Asparagine is a much less potent inhibitor, 4~ and thus its substitution for aspartic acid at a somewhat decreased concentration prevents the lag in the dose-response curve, as noted above. The process being inhibited is by-passed by supplying glutamine or appropriate peptides of glutamic acid; hence no lag in the response to these substances is observed. 4~,43 Similar antagonistic relationships between other amino acids appear to be the most common explanation for atypically shaped (sigmoidal) 41 j. C. Lewis and H. S. Olcott, J. Biol. Chem. 157, 265 (1945). 42 L. R. Hac, E. E. Snell, and R. J. Williams, J. Biol. Chem. 159, 273 (1945). 4s C. A. Lyman, K. A. Kuiken, L. Blotter, and F. Hale, J. Biol. Chem. 157, 395 (1945). 4~ W. Baumgarten, A. N. Mather, and W. Stone, Cereal Chem. 22, 514 (1945). 45 W. L. Brickson, L. M. Henderson, I. Solhjell, and C. A. Elvehjem, J. Biol. Chem. 176, 517 (1948).

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PROTEINS AND DERIVATIVES

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standard curves, which have been observed with various test organisms in several different assay media. These ~nhibitory relationships vary from one test organism to another, and, where such curves are observed, choice of an alternative assay organism may alleviate the difficulty. For example, L. arabinosus is not suited to determination of isoleucine by the procedure outlined here because its response to this amino acid is readily inhibited by leucine and valine. 4s This is not true for Leuco. mesenteroides or for any of four other organisms tested (S. faecalis R, L. delbriickii 3, L. casei, or Leuco. mesenteroides); one of the latter group is therefore employed for the determination. 14.~ Accuracy of the Microbiological Procedure. Recoveries of added amino acids and repeated assays of individual proteins indicate that the experienced operator should have no difficulty in obtaining values accurate to within _ 5 % of the true value. In some instances, reproducibility may be considerably better than this. 1-~,14,~s Summarized Methods Method of Henderson and Snell 14 (see detailed presentation above) Assay organisms:

L. arabinosus 17-5: For glutamic acid, leucine, phenylalanine, tryptophan, and valine. Leuco. mesenteroides P-60: For aspartic acid, glycine, histidine, isoleucine, lysine, proline, and tyrosine. S. faecalis R: For arginine, histidine, methionine, and threonine. Stock cultures: Carried as stabs by biweekly transfer in medium 1 (Table II) with glucose and sodium citrate reduced to one-half specified concentration and supplemented with 0.5% tryptone, 0.5% yeast extract, and 2.0% agar. Incubated for 24 to 48 hours at 37°; held at 4 to 12° between transfers. Basal medium: Medium 1, Table II, with appropriate amino acid omitted. Initial pH 6.8 (pH 6.0 for glutamic acid and proline assay). Inoculum medium: Medium 1, Table II, supplemented per 100 ml. (single strength) with 0.5 % tryptone and 0.5 % yeast extract and with glucose and sodium citrate lowered to one-half concentration specified. Concentration of amino acids for standard curves: Approximately five different points (in duplicate) within range specified in Table III. Sterilization: 10 minutes at 10 to I2 lb. of pressure. Inoculum: Incubated for 8 to 12 hours at 37 °. Cells centrifuged and resuspended in 5 to 25 vol. 46 of 0.9% saline. One drop of the resulting suspension is used per 2-ml. assay tube. 46 S. Saecalis diluted 25-fold; L. arabinoaus and Lvuco: m~6~atcriode~ 5- to 10-fold. The

[77]

MICROBIOLOGICALDETERMINATION OF AMINO ACIDS

491

Incubation time and temperature: 60 to 72 hours at 37 ° . Measurement of response: Acidimetric. Turbidimetric determinations at 24 to 48 hours are useful for many purposes. Method of Steele, Sauberlich, Reynolds, and Baumann 24

Assay organisms:

Leuco. citrovorum 8081 (Pediococcus sp.) : For alanine, arginine, cystine, glutamic acid, glycine, histidine, isoleucine, methionine phenylalanine, proline, threonine, tyrosine, and valine. Leuco. mesenteroides P-60 (Streptococcus equinus): For all amino acids except alanine. Stock cultures: Carried as stabs with transfers at least once a month in medium containing 1% yeast extract, 1% glucose, and 1.5 % agar. Basal medium: Medium 2, Table II, with the amino acid to be determined • omitted. Initial pH 6.8 to 7.0. Inoculum medium: Medium IV 47 supplemented with 0.2% yeast extract. This is essentially identical with the basal medium used here, but with all amino acids except cysteine and tryptophan replaced with acid hydrolyzed casein (1.0 g. per 100 ml. of double-strength medium). Concentrations of amino acids for standard curves: Approximately five different points in duplicate within range specified in Table III. Sterilization: Not specified, but apparently about 10 minutes at 15 lb. of pressure. Inoculum: Incubated for 20 to 24 hours at 37 °, centrifuged, and diluted with sterile 0.9% NaC1 solution to a standard turbidity (G = 65 to 70 in Evelyn tube and colorimeter, filter 660). One drop of this diluted suspension used to inoculate each 2-ml. assay tube, Incubation time and temperature: 72 hours at 37 ° for acidimetric assays, 20 hours for turbidimetric assays. Measurement of response: Electrometric titration after 72 hours, or turbidimetric estimation after 20 hours. Method of Stokes, Gunness, Dwyer, and Caswell

TM

Assay organisms:

Streptococcus faecalis R: For arginine, histidine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, and valine. Lactobacillus delbriickii 5: For phenylalanine and tyrosine. Stock cultures : Carried as stab cultures by monthly transfer in a medium containing 1 g. of glucose, 0.5 g. of Bacto peptone, 0.6 g. of anhydrous amount of inoculum is not critical. For glutamie acid and proline assay, the test organisms are resuspended in an equal volume of saline. 47H. E. Sauberlich and C. A. Baumann, J. Biol. Chem. 166, 417 (1946).

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PROTEINS AND D~.RrCATIVES

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sodium acetate, 0.5 ml. of salts A, 26 0.5 ml. of salts B, 2e and 1.5 g. of agar per 100 ml. of medium (pH 6.8). Stored in the refrigerator between transfers. Basal medium: Medium 3, Table II, with the amino acid to be determined omitted. Initial pH 6.8. Inoculum medium: Same as that given above for stock cultures, but with agar omitted. Concentrations of amino acids for standard curves: Several different concentrations of the standard in the range shown in Table III. Sterilization: Autoclaved at 15 lb. of pressure for 13 minutes. Inoculum: Incubated for 16 to 24 hours at 37 ° in 8 ml. of inoculum medium, centrifuged, washed with water, suspended in 100 ml. (S. faecalis), or 20 ml. (L. delbri~ckii) of water. One drop of this suspension used for each assay tube of 10 ml. Incubation time and temperature: 40 hours at 37 ° for S. faecalis, 72 hours at 37 ° for L. delbr~kii. Measurement of responses: Titration of acid produced.

[78] M e a s u r e m e n t of Amino Acids by Column Chromatography By

EDWARD L. DUGGAN

Amino acids may be fractionated into classes or individuals by chromatography on various adsorbents. 1-3 One method, analytical in design, is superior to others in simplicity of operation and versatility of application. The method developed by Stein and Moore originally required starch as the adsorbent. 4 The sensitivity of this fractionation to inorganic salts and the low capacity of such columns resulted in the application of ion exchange resins to the fractionation. The method in use in various laboratories during the past four years is that described by Moore and Stein. 6 Here the sulfonated polystyrene, cross-linked with 8% divinyl benzene (Dowex 50 X 8) is used. No prior desalting is required, and samples of 2 to 10 rag. of amino acids may be used. A scheme for the isolation of the 1 S. Moore and W. H. Stein, Ann. Rev. Biochem. 21, 521 (1952). E. Lederer and M. Lederer, "Chromatography," Chapter 26, Elsevier Publishing Co., Houston, Texas, 1954. s p. L. Kirk and E. L. Duggan, Anal. Chem. 26, 165 (1954). W. H. Stein and S. Moore, J. Biol. Chem. 176, 337 (1948). 5 S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951).