THE MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS*

II. ASS,IY AND UTILIZATION OF GLUTAMIC ACID AND GLUTAMINE BY LACTOBACILLUS ARABINOSUS

BY LUCILE R. HBC, ESMOND E. WELL, AND ROGER J. WILLIAMS

(From the Amino Products Division of the International Minerals and Chemical Corporation, Toledo, Ohio, and The University of Texas, Biochemical

Institute, a.nd the Clayton Foundation for Research, Austin)

(Received for publication, February 27, 1945)

In a discussion of the analytical data available on proteins, Vickery (2) in 1941 stated that few values for glutamic and aspartic acids were worthy of serious consideration. Since that time, however, several promising methods for the chemical analysis of glutamic acid have appeared. Chibnall and coworkers (3, 4), by improved methods of isolation, have reported values considered to be accurate within 2 per cent. Determinations have also been made by chromatographic adsorption (5, S), by isotope dilution (7), by enzymatic methods (8,9), by calculation from electrometric titration and gasometric ninhydrin data (lo), and by conversion to pyrrolidone- carboxylic acid (11). Although these methods are applicable to the study of pure proteins, they were too tedious and time-consuming for many routine applications. Microbiological methods of assay (1,12-14) which seemed to offer more assurance of usefulness in such studies were, therefore, undertaken.

Since initiation of this work, Dunn et al. (14) and Lyman et al. (15) have described microbiological methods for the determination of glutamic acid Ti-ith a basal medium composed of amino acids, vitamins, purine bases, and salts. Lewis and Olcott (16) used a similar technique except that a sup- plemented “glutamic acid-free casein hydrolysate” was substituted for the amino acid mixture. In each instance the assay organism was Lactobacillus arabinosus. All of these workers found the utilization of glutamic acid by this microorganism to be somewhat unusual and its assay slightly more difficult than that of other acids previously reported. It t,herefore seemed worth while to present additional data pertinent to this method.

Materials and Methods

Microorganism-Lactobacillus arabinosus 17-5 was used for all assays. Uti~er microorganisms studied were L. pentosus 124-2, L. casei, and Leu- conostoc mesenteroides P-60.

* For Paper I of this series, see McXnhan and Snell (1). 273

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274 DETERMINATION OF AMINO ACIDS. II

Inoculum-The stock stab cultures were maintained on yeast extract- glucose agar (1 per cent glucose, 1 per cent yeast extract, and 1.5 per cent agdr) as previously described (1). The inoculum for assay was grown in a medium which contained per liter 5 gm. of Bacto-peptone, 1 gm. of Bacto-yeast extract, 10 gm. of sodium acetate, 10 gm. of glucose, and 5 ml. each of inorganic Salt Solutions A and B. Daily transfers were made serially from the broth with but weekly reference to the stock culture. An 18 to 24 hour culture was centrifuged, washed once with physiological saline, and resuspended in 10 ml. of saline. 1 drop of this suspension per 2.5 ml. of assay medium was considered a “heavy” inoculum. When reference is made to a “light” inoculum, 3 to 7 drops of the above “heavy” suspension were added to 10 ml. of saline. 1 drop of this material, which was just faintly turbid, was used per 2.5 ml. of assay medium.

Basal Medium-The composition of the basal assay medium and the method of preparation of the stock solutions are given in Table I. This medium served as a basic one for study of the assay of several amino acids and differs from that of McMahan and Snell (1) primarily in the increased concentration of vitamins. Certain modifications in amino acid content which have been used in the assay of glutamic acid are also indicated.

Standard Xolution-Pure Z(+)-glutamic acid was used for all the stand- ard curves.

Preparation of Xamples-Thick walled Pyrex test-tubes which contained 100 mg. of sample and 2 ml. of 10 per cent hydrochloric acid were sealed in an oxygen flame and autoclaved at 15 pounds pressure for 4 to 36 hours. The tubes were cooled, opened, and the hydrolysates washed out with water. The solution was adjusted with sodium hydroxide to pH 6.1 to 6.3 and diluted to the desired volume, usually 50 or 100 ml.

Procedure-For assay, 125 ml. of the amino acid solution, 1 ml. of the vitamin supplement, 2.5 ml. of Salts A, and 2.5 gm. of glucose constituted a double strength medium. This amount was sufficient for 100 assay tubes if determinations were made turbidimetrically (final volume 2.5 ml.) or 50 tubes if analyses were made titrimetrically (final volume 5 ml.). Or- dinarily the amount of sample to be analyzed or the standard solution used was small enough so that its volume (less than 0.2 ml. per 2.5 ml. of medium) could be disregarded. The sample was pipetted into the bottom of the tube1 and 2.5 ml. of basal medium were added. If it was necessary to use samples larger than 0.2 ml., the volume in each tube was adjusted with water to 1.2 ml. and 1.2 m1.2 of the double strength basal medium

1 Serological Kahn pipettes graduated in 0.001 ml. were used for this purpose. The end of the pipette was drawn out to a fine capillary. Such pipettes could be operated easily and accurately with the finger.

2 Kolmer pipettes designed for use in the complement fixation test and graduated in 1.2 ml. were used.

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HAC, SNELL, AND WILLIAMS 275

TABLE I

Complete Basal Medium; Concentration per illl. of Final Medium

Amino acids

Z(+)-Argininc monohydrochloride I(-)-Cystine ........................ Glycine ............................. I(-)-Histidine monohydrochloride. Z(- )-Hydroxyproline ................ I(-)-Leucine ........................ I(+)-Lysine monohydrochloride. Z(-)-Proline. .................. .-. ... I(-)-Tryptophane*. ................. Z(-)-Tyrosine ...................... I(+)-Aspartic acid. ................. I(+)-Glutamic acidt. .............. dl-Alanine .......................... dl-Isoleucine ........................ dl-Methionine* ...................... dl-Norleucine* ...................... dl-Norvaline* ....................... dl-Phenylalanine*. ........... dl-Serine* ........................... dl-Threonine ........................ dl-Valine ............................

Vitamins

- mh-.

0.1 0.1 0.1 0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.4 1.0

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 (NH&SO4 3.0

-

Adenine sulfate.. Guanine hydrochloridt Uracil Xanthine.............

VW.

0.01

0.01

0.01

0.01

Salts A KHQPOd 1.0 K2HP04. 1.0

Salts B MgS04.7HzO 0.2

FeS0.7Hz0.. 0.01 MnSOa.4Hz0 . 0.01

NaCl 0.01

Y

Sodium acetate (anhy Biotin............................... 0.001 drous) 6.0 Folic acid.. 0.01 Glucose. 10.0 Calcium pantothenate.. 0.2 Nicotinic acid........................ 0.2 Riboflavin. . 0.2 p-Aminobenzoic acid. 0.3 Thiamine hydrochloride. 0.5 Choline chloride. 2.5 Inositol.............................. 2.5 Pyridoxine hydrochloride$. . 10.0

Stock solutions of the constituents were prepared as follows: Salts A contained 25 gm. each of KHzPOa and KsHPO., per 250 ml. of solution; Salts B contained 10 gm. of MgS04.7Hz0, and 0.5 gm. each of FeS04.7H,0, MnS04.4Hz0, and NaCl per 250 ml. of solution. The vitamin supplement contained 12.5 y of biotin, 125 y of folic acid, 2.5 mg. each of calcium pantothenate, nicotinic acid, and riboflavin, 3.8 mg. of p-aminobenzoic acid, 6.3 mg. of thiamine hydrochloride, 31.4 mg. each of choline chloride and inositol, and 125 mg. of pyridoxine hydrochloride dissolved by warming gently in 50 ml. of distilled water. Amino acid mixture, 1 liter of amino acid solution contained 400 mg. each of the dl-amino acids, 800 mg. of I(+)-aspartic acid, and 200 mg. each of the other Z-amino acids; 20 mg. each of adenine sulfate,

- I Purines and pyrimidiie

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276 DETERJIWATIOS OF AMINO ACIDS. II

TABLE I-Concluded

guanine hydrochloride, xanthine, and uracil; 6 gm. of ammonium sulfate; and 12 gm. of anhydrous sodium acetate. The materials were dissolved by boiling in about 000 ml. of w&r. After the solution had cooled, 10 ml. of Salts B were added, the pII of the solution was adjusted to 6.3 with sodium hydroxide, and the final volume adjusted to 1 liter. Such solutions were stored in the refrigerator, usually without preservative, but a thin layer of toluene may be used if necessary.

* Ha.lf the indicated amounts have been found satisfactory in assay of glutamic acid.

t Omitted in medium for assay of glutamic acid. 1 One-tenth the amount indicated is sufficient for assay with Lactobacillus ara-

binosus 17-S.

were added. For titration, double amounts of sample and medium were used. The tubes were capped with metal caps or well fitted glass vials (3 cm. long) and autoclaved at 15 pounds pressure for 15 minutes. After inoculation, the tubes were incubated at 34” for 72 hours.

For turbidity readings the contents of the tubes were diluted with 5 ml. of mater delivered from an automatic pipette. A thermocouple turbidimeter (17) was used almost exclusively in this study, but the Klett-Summerson calorimeter also gave satisfactory results.

Titration of the acid produced in the 5 ml. assay quantities was carried out with 0.05 N sodium hydroxide with a glass electrode or brom-thymol blue as an indicator.

The assay range used was 0.01 to 0.2 mg. of I(+)-glutamic acid per 2.5 ml. of medium. Final analytical values are the average of duplicate anal- yses at three or four levels of concentration.

Results

Standard Curve-Typical standard assay curves for glutamic acid are plotted in Fig. 1 with four test organisms, Lactobacillus arabinosus, L. pentosus, L. casei, and Leuconostoc mesenteroides. As others have noted with L. ara@noszls (15, IS), the shape of these curves differs from that re- ported for other amino acids. There is an initial plateau at which no growth above that of the blank occurs. Once growth is initiated, however, the curves break sharply and rise very rapidly, followed by a more gradual rise proportional to the amount of amino acid present. This latter portion of the curve was used for assay since it gave the most regular and reliable results. A fundamental similarity in assimilation of this amino acid by the four microorganisms is indicated by their growth curves which differ primarily in the concentration of glutamic acid at which growth is initiated.

This inability to utilize glutamic acid at low levels of concentration suggested that it was not itself assimilated by these organisms, but was slowly converted by them into a substance which was assimilable. Early

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HAC, SNELL, AXD WILLIAMS 277

in this investigation glutamine and glutamic acid were found t,o have equal activity for Lactobacillus arabinosus at relatively high concentrations, but at low levels at which no growth was observed with glutamic acid, glu- tamine was active. This observat,ion suggested that glutamine rather

60,

, I I I I I I 0 .a1 .02 aa .04 .OJ .06 .07 .08 .OP 0.1

MG.LI+l GLUTAMK ACID PERTVBE (2.5 ML. 1

FIG. 1. Comparative response of La.ctobacillus arabinosus, L. pentosus, L. casei, and Leuconostoe mesenteroides to I(+) -glutamic acid. Turbidimetric measurements.

60

72 HR. INCUBATION 50

P 540

I

5 b a0 8 48HR. INCUBATION

j20 HEAVY INOCULUM

IO LIGHT I NOCULUM

0 .Ol .02 .oa A4 .OI .06 .07 08 .OP I MG.Lt+l GURAMIC ACID PERTUBE l2.5ML.)

FIG. 2. The effect of the size of inoculum and the period of incubation on the response of Lactobacillus arabinosus to I(f)-glutamic acid. Turhidimetric meas- urements.

than glutamic acid might be the substance required for the growth of these microorganisms and that the initial plateau observed in the glutamic acid curve represented a region in which concentration of glutamic acid was too low t.o permit significant conversion t.o glutamine by the test organism. Recent,ly Lyman et al. (15) have advanced a similar view and have suggested

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278 DETERMINATION OF AMINO ACIDS. II

that small amounts of glutamine be added to the basal medium used for assay of glutamic acid.

If conversion to glutamine or some other more readily assimilable sub- stance is necessary before rapid growth can take place, such conversion

I I I I I 1 1 I I I 0 .01 .OZ .03 .04 .05 .06 .07 .08 .09

MG. L I+1 GLUTAMIC ACID PERTUBE (2.5 ML.1

FIG. 3. The effect of initial pH of the basal medium on the response of Lactobacillus arabinosus to I(+)-glutamic acid. Turbidimetric measurements.

FIG. 4. Effect of varying the concentration of phosphates in the basal medium on the response of Lactobacillus arabinosus to I(+)-glutamic acid. Turbidimetric measurements.

should be favored by increasing the size of the inoculum or the period of incubation. From Fig. 2 it is evident that growth of Lactobacillus czrabi- nosus occurred at lower concentrations of glutamic acid when a large in- oculum or a relatively long period of incubation was used.

Since growth of these organisms is accompanied by acid production, the effect of variation in the pH of the basal medium upon growth response

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HAC, SNELL, AND WILLIAMS 279

to glutamic acid was determined. As the initial pH of the medium was decreased, growth was initiated at lower and lower concentrations of glutamic acid until at pH 5.0 the initial plateau of the growth curve was almost completely eliminated (Fig. 3). In such a medium at high con-

50

is E40 ITH NH; 4 2 LUTAMINE WITHOUT NH:

$30 3 P NH:

220 3

10 GLUTAMIC ACID WITHOUT N Hi

-* ____ ~ _--- -i ----, -q--*----w

I I I I I I I I , I 0 .01 .02 .OS .04 .05 .06 .07 .OB a9

MG.LIC) GLUTAMIC ACID ORGLUTAMINE PERTU33 (2.5ML.l

FIG. 5. Comparative response of Lactobacillus arabinosus to glutamine and Z(+)- glutamic acid in the presence and absence of ammonium sulfate. A “heavy” in- oculum was used and the period of incubation was 24 hours.

2 j20

t

/ -GLUTAMINE WITH NH:

: .--*GLUTAMINE WITHOUT NH2

- GLUTAMIC ACID WITH NH: e---*GLUTAMICACIDWlTHOUT NH:

r-- l t 1 I I I I I t 0 .Ol .02 .os .04 .05 .06 .07 .08 A9 t

MG L I+) GLUTA~~~C *Cw 02 GLUT*MINE PERTUBE 12.5 ML.)

FIG. 6. Comparative response of Lactobacillus arabinosus to glutamine and Z(+)- glutamic acid in the presence and absence of ammonium sulfate. A “light” in- oculum was used and the period of incubation was 72 hours.

cenkations of glutamic acid, however, maximum growth was not as great as that obtained in mediums of pH 6.0 to 8.0. It might be expected that maximum levels of growth would be lower in a medium of relatively high initial acidity, since growth of these organisms is inhibited by relatively high concentrations of acid. Acid produced as a metabolic product of

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280 DETERMINATION OF AMINO ACIDS. II

growth would reach toxic levels earlier in a medium more acid initially. The pH selected for use in assay, therefore, was 6.3.

It was possible to initiate growth at lower concentrations of glutamic acid in a medium of pH 6.8 if the amount of ph0sphat.e in the basal medium was decreased (Fig. 4). This effect was probably due t,o the fact that smaller amounts of acid were required to shift the pH of the less highly buffered medium to levels permitting more ready conversion of glutamic acid to the assimilated product.

These experiments all tend to indicate that the mechanism for the con- version of glutamic acid to glutamine operates optimally in acidic solutions. Some type of ammonia transfer must also be involved. In order to determine whether the ammonium ion served this function, growth curves

-GLUTAMINE WITH NH: - -GLUTAMINE WITHOUT N Hf -GLUTAMIC ACID WITH NH: ‘---‘GLUTAMIC ACID WITHOUT -NH:

FIG. 7. Comparative response of Lactobacillus arabinosus to glutamine and Z(+)- glutamic acid in the presence and absence of ammonium sulfate. h “heavy” in- oculum was used and the period of incubation was 72 hours.

produced by glutamic acid and glutamine were compared with and without ammonium salts added to the basal medium. Both light and heavy inocula mere used and readings were made at 24, 48, and 72 hour intervals. The omission of ammonium salts from the basal assay medium had no effect upon the utilization of glutamine, but their absence delayed growth and prevented utilization of glutamic acid at low concentrations. This was particularly noticeable when the period of incubation was short (Fig. 5), or when the inoculum was small (Fig. 6). Even with a heavy inoculum, no growth was evident at 24 hours (Fig. 5). Growth occurred at high concentrations of glutamic acid at 48 hours and at lower concentrations at 72 hours, but the initial plateau at which no growth occurred was more pronounced than when ammonia was present (Fig. 7). The addition of ammonium sulfate to the glutamic acid medium resulted in growth curves

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HAC, SXELL, AND WILLIAMS 281

at 24 hours similar to those for glutamine except that the initial plateau was evident and growth was slower throughout the entire curve. At 72 hours, the glutamine and glutamic acid curves coincided except at the very lowest concentration of glutamic acid. These results indicate that

TABLE II

Comparative Growth-Promoting Activities of Glutamic Acid and Related Substances

Test organism, Lactobacillus arabinosus; galvanometer readings.

Organism per tube

mg.

0.0 0.01 0.02 0.04 0.07 0.10 0.13 0.16 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2.00

L ( 2lutamine (filtered)

5.0 12.0 20.0 31.5 38.8 41.5 52.6 57.0 64.0 72.2 74.0

dlutamic acid

I i+)-

3.0 4.0

18.5 30.3 38.4 41.5 52.5 57.0 63.5 i3.0 76.5 i6.5 ‘76.5 77.0 77.0 77.0 ii.0

dl-

4.0 4.0 4.0 4.0 4.0 4.0

21.5 4.0 35.0 4.0 43.0 4.0

75.5

77.0

4.0 9.0

23.5 40.5 47.0 54.8 60.2 64.5 67.0 77.0

a(-)-

r-K&o& taric acid

-

C

7.0

7.0 12.0 35.5 58.0 72.0 73.3

77.0

77.0

Xutathionf

9.0

9.0 24.5 34.2 43.2

59.0 66.5

76.5

77.0

yrrolidom carboxylic

acid

4.0

4.0

4.0

4.0

4.0

-

! - L

_-

r-Hydroxy- glutaric

acid

4.0

4.0

4.0

4.0

4.0

0.05 mg.2(+)-glutamic acid . . .._..............................._._.... 35.4 0.05 I‘ “ ‘I + 0.02 mg. or-ketoglutaric acid . 38.2 0.05 Cc (. ‘( + 0.01 Cc (’ ‘( ..t... 41.0 0.05 Ii ‘i “ + 0.12 I‘ ‘< “ 54.5 0.05 “ “ “ + 0.02 “ pyrrolidonecarboxylic acid 35.0 0.05 “ L: I( + 0.12 ‘I “ “ 35.8 0.05 <‘ C‘ ‘L + 0.02 “ oc-hydroxyglutaric acid 35.7 0.05 I‘ “ ‘I + 0.12 “ “ “ 34.8

conversion of glutamic acid to glutamine does occur and that ammonium salts are utilized in the conversion.

Identical values were obtained with four different preparations of glu- tamine which were sterilized by filtration through a Berkefeld filter and added aseptically to the sterile basal medium just before inoculation. Glutamine is rapidly decomposed, primarily to ammonium pyrrolidone-a- carboxylate or pyrrolidonecarboxylic acid (18), by heating it in an aqueous

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282 DETERMINATION OF AMINO ACIDS. II

solution. A similar conversion of glutamic acid takes place in slightly acid solutions, but the reaction is slower (19). Curves obtained with glutamic acid sterilized in the &utoclave or by filtration were identical.

Use has been made of these facts in the estimation of the glutamine content of various naturally occurring substances. Samples to be tested were analyzed directly and after hydrolysis. For direct analysis, aliquots of the samples to be tested were sterilized by filtration through a Berkefeld filter, and by autoclaving. The values so obtained compared with those determined after hydrolysis gave at least a rough indication of the pro- portions of the materials present as glutamine, glutamic acid, and pyrroli- donecarboxylic acid. Refinement of these techniques should result in a

60

I I I I 1 I 8 I t 0 .Ol .02 .03 .04 .05 .Ob .07 .oa .09

MG. GLUTAMIC ACID PERTUBE 12.5 ML.1

FIG. 8. Comparative response of Lactobacillus arabinosus and Lactobacillus casei to I(+)- and dl-glutamic acids. The concentration of the dl isomer is twice that of the naturally occurring antipode.

fairly accurate method for determining the proportions of these substances present in natural products.

Hydrolyzed samples of glutathione gave theoretical values for glutamic acid. Unhydrolyzed samples of this peptide showed some activity which increased with increasing concentration, but was less than that to be ex- pected from its glutamic acid content.

Several compounds which have a chemical structure similar to glutamine and glutamic acid were tested for their ability to promote growth. In Table II are listed the turbidimetric values for these substances compared with those obtained with glutamine and I(+)-glutamic acid.

d(-)-Glutamic acid3 initiated growth at much higher concentrations of glutamic acid than I(+)-glutamic acid. The activity of the unnatural

a Furnished by Dr. A. C. Kibrick.

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TABL

E III

Glut

amic

Acid

Co

nten

t (in

Pe

r Ce

nt)

of

Vario

us

Pure

Pr

otein

s*

Glut

amic

acid

re

cove

ry Ca

sein

(Lab

co)

Lact

oglo

bulin

t

Lact

oglo

bulin

§

Egg

album

in?

Egg

album

in11

Glia

dinl

]

Gelat

in (K

nox)

Silk

fibro

int

Ed&t

in

(Difc

o sta

ndar

dized

Hors

e he

mog

lobint

(re

crys-

talliz

ed

3 tim

es)

Hors

e ca

rbox

yhem

oglob

inl/

(recr

ysta

llized

2

times

)

No.

of

analy

ses

Glut

amic

acid

No.

of

analy

ses

Glut

amic

acid

No

. of

an

alyse

s Gl

utam

ic ac

id

No.

of

analy

ses

Glut

amic

acid

No

. of

an

alyse

s Gl

utam

ic ac

id

No.

of

analy

ses

Glut

amic

acid

No

. of

an

alyse

s Gl

utam

ic ac

id

No.

of

analy

ses

Glut

amic

acid

No

. of

an

alyse

s Gl

utam

ic ac

id

No.

of

analy

ses

Glut

amic

acid

No

. of

an

alyse

s Gl

utam

ic ac

id

i

0 hr

.

100

-- -

4 hr

s.

100 1

21.1

- 1 _

3 -

Hydro

lysis

time

Value

s rep

orted

in

literat

ure

‘6 hr

s. 1

. -

100 1

22.:

> -

LO hr

s. 16

hrs.

20

hrs.

24

hrs.

36

hrs.

Mi

crobio

logica

---

-- 100

100

100

98

97

10

5 5

8 22

.5

(14)

22

.7

21.5

21

.8

21.7

19

.7

(16)

21

.5

(15)

4

6 2

3 2

17.7

17

.9

18.9

18

.8

19.6

18

.7

(16)

3

1 2

2 18

.8

18.9

19

.0

18.6

5

1 2

14.9

15

.1

14.1

1

1 2

3 13

.7

(16)

15

.0

15.2

15

.0

14.8

14

.3

(15)

1

3 2

2 1

45.3

46

.3

47.3

45

.5

47.3

44

.2

(16)

2

1 1

1 10

.7

11.3

11

.9

11.2

10

.2

(16)

2

2 2.

2 (1

4)

2.2

2.4

2.1

(16)

2 21.3

19

.1

(16)

1

1 1

3 1

9.7

8.5

8.8

10.8

12

.4

2 2

I I

I 8.

91 9.

01

7 1 Ch

emica

l

22.0

(2

)

21.5

(3

) 19

.01

16.1

(3

) 17

.0

(10)

46

.9

(2)

45.7

(IO

) 11

.7

(10)

5.

8 (2

0)

3.5

(IO)

18.3

(IO

) 20

.7

(3)

6.3

(21)

6.

75

(2)

8.57

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284 DETERMINATION OF AMINO ACIDS. II

antipode was evidenced somewhat earlier, however, if a dl mixture was used. Several samples of dl-glutamic acid were tested and repeated assays were made. All of the samples showed approximately 50 per cent activity at low concentrations, but greater activity at higher concentrations. This

TABLE IV

Comparison of Values Obtained with Light and Heavy Inoculum*

Per cent glutamic acid

Hydrolysate

mg.

0.015 0.020 0.026

Average

Casein Lactoglobulin Egg albumin

Light Heavy Light Heavy inoculum inoculum

Light Heavy inoculum inoculum inoculum inoculum

21.00 21.33 21.25 22.11 20.19 21.75

__-

20.8 21.8

18.12 18.75 17.50 19.62 17.00 18.12

.-

- 17.5 18.8

14.16 15.00 13.96 14.40 13.55 13.60

-~

13.9 14.‘3

Gliadin

Light H&WY inoculum inoculum

-___

45.00 1 44.60 46.25 I 41.66 46.50 ’ 43.75

_--___

45.9 ~ 43.3

* Incubation period 72 hours. Each value is an average of duplicate determina- tions.

TABLE V Recovery of Glutamic Acid Added to Protein Hydrolysates

Protein

Casein.........................

Lactoglobulin .................

Egg albumin .................

-

-

mx. 0.0206 0.0206 0.0325 0.0325 0.0440 0.0272 0.0368 0.0288 0.0429

mg 711g,

0.02 0.0400 0.03 0.0510 0.02 0.0520 0.04 0.0725 0.03 0.0740 0.02 0.0470 0.03 0.0671 0.03 0.0580 0.02 0.0622

I(+)-Glutamic acid

Per cent recovery

98.6 100.7

99.0 100.0 100 .o

99.5 100.3

98.6 98.8

increasing activity with increasing concentrations of dl-glutamic acid for Lactobacillus arabinosus and Lactobacillus casei is shown in Fig. 8.

a-Ketoglutaric acid was utilized at lower concentrations than d(-)- glutamic acid, but at higher concentrations than Z(+)-glutamic acid. Here, as in the case of the unnatural antipode, the addit’ion of even small amounts of ac-ketoglutaric acid (0.02 mg.) to limiting cmantities of Z(+)- glutamic acid resulted in increased growth (Table II).

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HAC, SNELL, AND WILLIAMS 285

Pyrrolidonecarboxylic acid and cY-hydroxyglutaric acid were completely inactive.

Reliability of Assay. Agreement with Other Methods of AnalysisThe assay results obtained with several pure proteins are listed in Table III. Consistent values were obtained on repeated assay. Average values ob- tained after 4, 6, 10, 16, 20, 24, and 36 hours hydrolysis are compared with

9 72 HR. INCUBATION HEAVY INOCULUM 6 =a n

lR.INCUBATION i Zb

HEAVY INOCULUM

:5

g4 P LIGHT INOCULUM 213 8 22 4

:: .02 .04 .Ob as 0.1 0.12 0.14 0.16 0.18 0.2

MG.L~+lGL”TAMlC ACID PERTWE LSML.)

FIG. 9. The effect of size of inoculum and the period of incubation on the response of Lactobacillus arabinosus to I(+)-glutamic acid. Titrimetric measurements.

TABLE VI

Comparison of Turbidimetric and Titrimetric Values*

Protein Per cent glutamic acid

Turbidity I Titration

Casein................

Lactoglobulin .......... Egg albumin. .......... Gliadin. ...............

. . . .

22.6 20.8 21.1 21.4 20.7 20.6 18.7 18.1 14.4 14.0 44.9 44.0

* Incubation period, 72 hours. Each figure is the average of duplicate deter- minations made at three or four levels of concentration.

those obtained with pure Z(f)-glutamic acid similarly treated. The highest values for lactoglobulin, gliadin, and horse hemoglobin were ob- tained after 36 hours hydrolysis, even though pure Z(f)-glutamic acid had undergone slight racemization at a similar period. The values obtained are in good agreement with those obtained by similar microbiological t.echniques, and with the most reliable chemical analyses.

ilgreement of Values Calculated at Various Assay Levels-In Table IV

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286 DETERMINATION OF AMINO ACIDS. II

the values obtained at various assay levels are compared when light and heavy inocula were used. There is good agreement at different levels of concentration. Since a fairly heavy inoculum after 72 hours incubation gives slightly higher assay values as well as an increased range for assay, it is preferable to a very light inoculum.

Recovery Experiments-Recovery values for I(+)-glutamic acid added to hydrolysates of casein, lactoglobulin, and egg albumin are given in Table V. Although the recovery values obtained with these pure proteins were excellent, certain difficulties were encountered with some industrial materials in which both stimulatory and inhibitory substances were noted.

Turbidity Versus Titration ValuesIn Fig. 9 are plotted the titrimetric curves which correspond to the turbidimetric curves given in Fig. 2. Values obt,ained from some of the proteins tested by the two methods are compared in Table VI. The values obtained by turbidimetric measure- ments are usually slightly higher than those obtained by titration. Since the turbidimetric method was easier and less time-consuming than titra- tion, it was preferred provided the samples were not too highly colored and did not develop turbidity during incubation.

DISCUSSION

All of the data concerned with utilization of glutamic acid are readily explained by the assumption that glutamine, rather than glutamic acid, is the substance actually utilized by the test organism. Glutamine was the most active substance tested, and growth was proportional to the concentration. Conditions which increased the availability of glutamic acid caused the dose-response curve to become more similar to that ob- tained with glutamine. Such conditions included the use of heavy inocula, extension of the period of incubation, and a decrease in the pH of the medium. Especially suggestive was the fact that omission of ammonium salts greatly reduced the availability of glutamic acid, but failed to affect response to glutamine in any manner.

According to Pollack and Lindner (22), glutamine and glutamic acid had equal growth-promoting activity for Lactobacillus arabinosus, L. pentosus, L. casei, and Streptococcus la&is. Results presented above and those of Lyman (15), Lewis and Olcott (16), and Niven (23) fail to confirm these data at low concentrations of glutamic acid. Pollack and Lindner did not consider it likely that glutamic acid was converted to glutamine because the original medium contained no source of ammonia other than amino acids and organic nitrogen compounds. This view is hardly con- sistent with the known versatility of microorganisms. Although ammonium chloride added to their medium did increase growth obtained with glutamic

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HAC, SNELL, AND WILLIAMS 287

acid, they considered the effect to be too small to be accounted for by con- version of glutamic acid to glutamine.

McIlwain et al. (24) reported that glutamine could be replaced by high concentrations of dl-glutamic acid for the growth if Streptococcus hemo- lyticus and considered that the organism might be able to synthesize glu- tamine when sufficiently high concentrations of glutamic acid were present. It was suggested that the essential growth activity dependent upon glu- tamine was ammonia transfer. Later, however, in studies demonstrating the extreme specificity of glutamine for certain microorganisms, McIlwain retracted these suggestions (25).

Lyman et al. (15) considered it advisable to eliminate the initial plateau obtained with glutamic acid by the addition to the basal medium of sub- optimum quantities of glutamine. In the course of this investigation it was found that small amounts of a tryptic digest of casein also eliminated this initial plateau. Such digests are superior to glutamine for this purpose because they are stable to heat and can be autoclaved with the medium without loss in activity. They have been previously reported (26, 27) to contain substances, probably peptides, which replace glutamine and asparagine for lactic acid bacteria. Addition of such substances to the assay medium produced little or no difference in assay values, and they were therefore not used routinely.

When used alone in high enough concentration, d(-)-glutamic acid, a-ketoglutaric acid, and glutathione permitted attainment of the same maximum growth level achieved with glutamine or I(+)-glutamic acid, although they were less active than either of these compounds at low levels of concentration. When tested in the presence of suboptimum quantities of Z(f)-glutamic acid, d(-)-glutamic acid and oc-ketoglutaric acid showed activity at lower concentrations than when tested alone. This indicates that conversion to glutamine is also necessary for utilization of these com- pounds, and such conversion occurs at lower concentrations when the test organism is actively growing. This view is at variance with that ex- pressed by Dunn et al. (14), who postulated some essential role for d(-)- glutamic acid on the basis of similar data for dl-glutamic acid.

SUMMARY

A turbidimetric or titrimetric method for the quantitative determination of I(+)-glutamic acid in protein hydrolysates is described. Lactobacillus arabinosus is the test organism. The following values, corrected for moisture, were obtained with the proteins studied after 24 hours hydrolysis : Labco casein 21.7, lactoglobulin 18.8 and 19.0 (different preparations), egg albumin 15.0, gliadin 45.5, gelatin (Knox) 11.2, silk fibroin 2.2, edestin

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288 DETERMINATION OF AMINO ACIDS. II

21.3, horse hemoglobin 10.8, and horse carboxyhemoglobin 8.9 per cent. Recovery experiments, agreement of values calculated at various assay levels, and upon repeated assay, specificity studies, and agreement with other methods of analysis all indicate reliability of the proposed method. Occasional low values as compared with chemical analyses require further study for explanation. In some instances, both stimulatory and inhibitory substances have been encountered in crude natural materials, and further study of their assay is indicated.

Glutamine is more active than I(+)-glutamic acid; activity of the latter is increased toward that of glutamine as a limit by increasing the size of the inoculum, lengthening the incubation period, lowering the initial pK of the medium, and adding ammonium salts to the medium. These data indicate that glutamic acid is converted to glutamine before utilization. This is probably also true for d(-)-glutamic acid, cr-keto- glutaric acid, and glutathione, which are less active than I(+)-glutamic acid, but which permit maximum growth at high concentrations. Pyr- rolidonecarboxylic acid and hydroxyglutaric acid are inactive.

For some samples, the method appears adaptable to the determination of glutamine as well as glutamic acid.

BIBLIOGRAPHY

1. McMahan, J. R., and Snell, E. E., J. Biol. Chem., 152,83 (1944). 2. Vickery, H. B., Ann. Xew York Ad. SC., 41,87 (1941). 3. Bailey, li., Chibnall, A. C., Recs, M. W., and Williams, E. F., Biochem. J., 37,

360 (19A3). 4. Chibnall, A. C., Rees, &!I. W., and Williams, E. F,, Biochem. J., 37, 372 (1943). 5. Wieland, T., Ber. them. Ges., 75, 1001 (1942). G. Cannan, R. Ii., J. Biol. Chern., 152, 401 (1941). 7. Rittenberg. D., and Foster, G. L., J. Biol. Chem., 133, 73i (19-10). 8. Cohen, P. P., Biochem. J., 33,,651 (1939). 9. Woodward, G. E., R.einhart., F. E., and Dohan, .J. S., J. Viol. C’hern., 138,677 (1941).

10. Ribrick, rl. C., J. Biol. Chem., 152, 411 (1944). 11. Olcott, H. S., J. Biol. Chem., 153, 71 (1944). 12. Shankman, S., Dunn, M. S., and Rubin, L. B., J. Biol. Chem., 161, 511 (1943). 13. Iiuilcen, I<. -k., Sorman, IV. I-I., Lyman, C. M., Hale, F., and Blotter, L., J. Biol.

Cheni., 151, 615 (1943). 14. Dunn, 31. P., Cnmien, h4. S., Rockland, L. B., Shankman, S., and Goldberg, S.

C., J. BioZ. Chcm., 155, 591 (1944). 15. J,ymnn, C. AI., Ku&en, I<. -k,, Blotter, L., and Hale, F., J. Biol. Chem., 15'7.

395 (1945). 16. Len-is. J. C.! and Olcott, H. S., .I. Biol. Chem., 157, 265 (1945). 17. Williams, R. J., MdAlister, E. D., and Roehm, R. R., J. BioZ. Chem., 83,315 (1929). 1s. T’ickery, H. B., Pucher, G. W., C’lnrk, H. E., Chibnall, A. C., and Westall, R. G.,

Biochem. J., 29, 2710 (1935). 19. Wilson, H., and Cannan, It. Ii., J. BioZ. Chem., 119, 309 (1937). 20. Dal-+ I-1. D., Z. physiol. Chem., 130, 159 (1923).

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HAC, SNELL, AND WILLIAMS 289

21. Chibnall, A. C., and Bailey, K., cited by Cohn, E. J., and Edsall, J. T., Proteins, amino acids and peptides, American Chemical Society monograph series, New York, 358 (1943).

22. Pollack, M. A., and Lindner, M., J. Biol. Chem., 143, 655 (1942). 23. Niven, C. F., Jr., J. Buck, 47, 343 (1944). 24. McIlwain, H., Fildes, P., Gladstone, G. P., and Knight, B. C. J. G., Biochem. J.,

33, 223 (1939). 25. McIlwain, H., Biochem. J., 33, 1942 (1939). 26. Pollack, M. A., and Lindner, M., J. Biol. C/tern., 147, 183 (1943). 27. Wright, L. D., and Skeggs, H. R., J. Butt., 48, 117 (1944).

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WilliamsLucile R. Hac, Esmond E. Snell and Roger J.

LACTOBACILLUS ARABINOSUSBYGLUTAMIC ACID AND GLUTAMINE

II. ASSAY AND UTILIZATION OFDETERMINATION OF AMINO ACIDS:

THE MICROBIOLOGICAL

1945, 159:273-289.J. Biol. Chem. 

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