INVESTIGATIONS OF AMINO ACIDS, PEPTIDES, AND PROTEINS

XXIII. THE DETERMINATION OF PHENYLALANINE IN PROTEIN HYDROLYSATES WITH LEUCONOSTOC MESENTEROIDES

P-60 AND LACTOBACILLUS CASEI*

BY MAX S. DUNN, S. SHANKMAN, AND MERRILL N. CAMIEN

(From the Chemical Laboratory, University of California, Los Angeles)

(Received for publication, July 26, 1945)

During the past 2 years microbiological procedures’ have been reported for the determination in proteins of thirteen of the twenty-one amino acids which occur in protein materials. Seven amino acids (isoleucine, glutamic acid, methionine, leucine, phenylalanine, tryptophane, and valine) have been determined with Lactobacillus arabinosus 17-5, three (arginine, tyrosine, and valine) with Lactobacillus casei, two (histidine and lysine) with Leuconostoc mesenteroides P-60, two (aspartic acid and serine) with Lactobacillus delbriickii (4), and one (tryptophane) with Eberthella typhosa T-63 (5). Comparable methods (unpublished) have been developed in the authors’ laboratory for the determination of cystine, glycine, and threonine. It is possible, therefore, to determine microbiologically all of the accepted amino acids except alanine, proline, hydroxyproline, 3,5- diiodotyrosine, and thyroxine.

It has been shown, previously, that phenylalanine2 is essential for the growth of Clostridium sporogenes (7), Corynebacterium diphtheriae (8), Streptococcus hemolyticus (9)) Lactobacillus mannitopoeus (lo), Lactobacillus buchneri (lo), Lactobacillus lycopersici (lo), Lactobacillus delbriickii (II), Lac- tobacillus arabinosus 17-5 (12-16), Lactobacillus casei (3,17), and Leuconostoc mesenteroides P-60 (18). Although Hegsted (15) has assayed phenylalanine with Lactobacillus arabinosus 17-5, the present studies were undertaken

* For Paper XXII in this series see Carnien et al. (1). This Jvork was aided by grants from the Gelatin Products Company, Merck and Company, Inc., the Nutrition Foundation, Inc., Schering and Glatz, and the University of California. The authors are indebted to H. Block, W. Frankl, B. Merrifield, and L. B. Rockland for valuable suggestions and technical assistance.

1 Literature references have been given by Dunn et al. (2). According to Baum- garten et al. (3) cystine, glutamic acid, isoleucine, leucine, threonine, tryptophane, and valine can be determined with Lactobacillus arabinosus and arginine, glutamic acid, isoleucine, leucine, phenylalanine, serine, tryptophane, tyrosine, and valine with Lactobacillus casei.

2 It is of interest that Kidder and Dewey (6) have shown recently that phenylala- nine is essential for the growth of the ciliated protozoan, Tetrahymena geleii W.

643

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644 AMIKO ACIDS, PEPTIDES, AND PROTEINS. XXIII

because it appeared that the values reported for phenylalanine and other amino acids in casein and other proteins were less accurate than those obtained for the amino acids determined by analogous published methods. The determination of phenylalanine in protein hydrolysates with Lacto- bacillus casei and Leuconostoc mesenteroides P-60 is described in t,he present paper.

EXPERIMENTAL

The assay technique described in previous publications from this labora- tory was employed in the present experiments. Separate solutions of basal media, amino acid test mixtures, protein hydrolysates, standard amino acid, sodium chloride, and inocula were delivered to 4 inch test- tubes with the aid of an automatic pipette (Baltimore Biological Company instrument). The total volume in each case was 3 ml. All of the solutions mere adjusted to the same concentration of sodium chloride to compensate for any stimulatory or inhibitory salt effects. The standard (phenylal- anine) was run at eight levels (0 to 16 r), the amino acid test mixtures and protein hydrolysates were run at five levels, and from five to six tubes were employed at each level of sample and from eight to twelve tubes at each level of standard. By using numbers of tubes more nearly sta- tistically significant than those usually employed, intrinsic errors inherent in microbiological procedures were compensated and assay values of r’ela- tively high accuracy mere obtained. It was found as satisfactory to titrate the mixture of solutions from five or more tubes at a particular level of sample as to titrate the individual solutions and calculate the average titration value.

,4 basal medium essentially of the composition shown in Paper XVIII (18) (Medium D, Table I, with phenylalanine omitted) was employed for the assay of phenylalanine with Leuconostoc mesenteroides P-60. This basal medium was modified to contain 1.9 and 2 times the amounts of the amino acids given in the table, since it was found from experiments on multiple amino acid media that the quantity of acid produced at any given concentration of phenylalanine was nearly constant at both levels of total amino acids.

The basal medium utilized for the determinat,ion of phenylalanine with Lactobacillus casei was essentially the same as that originally described by Hutchings and Peterson (12) and that employed previously (17) in the authors’ laboratory for the assay of phenylalanine and other amino acids in an amino acid test mixture. The composition of this medium modified for use in the present experiments was dl-alanine 200 mg., natural asparagine 200 mg., I(+)-arginine monohydrochloride 200 mg., Z(+)-glutamic acid 200 mg., Z(-)-histidine monohydrochloride monohydrate 200 mg., dl-

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DUNN, SHANKMAN, AND CAMIEN 645

isoleucine 200 mg., I( -)-leucine 200 mg., I(+)-lysine monohydrochloride 200 mg., dl-methionine 200 mg., dl-threonine 200 mg., I(-)-tryptophane 200 mg., Z(-)-tyrosine 200 mg., dl-valine 200 mg., I(-)-cysteine hy- drochloride 275 mg., dl-serine 300 mg., and folk acid 6 y per liter of solution. An equivalent amount of cysteine hydrochloride was substituted for cystine

TABLE I

Results o.f Assaw of Phenvlalanine in Amino Acid Test Mixture I*

I Lerconostoc mesenteroides I Ladobacillus cod

.2Tisx- Titration volume of 0.028 N

tu;;by NaOH per tube Phenylalanine found1

Ran& Average Per tube Per ml. sample

Y nzl. ml.

84.4 3.40-3.75 3.56 168.8 5.07-5.46 5.27 253.2 6.M-7.00 6.81 337.6 7.82-8.33 8.14 422.0 9.20-9.74 9.42

2.36 4.75 7.30 9.50

12.00

Y ??d. ml. Y Y

4.72 3.51-3.70 3.60 2.38 4.76 4.75 4.92-5.15 5.03 4.60 4.60 4.86 5.95-6.39 6.20 7.00 4.67 4.75 6.97-7.19 7.05 9.25 4.62 4.80 7.69-7.78 7.74 11.62 4.67

Titration volume of 0.028 N Phenylalanine NaOH per tube founds

Average

* The composition of the test mixture simulating casein was the same as that given in Paper XVII (20) except that’ the mixture contained 2.83 per cent of I(-)-phenyl- alanine (as dl-phenylalanine). The basal medium X 2 and 5 days incubation time at 35” were employed with Leuconostoc mesenteroides and 3 days incubation time at 35” was employed with Lactobacillus casei.

t Five replicate tubes at each level of sample. $ An average of 4.78 y of phenylalanine was found per ml. of amino acid test mix-

ture. The average mean deviation from the mean at the different levels was 0.9 per cent. The percentage of phenylalanine recovered in the assay was 100. The following percentages of phenylalanine were recovered in analogous experiments: 100 and 100 (basal medium X 1.3, 4 days incubation), 101 and 108 (basal medium X 1.3,5 days incubation), 102 and 101 (basal medium X 2,4 days incubation), and 100 (basal medium X 2, 5 days incubation).

§ An average of 4.67 y of phenylalanine was found per ml. of amino acid test mix- ture. The average mean deviation from the mean at the different levels was 0.9 per cent. The percentage of phenylalanine recovered was 98. The recovery of phenylalanine in an analogous experiment was 105 per cent.

because of the low solubility of the latter amino acid. The amounts of serine and folic acid were increased, because the need for larger quanti- ties of these constituents was shown in unpublished experiments. Am- monium chloride was omitted and the remaining constituents (except amino acids) were the same as those give in Medium D, Table I, of Paper XVIII (18).

It was shown in a series of experiments with Lactobacillus casei and Leuconostoc mesenteroides P-60 that dl-phenylalanine had 50 per cent of

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646 AMINO ACIDS, PEPTIDES, AND PROTEINS. XXIII

the activity of I( - )-phenylalanine. The mean deviations from the theo- retical response of dl-phenylalanine were f2.6 per cent at fourteen con- centrations (0 to 30 y) with Leuconostoc mesenteroides and ~3.0 per cent at twelve concentrations (0 to 50 -y) with Lactobadlus casei. The

TABLE II

Results of Assays of Phenylalanine in Amino Acid Test Mixture 2%

Amino acid mix- ‘“t:“b”,’

Y 98.4

196.8 295.2 393.6 492.0

Titration volume of 0.028 N NaOH per tube

Ran& AWE&%

ml. ml.

4.2% 4.47 4.37 6.20- 6.48 G.34 7.89- 8.09 8.01 9.30- 9.79 9.50

10.71-11.09 10.90

Phenylalanine found1 Titration volume of 0.028 N NaOH per tube

Phenylalanine founds

Per tube Per ml. sample

___~ Y Y

2.47 12.35 4.97 12.42 7.34 12.23 9.79 12.24

12.19 12.19

Ranget

ml.

4.30-4.50 5.60-5.78 6.40-6.71 6.98-7.34 7.55-8.00

Average Per tube --

mz. Y

4.43 2.49 5.67 4.87 6.55 7.20 7.17 9.44 7.79 12.00

Per ml.

Y 12.45 12.18 12.00 11.80 12.00

* Test Mixture 2 contained 100 mg. per 100 ml. of each of the amino acids listed in Test Mixture 1 except that the mixture contained 2.44 per cent of I(-)-phenylalanine (as dl-phenylalanine). The basal medium X 2 and 5 days incubation at 35” were employed with Leuconostoc mesenteroides and 3 days incubation at 35” was employed with Lactobacillus casei.

t Six replicate tubes at each level of sample. $ An average of 12.28 y of phenylalanine was found per ml. of amino acid test

mixture. The average mean deviation from the mean at the different levels was 0.49 per cent. The recovery of phenylalanine was 102.1 per cent. The recoveries of phenylalanine in analogous experiments with test mixtures containing lower per- centages of phenylalanine were 104 per cent (0.99 per cent of phenylalanine, basal medium X 2 and 5 days incubation time), 105 per cent (0.50 per cent phenylalanine, basal medium X 2 and 5 days incubation time), and 108 and 112 per cent (0.17 per cent phenylalanine, basal medium X 2 and 5 days incubation time).

$ An average of 12.08 y of phenylalanine was found per ml. of amino acid test mixture. The average mean deviation from the mean at the different levels was 1.5 per cent. The recovery of phenylalanine was 100.5 per cent. The recoveries of phenylalanine in analogous experiments with test mixtures containing lower per- centages of phenylalanine were 102 per cent (0.99 per cent phenylalanine), 106 per cent (0.50 per cent phenylalanine), and 117 and 120 per cent (0.17 per cent phenyl- alanine).

I(-)-phenylalanine employed was prepared by Dr. A. Recsei in this laboratory by resolution of dl-phenylalanine according to the procedure of Fischer and Schoeller (19). Stokes and Gunness (16) reported in 1944 that only the natural antipode of phenylalanine is active in promoting growth of Lactobacillus casei.

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DUNN, SHANKMAN, AND CAMIEN 647

The casein, the silk fibroin, and the hydrolysis procedure were the same as those described earlier (20). The experimental data are given in Tables I to VII.

DISCUSSION

It has been found that phenylalanine could be determined most accur- ately by incubating Leuconostoc mesenteroides P-60 for 5 days at 35” in a

TABLE III

Results of Assays of Phenylalanine in Amino Acid Test Mixture S*

Amino acid mix ture per tube

Y

250.7 501.5 752.2

1003.0 1253.7

Leuconostoc mesenteroides

Titration vol- Phenylalanine foundt ume of 0.111 N

NaOHt Per tube Per ml. SamplC

ml. -/ y

7.19 2.44 ~ 4.88 9.80 4.72 4.72

12.10 7.25 4.83 13.92 9.49 4.75 15.85 12.25 / 4.90

I Lactobacillus casei

Titration vol- umeof 0.111 N

: NaOHt

ml.

9.07 11.25 13.23 15.02 16.39

Phenylalanine found5

Per tube Per ml. sample

Y Y

2.30 4.60 4.79 4.79 7.55 5.03

10.05 5.02 12.40 4.96

* The composition of the test mixture simulating silk fibroin was the same as that given in Paper XIX (21), except that the mixture contained 0.96 per cent of 1(-)- phenylalanine (as dl-phenylalanine). The basal medium X 2 and 4 days incubation at 35” were employed with Leuconostoc mesenteroides and 3 days incubation at 35” was employed with Lactotacillus casei.

i The solutions in the six replicate tubes employed at each level of sample were mixed and the mixture titrated. The average value given per tube at each level of sample was calculated from these data.

$ An average of 4.82 -y of phenylalanine was found per ml. of amino acid test mix- ture. The average mean deviation from the mean at the different levels was 1.3 per cent. The recovery of phenylalanine was 99.5 per cent. The recoveries of phenylalanine in analogous experiments were 103 and 104 per cent (basal medium X 1.3, 4 days incubation), 105 and 111 per cent (basal medium X 1.3, 5 days incuba- tion), 103 and 103 per cent (basal medium X 2, incubation 4 days), and 101 per cent (basal medium X 2, 5 days incubation).

0 An average of 4.85 y of phenylalanine was found per ml. of amino acid test mixture. The average mean deviation from the mean at the different levels was 3.1 per cent. The recovery of phenylalanine was 101.6 per cent in this experiment and 99 per cent in an analogous experiment.

basal medium with total amino acids at twice the level employed previously for the assay of histidine and lysine. Under these conditions the average mean deviations from the mean values (five levels of samples) averaged about 1 per cent, the assay values for phenylalanine in casein agreed closely, and the recoveries of phenylalanine in the amino acid test mixtures and the casein hydrolysates were approximately 100 per cent.

It appears significant that the accuracy with which phenylalanine could

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648 AMINO ACIDS, PEPTIDES, AND PROTEINS. XXIII

be recovered from amino acid test mixtures decreased markedly as the percentage of phenylalanine decreased from 2.44 to 0.50 per cent and that the recoveries were unsatisfactory from such mixtures containing 0.17 per cent of this amino acid.3 Since the accuracy of microbiological assays may vary inversely with the proportion of the amino acid in the sample,

TABLE IV

Results of Assay of Phenylalanine in Casein Ilydrolylsate

Casein per tubf (corrected for moisture and

ash)

Y ml. Y per cm6 ml. Y

46.3 7.00 2.28 4.92 9.20 2.45 92.7 9.61 4.56 4.92 11.39 4.95

139.0 11.84 6.96 5.01 13.12 7.41 185.4 13.56 9.01 4.86 14.50 9.32 231.7 15.20 11.26 4.86 16.00 12.00

Leuco?wstoc meselzteroides

‘itration vol- me of 0.111 h

N’aOW

Phenylalanine found+

Per tube

Average.. -

Casein (corrected for moisture 2nd

ash)

4.91

u

-

T I-

ritration vol- me of 0.0814 I

NaOW

Lactobacillus casei

Phenylalanine foundt

Per tube

-

I

_

/ -

Casein :corrected for moisture and

ash)

ger cent

5.30 5.34 5.32 5.03 5.18

5.23

* The solutions in the six replicate tubes employed at each level of sample were mixed and the mixture titrated. The average value given per tube at each level of sample was calculated from these data.

t The basal medium X 2 and 5 days incubation were employed. The average mean deviation from the mean at the different levels was 0.9 per cent. The following corrected values for phenylalanine were found from comparable assays: 4.94 and 4.96 per cent (basal medium X 1.3, 4 days incubation), 4.88 and 5.04 per cent (basal medium X 1.3, 5 days incubation), 4.97 and 4.99 per cent, (basal medium X 2,4 days incubation), and 4.91 per cent (basal medium X 2, 5 days incubation).

$ The average mean deviation from the mean at, the different levels was 1.9 per cent. The basal medium X 1 and 3 days incubation were employed. The corrected value, 5.02 per cent, was found in a comparable assay.

there may be a limiting accuracy with which a given amino acid may be determined in a particular protein with a particular microorganism.

Phenylalanine in Casein

The percentage of phenylalanine determined under the best conditions (basal medium4 X 2, 5 days incubation of Leuconostoc mesenteroides at

3 Since the phenylalanine content of amino acid Test Mixture 2 (with phenylala- nine omitted) was found by assay to be less than 0.01 per cent, contaminationof these amino acids with phenylalanine could not be a significant factor.

4 The expression “basal medium X 2” signifies that the composition of the basal

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DUNN, SHANKMAN, .4ND CAMIEN 649

35”) was 4.90 (4.89 and 4.91), corrected for the 6.21 per cent moisture and the 0.55 per cent ash reported previously (20). The average corrected value, 4.97 (4.89 to 5.05, range in six determinations) per cent, was ob- tained with Leuconostoc mesenteroides under the other stipulated conditions. The average corrected value found with Lactobacillus casei was 5.1 (5.1, 5.0, and 5.2) per cent.

It seems probable that the true value for phenylalanine in casein is

TABLE V

Recovery of Phmylalanille Added to Casein Hydrolysate*

Casf;&per I(-)-Phenylalanine

(corrected for l( -al&;+~Yl-

moisture and added per ash) tube

(1) (II)

’ Y ‘-

Y Y Y per cent Y 23.2 1.20 1.14 2.41 105.8 1.21 46.3 2.40 2.28 4.68 100.0 2.42 69.5 3.60 3.42 6.99 99.2 3.63 92.6 4.80 4.56 9.34 99.6 4.84

115.8 6.00 5.70 11.45 95.8 6.05 I~

Average............................... 100.1

Y

2.36 4.95 7.27 9.60

12.51 _

-

Recovery,I

(VIII)

ger cent

95.8

105.4 101.1

99.2 107.7

101.8

* The casein hydrolysate sample assayed was prepared to contain 2.40 y of Z(-)- phenylalanine per ml. (added as twice the quantity of dl-phenylalanine) and 46.3 y of hydrolyzed casein (corrected for moisture and ash) per ml. Volumes of this solu- tion varying from 0.5 to 2.5 ml. were taken for the assays. The solutions from the six replicate tubes at each level were mixed and titrated.

i Estimated on the basis of the 4.91 per cent of phenylalanine in casein given in Table IV. The basal medium X 2 and 5 days incubation were employed.

$ Calculated from the relation, ((IV - III)/II) X 100 = per cent recovery. The recoveries of phenylalanine in comparable experiments were 103 per cent (basal medium X 1.3, 4 days incubation), 110 per cent (basal medium X 1.3, 5 days incu- bation), and 100 per cent (basal medium X 2,4 days incubation).

0 Estimated on the basis of the 5.23 per cent of phenylalanine in casein given in Table IV. The unaltered basal medium and 3 days incubation were employed.

11 Calculated from the relation, ((VII - VIj/II) X 100 = per cent recovery.

5.0 f 0.2 per cent. The uncorrected value, 4.77 per cent, has been reported recently by Albanese (22) who determined phenylalanine by the Kapeller- Adler (23) calorimetric procedure after eliminating t,hree interfering amino

medium (Medium D, Table I, described in Paper XVIII (18)) has been modified to contain twice the stated concentrations of amino acids but the same concentrations of all other constituents. The expression “basal medium X 1.3” is employed in this paper in a corresponding sense.

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650 AMINO ACIDS, PEPTIDES, AND PROTEINS. XXIII

acids. Tryptophane was destroyed by acid hydrolysis of the casein, histidine was removed by adsorption on permutit,, and tyrosine was de- stroyed by oxidation with acid KMn04 solution. It was shown that from 100 to 102 per cent of phenylalanine added to a mixture of histidine and tyrosine and to hydrolysates of casein and gelatin was recovered. It is

TABLE VI

Results of Assay of Phenylalanine in Silk Fihroin Hydrolysate

I Le¶Konosloc mesente7oides

Silk fibroin per tube (corrected

Phenylalanine found1

formoisture Titration vol- and ash) ume of 0.111 N

NaOH* Per tube i Silkfibroin

corrected for noisture and

ash)

Y ml. Y

118.8 6.04 1.54 237.5 7.87 3.05 356.3 9.49 4.45 475.1 11.05 6.09 593.9 12.48 7.68

Average..........................

-

. -

7 u Xtrationvol- ,me of 0.111 N

NaOH Per tube

per cent ml. Y

1.30 8.32 1.54 1.28 10.10 3.45 1.25 11.49 5.10 1.28 12.69 6.80 1.29 14.00 8.61

1.28

Lactobacillus casei

T Phenylalanine foundf

Silk fib&n corrected for noisture and

ash)

per cent

1.30 1.45 1.43 1.43 1.45

1.41

* The solutions from the six replicate tubes at each level of sample were mixed and titrated.

t The basal medium X 2 and 5 days incubation were employed. The average mean deviation from the mean at the different levels was 1.0 per cent. The following corrected values for phenylalanine were found from comparable assays: 1.29 per cent (basal medium X 1.3, 4 days incubation), 1.37 per cent (basal medium X 1.3, 5 days incubation), and 1.28 per cent (basal medium X 2,4 days incubation).

$ The average mean deviation from the mean at the different levels was 3.3 per cent. The basal medium X 1 and 3 days incubation were employed.

of interest that Kapeller-Adler (23) found values from 4.5 to 5.3 per cent (presumably uncorrected) for phenylalanine in casein essentially by the same procedure except that histidine was removed as its phosphotungstate. Kuhn and Desnuelle (24) determined phenylalanine by the Kapeller- Adler method in an acid hydrolysate of Hammarsten casein (15.66 per cent nitrogen after drying 24 hours at SO”) from which the basic and di- carboxylic amino acids had been removed by electrodialysis. Although only 4.1 per cent of phenylalanine was found, about 17 per cent of the nitrogen in the casein sample was lost through formation of humin, con- version to ammonia, and adsorption on the BaS04 precipitates.

It is difficult to understand why the values (Table VIII) for phenylalanine

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DUNN, SHANKMAN, AND CAMIEN 651

determined by calorimetric analysis of casein hydrolyzed with NaOH, HCI, HzS04, HI, and mixtures of some of these acids should be so divergent and why the values for phenylalanine in casein hydrolyzed for different times with a given acid or base should fluct’uate so widely. Although Schein and Berg (38) found that “neither destruction nor racemization

TABLE VII

Recovery of Phenylalanine Added to Silk Fibroin Hydrolysate*

Silk fibroin per tube

(corrected for moisture and

ash)

(1)

Y

59.4

118.8 178.2 237.5 297.0

I I(-)-Phenylalanine T-

1 added ner I tube In silk fibroin

per tubet (11) (III)

Y Y

1.20 0.76 2.40 1.52 3.60 2.28 4.30 3.04 6.00 3.80

Average

Y 1.91 3.76 5.64 7.63 9.46

Recoveryt

(V)

per cent

95.8 93.3 93.3 95.6 94.3

94.5

f

.-

-

In silk lbroin per

tube5 F”“t”uiLper

(VI) (VII)

T

___~

Y Y

0.84 2.13 1.67 4.30 2.51 6.80 3.35 8.57 4.19 10.80

~-

Recovery;l

(VIII)

per cent

107.5 109.6 119.2 108.8 110.2

111.1

* The silk fibroin hydrolysate sample assayed was prepared to contain 2.40 y of I(-)-phenylalanine per ml. (added as twice the quantity of dl-phenylalanine) and 118.8 y of hydrolyzed silk fibroin (corrected for moisture and ash) per ml. Vol- umes of this solution varying from 0.5 to 2.5 ml. were taken for the assays. The solutions from the six replicate tubes at each level were mixed and titrated.

t Estimated on the basis of the 1.28 per cent of phenylalanine in silk fibroin given in Table VI. The basal medium X 2 and 5 days incubation were employed.

3 Calculated from the relation, ((IV - III)/II) X 100 = per cent recovery. The recoveries of phenylalanine in comparable experiments were 97.1 per cent (basal medium X 1.3, incubation time4 days), 96.2 per cent (basal medium X 1.3, incubation time 5 days), and 99.6 per cent (basal medium X 2, incubation time 4 days).

$ Estimated on the basis of the 1.41 per cent of phenylalanine in silk fibroin given in Table VI. The unaltered basal medium and 3 days incubation were employed.

/j Calculated from the relation, ((VII - VI)/II) X 100 = per cent recovery.

of the basic amino acids occurs until the minimum t,ime required [18 hours at 120”] for the acid [8 N HzS04] hydrolysis of casein has been greatly exceeded,” it may be that phenylalanine behaves in an entirely different manner under these conditions. It is recognized that amino acids differ markedly in stability towards acids and bases and that additional studies should be made of phenylalanine and other amino acids under different experimental conditions. This problem is under investigation in the authors’ laboratory.

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652 AMINO ACIDS, PEPTIDES, AND PROTEINS. XXIII

TABLE VIII

Percentage of Phenylalanine in Casein (Literature VaZues)*

Source of casein sample

Harris.. . . . . Labco, . . .

“ hydrolyzed Difco “ Source not given..

“ “ “ . “ “ “

“ “ “ .

“ “ “ “ “ “ “ “ “

“ “ ‘I .

L‘ “ “

Labco, vitamin- free.

Authors’ prepara- tion**.

Hammarsten, fat- free.

H. B. Vickery..

-

t

-

.

Hydrolysisprocedure

5 N NaOH for 5 hrs. at 125” Same

“ “ “

8 N HZSO, for 2 hrs. at 125“ 20% HCl-HCOOH for 2

hrs. at 125” Concentrated HI for 2 hrs.

at 125” 20% HCl for 2 hrs. at 125” 5 N NaOH for 2 hrs. at 125” 207, HCI for 2,4,8,18 hrs.

5 N NaOH for 2,4,6,18 hrs.

7 “H&O 4 “ 2,4,8,18 “

5 “ XaOH “ 24 hrs.

250/, HZSO., “ 20 “

H&34

6 N HCl for 24 hrs.

-

_ _

K-A$ I‘ 1 iL : “ i

+ “ + + (‘ : ‘L $

“ :

“ ‘C i “ II

“ I/

“ II

“ 1:

“ $

Benzoic acid$$

Microbio- logical§j

-

-

Phenylalsnine

- kf- er- rice x0. -

per cent 6.3s 4.88 5.6,s 6.25 4.3,s 4.65 5.8 3.9 4.9

31 31 31 31 32 33 33

4.3

5.0 5.5 5.03,4.39,4.81,

3.93 4.97,4.73,5.71,

5.52 3.89,5.69,5.46,

4.71

33

33 33 34

34

34

3.997 35

5.5tt 36

2.68,3.04,3.2,3.4 37

3.7Ojj i[ 15

- -

* In 1919, Foreman (25) isolated 3.9 per cent of phenylalanine as the hydrochloride from an ester fraction of a sulfuric acid hydrolysate of casein. Lower percentages of phenylalanine were isolated earlier by Fischer (26), Fischer and Abderhalden (27), and Osborne and Guest (28) essentially by the same method. Boyd (29) isolated 2.2 per cent of phenylalanine as its hydantoin and Baptist and Robson (30) isolated 0.3 per cent of phenylalanine as its picrolonate.

t No physical or chemical characteristics of the casein samples were reported by the authors quoted other than those described in the foot-notes to this table.

f The Kappeler-Adler (23) calorimetric procedure. § Calculated for casein containing 16 per cent nitrogen. // Modified Kappeler-Adler calorimetric procedure. 7 Average of several determinations (no values listed) corrected for moisture and

ash. Tryptophane was removed as its HgSOa complex and phenylalanine deter- mined in the mercury-free filtrate. Recovery of phenylalanine added to a mixture containing ten amino acids was 102.5 per cent.

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DUNN, SHANKMAN, AND CAMIEN 653

TABLE VIII-Concluded

** Precipitated from milk at the isoelectric point of casein. tt Corrected for moisture and ash. $1 Gravimetric determination of benzoic acid formed by oxidation of casein with

potassium dichromate. $I Lactobacillus arabinosus 17-5. /j/j Recovery of phenylalanine added t’o casein 92.5 (74 to 110) per cent.

Phenylalanine in Silk Fibroin

On the whole the assays of phenylalanine in silk fibroin hydrolysates and the recoveries of this amino acid from these preparations were some- what less accurate than those obtained with casein. These results were not unexpected in view of the relatively low percentage of phenylalanine and the unusual distribution of amino acids in this protein.

The phenylalanine content of silk fibroin determined under the most satisfactory experimental conditions (assay with Leuconostoc mesenteroides incubated 5 days at 35” with the amino acids X 2 in the basal medium) was found to be 1.28 per cent, corrected for the 5.68 per cent moisture and the 0.25 per cent ash reported previously (20). The average corrected value, 1.31 (1.29, 1.37, and 1.28) per cent, was obtained with Leuconostoc mesenteroides under the other stipulated conditions. The corrected value, 1.41 per cent, was obtained with Lactobacillus casei. Abderhalden and coworkers (39-47) found that silk fibroin prepared from silk of different sources contained from 0.6 to 1.6 per cent of phenylalanine and other investigators (48-51) have reported values ranging from 0.25 to 1.3 per cent..

SUMMARY

Microbiological procedures have been described for the det,ermination of phenylalanine in protein hydrolysates with two microorganisms, Leu- conostoc mesenteroides P-60 and Lactobacillus casei. Closely agreeing values for the percentage of phenylalanine in casein and in silk fibroin were found by assays with these organisms. The corrected percentages of phenylalanine in casein and silk fibroin, respectively, were found to be 4.91 and 1.28 per cent under the conditions considered to be most reliable (assay with Leuconostoc mesenteroides incubated for 5 days at 35” in a basal medium identical with that reported previously (18) for the determination of lysine and histidine except that the concentration of total amino acids was doubled).

It is considered that the probable true value for phenylalanine in casein is 5.0 f 0.2 per cent, since data of high accuracy were obtained in the recovery of phenylalanine from three amino acid test mixtures of different composition and from casein hydrolysates. This figure is in approximate

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654 -4MINO SCIDS, PEPTIDES, AND PROTEINS. XXIII

agreement with the values obtained by investigators who employed what appeared to be dependable modifications of the Kapeller-Adler colori- metric procedure. It is recognized, however, that the extent to which phenylalanine is destroyed or alt,ered during hydrolysis of casein must be measured before the true value for phenylalanine in this protein can be determined with certainty.

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CamienMax S. Dunn, S. Shankman and Merrill N.

AND LACTOBACILLUS CASEIP-60LEUCONOSTOC MESENTEROIDES

HYDROLYSATES WITHPHENYLALANINE IN PROTEIN

DETERMINATION OFPEPTIDES, AND PROTEINS: XXIII. THE INVESTIGATIONS OF AMINO ACIDS,

1945, 161:643-655.J. Biol. Chem. 

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