QUANTITATIVE INVESTIGATIONS OF AMINO ACIDS AND PEPTIDES

IX. SOME PHYSICAL PROPERTIES OF I(-)-HISTIDINE*

BY MAX S. DUNN, EDWARD H. FRIEDEN, M. PALMER STODDARD,axn HAROLD V. BROWN?

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

(Received for publication, March 23, 1942)

The investigation of some physical properties of amino acids, undertaken in the authors’ laboratory, has been extended in the present report to Z( - )- histidine. The solubility and specific rotations of I( - )-histidine were es- tablished by methods which differ somewhat from those employed pre- viously T;iith I( - )-leucine (1). Studies were made of the variation of specific rotation with temperature, concentration of solute, and character qf the solvent.

Preparation of PuriJied Natural 1( - )-Histidine

100 gm. of commercial I( - )-histidine monohydrochloride monohydratel were dissolved in 125 ml. of boiling water and 85 ml. of concentrated am- monium hydroxide were added to the solution. Crystallization of the histidine began as the solution cooled. When the temperature of the solution reached 50”, 100 ml. of 96 per cent ethanol were added and the mixture was allowed to stand overnight in the refrigerator. The sus- pension was filtered and the crystals were washed free of chloride with 96 per cent ethanol. The histidine, recrystallized from water with the aid of ethanol and dried overnight in air, contained less than 0.05 per cent moisture determined by drying the product to constant weight in a partial vacuum at 75”.

Purity of Purijied Natural I( -)-Histidine

Nitrogen Analysis-The nitrogen values were low and inconsistent when determined by a conventional Kjeldahl method. Vickery2 has shown that

* Aided by grants from the University of California, the Rockefeller Foundation, and Merck and Company, Inc. For t,he preceding paper in this series, see Stoddard and Dunn (1). Some of t.he material in t.his paper is taken from a thesis submitted by Edward H. Frieden in partial fulfilment of the requirements for the degree of Master of Arts in the Graduate School of the Universit,y of California, I,os Angclcs. The authors are indebted to Dr. C. D. Coryell for helpful suggestions.

t Present address, Fitzsimmons Hospital, Denver, Colorado. 1 Purchased from A. D. Mackay, New York. 2 Private communication from Dr. H. B. Vickery, Connecticut Agricultural Ex-

periment Station, New Haven, Connecticut, November, 1941.

487

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

488 AMINO ACIDS AND PEPTIDES. IX

nitrogen in histidine may be determined quantitatively by Kjeldahl analysis if a mercury catalyst and an 8 hour digestion period are employed. This information was not available at the time of the authors’ experiments.

Equivalent Weight-The perchloric acid titration procedure of Harris (2)) as modified by Toennies and Callan (3), was first used. Glacial acetic acid was purified by fractional distillation and crystallization of the commercial 99 per cent product. The fraction boiling at 116-117” was recrystallized until the freezing point was 16.5”, the density at 20” was 1.0497, and the equivalent weight was 100.4 per cent of the theoretical value. Acetic acid solutions of perchloric acid were prepared by adding to 65 per cent aqueous perchloric acid solution a quantity of redistilled acetic anhydride exactly equivalent to the water present and diluting the mixture with purified glacial acetic acid. The end-point of the perchloric acid titration of amino acids with crystal-violet indicator is unsatisfactory ynless the water content of the acetic acid is less than 0.2 per cent. Under anhydrous conditions the color changes from dark green to light green and thence to yellow are sharp and reproducible with high precision.

The 0.2 N perchloric’ acid-acetic acid solutions were standardized against analytically pure glycine.3 The precision and accuracy of the method were found to be 100.6 f 0.3 per cent in analyses of analytically pure dl-serine,J dl-phenylalanine,5 and dl-valine.6 All volumetric apparatus was calibrated for acetic acid delivery.

In the perchloric acid titration of histidine, which has not been applied previously to the analysis of this amino acid, it is necessary to add an excess of the perchloric acid and titrate back with standard glycine solution. This procedure, described by Toennies and Callan (3), is necessary, since histi- dine forms a monoperchlorate and diperchlorate, the former being only slightly soluble in glacial acetic acid. It was found that histidine may be analyzed with fair precision and accuracy by the perchloric acid back titra- tion technique. The purity of the purified I( - )-histidine was shown to be 100.13 per cent (average of six determinations with a probable error of the mean of 0.14) by the analysis of six samples ranging in weight from 0.0911 to 0.1418 gm.

The behavior of the basic amino acids in the form01 titration has been investigated by Levy (4) who concluded that values about 7 per cent high are obtained with histidine under optimum conditions. The present authors’ finding that the location and sharpness of the end-point vary with the concentration of formaldehyde is in agreement with the observation of

3 Amino Acid Manufactures, Lot No. 8, A. P. grade. 4 Amino Acid Manufactures, Lot No. 6, A. P. grade. 5 Amino Acid Manufactures, Lot No. 12, A. P. grade. 6 Amino Acid Manufactures, Lot No. 9, A. P. grade.

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

DUNN, FRIEDEN, STODDARD, AND BROWN 489

Levy. The maximum sensitivity (maximum value of AE/AV’ at the cnd- point of the Ctration) occurs at 0.3 M formaldehyde concentration. Under these conditions the apparent equivalent weight of histidine is 100.9 f 0.1 per cent of the theoretical value. Adjustment of the pH of the amino acid solution to any given value is unnecessary in the titration.

Criteria of Optical Purity-The solubility of Z( - )-histidine in water was measured with large and small excesses of solute. Under these conditions, the presence of d(+)-histidine, dl-histidine, or other amino acids would be revealed by significant differences in the solubility values provided that mixed crystals were not formed.

Samples of the purified I( - )-histidine which were 7 per cent and 150 per cent in excess of the accepted solubility* (4.29 gm. per 100 gm. of water at 25” (5))were placed in separate oil sample bottles. 40 ml. of distilled water were added to each bottle, the solutions were warmed to 50”, and the bottles were tightly stoppered and rotated in a thermostat at 25.10” f 0.05”. At intervals, samples of each solution were drawn from the bottles by means of a 5 ml. pipette equipped with a cotton filter. Each solution was trans- ferred to a weighed Petri dish and evaporated to constant weight at 95”. It was assumed that equilibrium had been attained when the weight of resid- ual solid in successive 5 ml. volumes was constant. The solubility values found, 4.091 and 4.104 gm. per 100 gm. of solution, differ by only 0.34 per cent.

In a second series of determinations solubility was measured by form01 titration with the glass electrode. In calculation of the solubility values from the experimental data account was taken of the observation, referred to previously in this report, that the apparent weight of histidine de- termined by form01 titration is 100.9 per cent of the theoretical value. The corrected solubility values found with small and large excesses of solute, 4.115 and 4.127 gm. per 100 gm. of solution, differ by only 0.29 per cent. The average of the solubility values determined by the gravimetric and titrimetric methods, 4.098 and 4.121 gm. per 100 gm. of solution, differ by only 0.56 per cent.

In another method employed to establish the degree of purity of the purified I(-)-histidine, advantage was taken of the effect which any im- purity would have on the specific rotation of a given sample of the histidine. The specific rotation of a sample (A) of the purified I( - )-histidine was de- termined at 25”. 3 gm. of this material were shaken at 35-40” with 25 ml. of distilled water and the suspension was rotated for 4 days in a thermo- stat at 25’. The suspension was filtered and the precipitate was washed

7 The change in voltage per unit change in volume of base. 8 The value, 4.19 gm. per 100 gm. of water at 25’, given by Dunn et al. (5), is in

error.

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

490 AMINO ACIDS AND PEPTIDES. IX

with ethanol and dried in air. The specific rotation of this material (Sample B) was measured at 25”. The specific rotation of Sample A was -38.87” (t = 25.0”, c = 2.670, 1 = 4.000 dm., X = 5893 A., a! = -4.151”) and that of Sample B was -38.78” (t = 25.0”, c = 2.671, 1 = 4.000 dm., X = 5893 A., a = -4.143”). The specific rotation values of the two samples differ by only 0.23 per cent.

The extent to which impurities, other than amino acids which have es- sentially the same rotation in water as I( -)-histidine, may be detected by this procedure is indicated by the following experiment with a mixture con- taining 99.6 per cent I(-)-histidine and 0.4 per cent dl-histidine. The latter substance was prepared by racemizing Z(-)-histidine according to the method of du Vigneaud and Hunt (6). A purified suspension of un- dissolved material was obtained as described in the preceding experiment. The specific rotation of the original mixture was -38.45” (t = 25.0”, c = 2.670,l = 4.000 dm., X = 5893 Ai., LY = -4.106”) and that of the puri- fied sr+spension was -38.72” (t = 25.0”, c = 21672, 1 = 4.000 dm., X = 5893 A., a! = - 4.138”). These rotations differ by 0.69 per cent. It may be concluded from this experiment that I( - )-histidine and dl-histidine prob- ably do not form mixed crystals and that the presence of 0.4 per cent, and probably a smaller percentage, of dl-histidine in Z( - )-histidine may be de- tected by the polarimetric method.

Variation of Specific Rotation of I( -)-Histidine with Temperature, Concen- tration of Solute, and Character of Solvent

A 0.01” Schmidt and Haensch polarimeter, a 4.000 dm. water-jacketed polarimeter tube, and a General Electric sodium vapor lamp were used to determine optical rotations. pH measurements were made with a Beck- man pH meter. It was found by qualitative spectroscopic analysis that practically all of the light from the sodium lamp was emitted at 5893 A., with one faint line in the green and one in the red. The temperature of the water in the jacket of the polarimeter tube ranging from O-80” was regulated within 0.05-0.5” by circulating water through an ice and water bath, a ther- mostat, or a hollow porcelain wire-wound rheostat. Fogging of the end plates of the polarimeter tube, which occurred when water near 0” was cir- culated through the water jacket, was prevented by directing a jet of dry air against each end plate. Temperatures were measured with three ther- mometers of suitable ranges, calibrated against a Bureau of Standards thermometer.

Carbon dioxide-free aqueous solutions of I( - )-histidine were prepared by successive dilutions of a standard solution. Absolute ethanol was pre- pared by a standard procedure and its water content was determined by the paraffin oil method (7). Methanol, acetone, and dioxane were purified by

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

DUNN, FRIEDEN, STODDARD, AND BROWN 491

fractional distillation and their water content estimated from their densi- ties. None of these solvents contained more than 0.1 per cent water. In preparing the solutions used in the polarimetric studies, the solvent was transferred to a volumetric flask by means of a burette or a weight pipette,

TABLE I

SpeciJic Rotation in Water of I(-)-Histidine As Function of Concentration of Solute

Temperature a (observed optical rotation)

m. “C.

3.770 24.90 2.290 25.00 1.882 24.97 1.128 25.00 0.941 25.00 0.752 25.02

degrees

-5.904 -3.647 -2.941 -1.714 -1.465 -1.169

degrees

-39.20 -38.80 -39.05 -39.01 -38.85 -38.80

3.826 0.47 -6.573 -42.90 3.065 0.47 -5.360 -43.70 2.296 0.41 -3.967 -43.20 1.913 0.47 -3.281 -43.00 1.529 0.49 -2.617 -42.75 1.147 0.49 -1.966 -42.90 0.765 0.42 -1.308 -42.90 0.383 0.46 -0.641 -41.85

TABLE II

Specific Rotation in Wa.ter of Z(-)-Histidine As Function of Temperature

p (solute per 100 gm. solution)

c (solute per 100 ml. solution)

Density of solution

gm. gm.

0.551 0.552 0.551 0.551 0.551 0.551 0.551 0.549 0.551 0.547 0.551 0.542 0.550 0.537

1.002 1.000 1.000 0.996 0.992 0.984 0.976

Temperature r (observed opti- cal rotation) Cl:,

“C. degrees degrees

18.4 -0.902 -40.8 22.9 -0.881 -39.9 23.4 -0.861 -39.0 35.7 -0.827 -37.7 44.2 -0.799 -36.5 63.9 -0.768 -35.4 78.8 -0.761 -35.4

-

the required volume of standard histidine solution was added, and the flask was filled to the mark with carbon dioxide-free water.

The experimental data are shown in Tables I to VII.

DISCUSSION

It would appear that the authors’ purified Z(-)-histidinc contains less than 0.5 per cent of amino acids and other impurities and that the physicai

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

492 AMINO ACIDS AND PEPTIDES. IX

data reported for this amino acid are accurate within a small percentage w probable error. The solubility of I(-)-histidine at 25.10” f 0.05” was shown to be 4.110 f 0.012 gm. per 100 gm. of aqueous solution and 4.286 f 0.013 gm. per 100 gm. of water. No solubility data with which to com- pare these figures could be found in the literature.

The average specific rotations of E(-)-histidine in water (Table I) we’ shown to be -38.95” (0.06”, probable error of the mean) at 25.00” f O.(i and -43.05” (O.OS”, probable error of the mean) at 0.46“ f 0.03”. Tl,t

TABLE III

SpeciJic Rotation Data for I(-)-Hi&dine in ‘Water at 95” *

m.

2.08 3.234 0.775 3.55 2.30 2.22 2.000

0.752-3.770

- 1

am. 0.5 2.0 2.0 1.0 2.0 0.5 4.001 4.000

- “C.

20 20 20 20. 26 20 24.0 25.0 1 -1.169 to -5.904

-

a (observed o tical rotation P

- degrees

-0.40 -2.57

-1.40 -1.75 -0.44 -3.121

degrees

-38.46 -39.27 -39.3 -39.44 -38.1 -39.65 -39.01 -38.95

- [mp .O D

degrees

-37.32 -38.12 -38.16 -38.30 -38.33 -38.51 -38.81

Bibliographi reference N

15 16 17 15 18 15

t -38.95 / This paper

* The specific rotation, [oL]: = -40.70” (c = 3.898, 1 = 1, Ly= - 1.45”), was re- ported by Bergmann and Zervas (23) in 1928. This paper was not found until afte, the present manuscript was in the proof form. It is apparent that the I (-) -histidine used by these investigators was of high purity and that the specific rotation, -40.34” (corrected to 25”), is higher than the value -38.95” found by the present author. It may be assumed, however, that the specific rotation of Bergmann and Zervas histidine may have been as low as -38.97” (at 25”) if the maximum probable error in the observed rotation, -1.45”, were 0.05”. It is of interest, also, that an [cY]~ = -39.74” (1 = 6, c = 3.183, LY. = -7.59”) for I(-)-histidine was reported by Kossel and Kutscher (24) in 1899. The reliability of this value is uncertain, however, since neither the chemical purity of the Z(-)-histidine sample nor the temperature at which the observed rotation was measured was stated.

t Dunn, M. S., and Stoddard, M. P., unpublished results.

most reliable data in the literature for the specific rotation of I( - )-histidine in water are given in Table III. The specific rotations reported by these investigators were corrected to 25” with the aid of the temperature co- efficients, given in Table VI, which were derived from the data recorded in Table II.

It may be observed that, in every case, the values reported by the authors cited are smaller than that found in the present study. It is of further in- terest that (with the exception of the first value, -37.32”) -38.45” is the

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

DUNN, FRIEDEN, STODDAHD, AND BROWN 493

iAverage of all of the listed specific rotations. It is evident, therefore, that the accuracy of all of these values is relatively high and that I( - )-histidine of high purity is readily prepared by the fractional crystallization pro- cedures commonly employed in the isolation of this amino acid from protein hydrolysates. is, It may be shown that the observed optical rotations given in Tables I, +#, and V of I( - )-h is i t d ine in water, 42 weight per cent ethanol, and 6 N

&ydrochloric acid fall on, or very close to, straight lines relating cx and gm. of

TABLE IV

Specific Rotations of I(-)-Histidins in Aqueous Solutions of Organic Solvents

Ethanol

Methanol

Acetone

Dioxane

p (solute per 100 gm.

solution)

gm. A-m.

1.004 0.920 0.803 0.736 0.610 0.551 0.403 0.368 0.813 0.750 0.608 0.561 0.403 0.374 0.573 0.551 0.578 0.561 0.574 0.557 0.572 0.553 0.572 0.551 0.572 0.551 0.572 0.547 0.572 0.545 0.573 0.540 0.594 0.551 0.594 0.558 0.540 0.551 0.540 0.555

c (solute per 100 ml.

solution)

weight per cen1

42.3 42.1 42.1 42.2 42.1 42.1 42.2 20.0 20.0 24.2 24.2 24.2 24.2 24.2 24.2 24.2 41.9 41.9 28.4 28.4

Density of solution

Temper- ature

“C.

0.916 25.00 0.916 25.00 0.904 25.00 0.913 25.00 0.923 0.80 0.923 0.80 0.928 0.80 0.962 25.05 0.971 1.00 0.970 0.7 0.967 17.8 0.963 25.0 0.963 25.3 0.957 39.7 0.953 57.1 0.943 70.9 0.928 25.0 0.940 0.85 1.020 25.0 1.028 0.75

I (observed optical

rotation) Ial;

-1.485 -40.35 -1.200 -40.70 -0.904 -41 .oo -0.601 -40.85 -1.300 -43.40 -0.980 -43.60 -0.653 -43.65 -0.860 -39.00 -0.955 -42.50 -1.011 -45.45 -0.949 -42.45 -0.923 -41.85 -0.912 -41.40 -0.861 -39.32 -0.837 -38.35 -0.824 -38.10 -0.968 -43.92 -1.093 -49.00 -1.155 -52.45 -1.259 -56.60

solute per 100 ml. of solution in plots of these data. It may be concluded from this observation, as well as by an inspection of the data in these tables, that the specific rotation of I( - )-histidine does not vary materially with different concentrations of solute under the stipulated conditions.

It has been reported that the specific rotations of other amino acids are similarly unaffected by changes in concentration of solute. The amino acids investigated include Z( - )-cystinc in orthophosphoric acid, trichloro- acetic acid, and 0.5 to 2.5 M hydrochloric acid (Andrews (S)), Z( +)-arginine in water at the isoelectric pH, hydrochloric acid, and sodium hydroxide

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

TABLE V

Specific Rotations of I(-)-Histidine in Acid Solutions

Solvent

6.08 N Hcl 6.08 “ “ 6.08 “ ‘I

6.08 ” “ 6.08 ‘[ “ 6.08 ‘( “ 6.08 ‘( Ii 6.08 ii IL 0.63 “ “ 0.239 (‘ “ 0.095 L( (‘ 0.0359 (( “ 0.0136 “ “

1.026 ” Ii 0.592 I( (( 0.335 ci [‘ 0.143 “ ‘( 0.0548 “ ”

1.026 ” ” 0.532 “ ‘( 0.344 “ (‘ 0.272 ii ” 0.245 “ ”

0.097 (‘ (‘ 0.140 (( (( 0.324 ” “ 1.003 (( Lc 4.51 (I ‘( 6.08 (( (( 8.0 ” ”

10.0 (( ‘( 0.594 M HSO 2.14 ” ” 4.63 “ ” 9.04 (‘ “

14.40 Ii (‘

4

~__ m. gm.

1.00641.0986 0.9160 1.525 1.1000 1.386 4.05331.1059 3.6653

1.540 1.1108 1.386 1.531 1.1044 1.386 1.525 1.1000 1.386 1.519 1.0956 1.386 1.512 1.0912 1.386 9.726 1.0309 9.4345 3.705 1.0105 3.6659 1.475 1.0021 1.4718 0.55710.9992 0.5574 0.21040.9981 0.2108

2.367 1.058911.678 7.12711.0326 6.9018 4.032 1.0271 3.9257 1.71841.0060 1.7082 0.65621.0007 0.6558

2.367 1.058911.678 4.69131.0226 4.5878 1.78731.0096 1.7704 0.6658 1.0159 0.6383c 0.25261.0027 0.25191

1.50 I I 1.50 1.50 1.63951.0203 1.6069 1.50331.0760 1.3971 1.525 1.1000 1.386 1.50031.1337 1.3234 1.53131.1719 1.3066 1.5126 1.0391 1.456 1.606 1.1324 1.418 1.492 1.2650 1.179 1.539 1.4885 1.034 1.496 1.7338 0.8628

a

h

i

Moles t, (I! (ob- .cid er

P tem- served k4;

moe pera- istidine ture

0;;;:1 ‘s,po;‘ic Series

tion) tion) - --

“C. degrees degrees

93.1 24.8 $0.537 +13.34 A (c varied at 61.0 24.8+0.815+13.36 constant 22.6 24.8+2.159+13.32 temperature

and acid concentra-

tion) 61.0 0.5+0.547 +8.88 B (t varied at 61.0 15.0+0.709+11.58 constant p 61.0 24.8+0.815+13.36 and acid 61.0 35.1 +0.923 +15.20 concentra- 61.0 45.1 f1.016 f16.80 tion)

1.00024.8+0.282 +0.73 C1 (cvariedat 1.00024.8+0.287 $2.39 constant 1.00024.8+0.150 +2.54 temperature 1.00024.8+0.065 +2.92 and molal 1.00024.8 f0.021 +3 ratio of acid

to histidine) 1.29 24.8 +I.199 $2.42 CZ (c varied at 1.29 24.8+1.023 $3.59 constant 1.29 24.8f0.718 +4.45 temperature 1.29 24.8 +0.369 +5.37 and molal 1.29 24.8+0.145 +5.52 ratio of acid

to histidine) 1.29 24.8f1.199 +2.42 D (c and acid 1.76 24.8+1.600 +8.53 concentra- 2.99 24.8+0.852+11.92 tion varied 6.34 24.8 f0.342 +12.83 at constant

15.0 24.8+0.126+12.44 tempera- ture)

1.00 24.8-tO.163 +2.71 E* (acid con- 1.29 24.8 +0.319 +5.32 centration 3.13 24.8+0.721+12.02 varied at 9.37 24.8 f0.834 +12.72 constant c

46.0 24.8+0.815+13.55 and temper- 61.0 24.8 +0.815 f13.36 ature) 80 24.8+0.661+11.01 DO 24.8f0.432 +7.05

6.04 24.8f0.838f13.85 F (acid con- 20.4 24.8+1.016+15.82 centration 47.6 24.8+1.063+17.81 varied at DO.1 24.8 +0.9s2 +15.95 constant c 48 24.8+0.890+14.87 and temper-

j 1 1 1 atwe)

494

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

DTJKN, FRIEDEX, STODDARD, AND BROWN 495

TABLE V-Concluded

* The values +2.71, +5.32, and +12.02 and their dependent data were derived by interpolation from curves relating the figures given in Series Cl, CL, and D.

(Miller and Andrews (9)), I(+)-glutamic acid in hydrochloric acid (Pertzoff (lo)), and Z( +)-lysine in hydrochloric acid (Lawrow (11)). On the other hand, Pertzoff (10) found that the specific rotation of I( +)-glutamic acid in an alkaline medium is linear in respect to the concentration of the amino acid, while that of Z(+)-aspartic acid in both acid and alkaline media is linear with respect to the square root of the concentration of the amino acid. This dissimilar behavior of amino acids which resemble each other so closely in other respects is unexpected.

By comparable investigations with solutes other than amino acids, it has been shown that the specific rotations of certain optically active sugars, esters,‘and alcohols exhibit definite concentration effects, especially at high concentrations of solute. This phenomenon is illustrated by the ob- servation of Clough (12) that there is a change of several hundred per cent in the specific rotation of Z-methyl lactate over a concentration range of solute of 5 to 100 per cent.

The specific rotations at 25” of I( -)-histidine in 0.01 to 1 N hydrochloric acid solutions containing acid and histidine in a molal ratio of about 1: 1 vary with respect to the concentration of the amino acid. It may be shown, however, by curves derived from plots of the data given in Table V, Series Ci and Cz, that this relationship is not linear. These concentration effects must necessarily be explained on some basis other than ionic dis- sociation, since there is negligible change in the proportion of the dicationic, monocationic, and zwitter ionic species. From a consideration of the acidic dissociation constants, pK1 = 1.77 and pK2 = 6.10, of histidine at 25” and the curve (derived from the data in Table VII) relating the concen- tration of histidine monohydrochloride and pH of its aqueous solution, it may be calculated that the percentage distribution of total histidine among the three ionic species is 98.8, 0.6, and 0.6 in 0.626 M Z( -)-histidine mono- hydrochloride and 98.6,0.4, and 1 .O in 0.0645 M Z( -)-histidine monohydro- chloride for the monocation, the dication, and the zwitter ion, respectively.

It is noteworthy that the observed concentration effects of Z( - )-histidine occur only in dilute hydrochloric acid solutions of widely varying ionic strength. In water, histidine exists primarily as the zwitter ion which con- tributes little to the ionic strength of the solution, while, in concentrated acid solutions, histidine is present almost entirely as the dication whose contribution to the ionic strength of the solution is masked by that of the acid. Further experiments on the influence of inorganic salts on the specific rotations of amino acids, suggested by these conclusions, are to be under-

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Amiio

ac

id

Z( -)

-Asp

arag

inei I‘ L‘

I( +)

-A

spar

tic

acid

I‘

“ “

‘I I(

I‘ I‘

“ ‘I

‘I ‘C

“

‘I ‘I

‘I I‘

I(-)-C

ystin

e ‘I

Z(f)-

Glut

amic

acid

“

‘I ‘I

‘l “

‘I

I(-)-H

istidi

ne ‘I “ ‘I

TABL

E VI

InjZ

uenc

e of

Te

mpe

ratu

re

on

[cY]~

of

I(-)-H

istidi

ne

and

Som

e Ot

her

Amino

Ac

ids*

Solve

nt

N HC

l W

ater

I‘

6.0

N HC

l 1

N HC

l W

ater

I‘ I‘

0.2

N Na

OH

1 N

NaOH

1

“ (I

2 cl

ii

1.02

N

HCl

1.00

“

“ 6.

0 “

“ 1

N HC

l W

ater

‘I

6.08

N

HCl

0.09

6 “

“ $

Wat

er

Acet

one

(41.

9 we

ight

%)

p (s

olute

pe

r lo

o

sol%

n)

Tem

pera

ture

range

(A

)

m.

“C.

8.942

14

-4

6 1.

961

34

-60

7.40

7 38

-6

8 1.

820

0.7

-45.

0 7.

984

12

44

0.99

0 14

-4

8 1.

872

32

-90

1.96

1 48

-7

8 2.

584

18

-46

6.00

17

-5

4 1.

30

12

40

9.76

12

-5

8 0.

9797

20

.54-

30.2

5 1

20

-29

1.81

5 0.

7 -4

5.0

8.75

2 13

-4

3 0.

99

15

-60

2.91

44

-7

5 1.

386

0.5

-45.

1 1.

481

0 -4

5 0.

551

18.4

-7

8.8

0.59

4 0.

89-2

5.0

(4

-0.1

25

-0.1

46

-0.1

37

-0.0

77

-0.1

03

-0.1

12

-0.0

97

-0.1

27

-0.1

82

-0.0

49

-0.1

93

-0.1

80

-2.0

4 -1

.7

-0.0

61

-0.0

80

-0.0

76

-0.0

81

+o

,179

+o

,0

89

f0.0

89

+0.2

10

- _

Tem

pera

ture

coe5

cient

15”

- 23

0

-0.12

5 -0

.125

-0

.125

-0.0

81

-0.o

s5

-0.1

43

-0.0

7s

-0.0

92

-0.1

33

-0.0

75

-0.0

99

-0.1

23

-0.0

96

-0.2

24

-0.2

15

-0.2

03

-0.0

42

-0.0

43

-0.0

45

-0.2

02

-0.1

9s

-0.1

94

-0.2

28

-0.2

14

-0.2

00

-2.0

4 -2

.04

-2.0

4

-0.0

61

-0.0

78

-0.1

04

-0.0

61

-0.0

77

-0.0

94

i-0.1

84

to.

094

to.3

08

-0.0

61

-0.0

77

-0.0

99

to.1

80

to.0

92

to.2

57

f0.1

76

to.o

ss

f0.2

00

3o”

-0.1

25

-O.ls

o

-0.0

73

-0.1

06

-0.1

13

-0.0

92

-0.1

88

-0.0

47

-0.1

91

-0.1

87

-2.0

4

-0.0

61

-0.0

76

-0.0

86

to.1

72

t-0.0

85

to.

158

400

-0.1

25

-0.1

80

-0.0

69

-0.1

20

-0.0

93

-0.0

84

-0.1

45

-0.0

51

-0.1

87

-0.0

89

-2.0

4

-0.0

61

-0.0

75

-0.0

55

to.1

64

to.0

80

to.

102

--

, Biblio

graph

ic ref

erenc

e No

.

19

19

19 2 19

19

20

19

19

19

19

19

21 f 12

12

12

Ihis

pape

r

$ I’h

is pa

per

‘I “

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Z( -)

-H

istid

ine

‘C

‘I ‘I

I(-)-L

eucin

e ‘I I‘ ‘I “ I‘ “ “ “ ‘I Z(

-)-Ty

rosin

e “

Diox

ane

(28.

4 we

ight

. y’

,) Et

hano

l (2

0.0

weigh

t ‘%

) Et

hano

l (4

2.1

weigh

t 70

) M

etha

nol

(24.

2 we

ight

o/o)

12

.0 N

H

Cl

7.00

li

;‘ 6.

00

“ “

5.00

[‘

(‘ 4.

00 “

“

3.00

“

“ 2.

00

‘I li

1.00

“

“ W

ater

1.

05 N

Na

OH

1.1

“ H

Cl

6~Hc

l

0.54

0 0.

75-2

5.0

0.57

5 1.

00-2

5.05

0.61

0 0.

80-2

5.0

0.57

2 0.

7 -7

0.9

1.69

4 0

-35

1.80

0

-45

1.82

0

-45

1.84

0

-45

1.86

0

-45

1.89

0

-45

1.92

0

-45

1.94

0

-45

2.08

5 0

-45

1.89

1 0

-45.

5/

l 16

-2

6 41

1 16

-2

6

to.1

71

j-0.1

46

to.0

99

to.

105

j-0.1

66

+0.1

65

to.1

62

to.1

56

$0.1

16

to.0

45

to.0

48

+0.0

47

to.0

45

to.0

42

+0.0

35

f0.0

58

to.0

60

+0.0

59

to.0

57

to.0

55

+0.0

49

i-0.0

62

to.0

64

+0.0

63

to.0

61

t-0.0

59

+0.0

54

to.0

68

to.0

68

$0.0

67

to.0

65

to.0

63

$0.0

58

to.0

75

to.0

75

+0.0

73

to.0

72

to.0

69

+0.0

64

i-0.0

82

to.0

85

+0.0

82

i-0.0

80

to.0

77

+0.0

72

to.0

91

to.1

01

+0.0

95

to.0

92

i-0.0

87

+o.o

sz

to.1

0 to

.13

+0.1

2 to

.11

to.1

0 +0

.10

to.0

64

to.0

74

+0.0

66

f0.0

58

to.0

50

+0.0

34

-0.0

87

-0.0

90

-0.0

87

-0.0

85

-0.0

84

-0.0

82

-0.2

70

-0.3

86

-0.2

77

-0.2

41

-0.2

20

-0.2

60

-0.3

06

-0.2

69

-0.2

33

-0.2

10

-

This

pape

r

‘I I‘

* Nu

mer

ous

data

on

th

e in

fluen

ce

of t

empe

ratu

re

on t

he

spec

ific

rota

tions

of

am

ino

acid

s at

wa

ve-le

ngth

s of

lig

ht

othe

r th

an

x =

5893

A.

repo

rted

in

the

litera

ture

we

re

not

cons

ider

ed.

t Th

e ob

serv

ed

rota

tion

of p

urifie

d I(-

)-asp

arag

ine

diss

olve

d in

6.0

8 N

hydr

ochl

oric

acid

de

crea

sed

from

+2

.383

” to

+1.

748”

in

34

hour

s at

25”

. Si

nce

the

spec

ific

rota

tion,

+2

4.6”

, ca

lcula

ted

(see

belo

w)

for

I(+)-a

spar

tic

acid

is

the

ac

cept

ed

valu

e (5

), it

seem

s ce

rtain

th

at

the

aspa

ragi

ne

was

hydr

olyz

ed

to a

spar

tic

acid

. It

is n

ot

impr

obab

le,

ther

efor

e,

that

as

para

gine

m

ay

be h

ydro

lyzed

ap

prec

iabl

y in

1 N

hyd

roch

loric

ac

id

at e

leva

ted

tem

pera

ture

s.

[a]::

=

+1.7

4s”

x 10

0.0

2.01

5 X

132.

1/15

0.1

X 4.

000’

= +2

4.6”

in

6.

08 N

H

Cl

$ Du

nn,

M.

S., a

nd

Stod

dard

, M

. P.

, un

publ

ished

da

ta.

§ N

orm

ality

of

the

acid

in

the

am

ino

acid

sol

utio

n.

11 G

m.

of s

olut

e pe

r 10

0 m

l. of

sol

utio

n.

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

498 AMINO ACIDS AND PEPTIDES. IX

taken. An interpretation of the interesting data on the specific rotations of I( -)-histidine at varying concentrations of hydrochloric acid and sulfuric acid, given in Table V, Series E and F, is to be presented elsewhere.

The specific rotations of I(-)-histidine (Table IV) in 42.2 per cent eth- anol, 24.2 per cent methanol, 41.9 per cent acetone, and 28.4 per cent dioxane are all different, even though these solvents have identical dielec- tric constants. It would appear, therefore, that the rotation of I( -)-histi- dine in solvents other than water is influenced inappreciably by the dielectric constant. On the other hand, the authors’ experiments are con- sistent with the observation of Lowry (13) that the specific rotations of a number of optically active solutes in a series of solvents appear to be de- pendent upon the dipole moment of the pure solvent. The present authors have found that the specific rotations of Z(-)-histidine increase linearly

TABLE VII

pH of Aqueous Solutions of I(-)-Hi&dine Monohydrochloride*

Histidine monohydrochloride PH

noles per 1.

0.600 3.95 0.191 4.00 0.153 4.01 0.122 4.01 0.061 4.01 0.0305 4.02 0.0158 4.09 0.0079 4.21 0.000 6.60

* Amino Acid Manufactures, I(-)-histidine monohydrochloride monohydrate Lot No. 5, A. P. grade

with the decreasing average solvent dipole moments which were calculated from the dipole moments and the mole fractions of the components of the mixed solvents.

The rotation of I( -)-histidine in aqueous solutions of acetone is not in harmony with the foregoing observation. From a consideration of the polarity of acetone (p = 2.48), it would be expected that the specific rota- tion of I( - )-histidine in 42 per cent acetone solution would be considerably less than that in water. It was assumed that this apparent abnormality might be explained by a reaction of histidine with acetone analogous to that of this and other amino acids with formaldehyde. In order to test this hypothesis, the rotation of I( - )-histidine in water as a function of acetone concentration was measured by the apparatus and technique described in Paper VII of this series (14). It was found, however, that the rotation of

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

DUNN, FRIEDEN, STODDARD, AND BROWN 499

histidine decreases regularly as acetone is added to its aqueous solution. Although there is no evidence from this experiment of complex formation, it cannot be regarded as settled that optical activity of amino acids is not a function of solvent polarity. An effect similar to that observed in the present experiments was reported by Clough (12) who found that the rota- tion of Z-methyl aspartate in benzene, chloroform, methanol, and water de- creased regularly, while the rotation in acetone was similar to that in chloroform.

According to Lowry (13), concentration effects are explained by the orienting influence of the optically active molecules of the solute upon one another. Since maximum orientation of molecules occurs in the solid (crystalline) state, it may be inferred that the rotations exhibited by in- creasing concentrations of an optically active solute in different solvents should approach the same value. The observation that there is no ap- preciable change in the specific rotation of certain amino acids with vary- ing, but relatively low, solute concentration may be explained if there is negligible interaction between solute molecules.

Pertzoff (10) advanced the view that concentration effects are due to variations in the electrical field of the solvent, resulting from changes in the effective molar volume of the solute. That the latter is a function of glu- tamic acid and aspartic acid concentrations was demonstrated by Pertzoff who observed that the maximum change in the molar volume and in the optical rotation with concentration occurs with the ion, +NHsR(COO-)2.

The effect of temperature upon the specific rotation of amino acids has been studied by a number of workers. The results of these investigations are summarized in Table VI. The temperature coefficients at 15”, 20”, 25”, 30”, and 40” were determined by interpolation of curves derived from plots of the data given by the authors cited. It is of interest that temperature coefficients of the amino acids are both positive and negative, increase and decrease in magnitude with increasing temperature, and (with the excep- tion of the large value, -2.04, for I(-)-cystine) range from -0.042 to +0.308 at 15” and +0.034 to -0.187 at 40”.

SUMMARY

1. It has been shown that I( -)-histidine of 99.5 per cent or higher purity may be prepared by the fractional crystallization from water and alcohol of material isolated by standard methods from protein hydrolysates.

2. The solubility of I( - )-histidine at 25.10” f 0.05” is 4.286 f 0.013 gm. per 100 gm. of water. The specific rotation (1 4 dm. and X 5893 A.) of I(-)-histidine” in water is -38.95” f 0.06” at 25.00“ f 0.02” (c 0.752 to

’ The value +39.01’, given by Dunn et al. (5) as the specific rotation of Z(-)- histidine in water at 25”. is in error.

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

500 AMINO ACIDS AND PEPTIDES. IX

3.770) and -43.05” f 0.08” at 0.46” f 0.03“ (c 0.765 to 3.826) and in 6.08 N hydrochloric acid is +13.34” f 0.02” at 24.80” f 0.05” (c 1.0064 to 4.053).

3. The specific rotation of Z(-)-histidine in water, aqueous ethanol, and 6 N hydrochloric acid does not vary materially with different concentrations of solute. In 0.01 to 1 N hydrochloric acid solutions at con- stant molal ratio (1: 1) of acid to solute, the specific rotation of Z( - )-histi- dine is dependent upon the concentration of the solute, although the func- tion is not linear.

4. The specific rotations of Z(-)-histidine in solvents other than water have been found to be dependent upon the dipole moment, but not the dielectric constant, of the solvent.

5. Temperature coefficients of I(-)-histidine and certain other amino acids have been compared.

6. Hypotheses advanced to explain the described physicochemical be- havior of I( - )-histidine have been discussed.

BIBLIOGRAPHY

1. Stoddard, M. P., and Dunn, M. S., J. Biol. Chem., 142, 329 (1942). 2. Harris, L. J., J. Biol. Chem., 84, 296 (1929); Biochem. J., 29, 2820 (1935). 3. Toennies, G., and Callan, T. P., J. Biol. Chem., 126,259 (1938). 4. Levy, M., J. Biol. Chem., 109, 365 (1935). 5. Dunn, M. S., Ross, F. J., and Stoddard, M. P., Handbook of chemistry and

physics, Cleveland, 1344 (1941). 6. du Vigneaud, V., and Hunt, M., J. Biol. Chem., 116,93 (1936). 7. Robertson, G. R., Laboratory practice of organic chemistry, New York, 162

(1937). 8. Andrews, J. C., J. Biol. Chem., 66, 147 (1925). 9. Miller, H. K., and Andrews, J. C., J. Biol. Chem., 87,435 (1930).

10. Pertzoff, V. A., Pouvoir rotatoire des ions de l’acide d-glutamique, Montpellier (1937).

11. Lawrow, D., 2. physiol. Chem., 28,388 (1899). 12. Clough, G. W., J. Chem. SOL, 113,526 (1918). 18. Lowry, T. M., Optical rotatory power, London, 349-351, 353,358-365 (1935). 14. Frieden, E. II., Dunn, M. S., and Coryell, C. D., J. Physic. Chem., 46,215 (1942). 15. Abderhalden, E., and Weil, A., 2. physiol. Chem., 77, 435 (1912). 16. Ehrlich, F., Biochem. Z., 63, 379 (1914). 17. Lutz, O., and Jirgensons, B., Ber. them. GES., 64, 1221 (1931). 18. Pyman, F. L., J. Chem. Sot., 99, 1386 (1911). 19. Clough, G. W., J. Chem. Sot., 107, 1509 (1915). 20. Cook, E. P., Ber. them. Ges., 30, 294 (1897). 21. Toennies, G., and Lavine, T. F., J. BioZ. Chem., 89, 153 (1930). 22. Stein, W. H., Moore, S., and Bergmann, M., J. Am. Chem. Sot., 64,724 (1942). 23. Bergmann, M., and Zervas, L., Biochem. Z., 203, 280 (1928). 24. Kossel, A., and Kutscher, F., Z. physiol. Chem., 28, 382 (1899).

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from

Stoddard and Harold V. BrownMax S. Dunn, Edward H. Frieden, M. Palmer

l(-)-HISTIDINESOME PHYSICAL PROPERTIES OFAMINO ACIDS AND PEPTIDES: IX.

QUANTITATIVE INVESTIGATIONS OF

1942, 144:487-500.J. Biol. Chem. 

  http://www.jbc.org/content/144/2/487.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/144/2/487.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on Decem

ber 13, 2018http://w

ww

.jbc.org/D

ownloaded from