The BERTHELOT Reaction (1) Utilizing Phenol and Alkaline Hypochlorite

The Journal of Biochemistry, Vol. 66, No. 1. 1959

Colorimetric Determination of a)-Amino Acids

By SHOZABURO KITAOKA* and YOSHIHISA NAKANO"

(From the Department of Agricultural Chemistry, College of Agriculture, Shimane University, Matsue, Shimane)

(Received for publication. January 14, 1969)

A colorimetric method of determining minute amounts of u>-amino acids with high

sensitivity, accuracy and reproducibility utilizing a modification of the Berthelot reac tion is proposed, and the experiments leading to the establishment of the standard

procedure are described. Sample solution (0.1 -0.3 ml) is mixed with 0.2m borate buffer (0.2m1) and 6°'v phenol (l.0 ml). After cooling in ice water and adding 7.5;,c

sodium hypochlorite solution (0.4m1), the mixture is heated at 100°C for 10min and

promptly cooled. The optical density is read at 630mƒÊ. Most ƒ¿-amino acids, except some basic ones, give no color reaction but interfere with the reaction if present in significant amounts. Primary amines, but not secondary ones, and ammonium salts

also give positive reactions. Determination of the reaction velocity of pumpkin glut amate decarboxylase [EC 4.1.1.15] is reported as an example of the application of this method for biochemical studies.

The BERTHELOT reaction (1) utilizing MATERIALS AND METHODS phenol and alkaline hypochlorite reagents has been applied under different conditions for Ammonia-free, distilled water was used throughout. the micro-determination of free ammonia in Phenol Reagent-Six grams of reagent grade phenol the blood and urine ( 2, 3), that liberated was dissolved in about 90 ml of water and diluted to 100 ml. This solution was stable for at least one from urea by the action of urease [EC 3.5.1.5] month when stored in a refrigerator. ( 4-7), and that released from adenosine by Sodium Hypochlorite Reagent-Commercialy available the action of serum adenosine deaminase [EC sodium hypochlorite solution (Antiformin) containing 3.5.4.4] (8). A modification of the reaction 10 to 15% available chlorine was used as the stock was also used to determine taurine (9). We solution. After determining the exact content of found that under specific conditions, quite available chlorine by iodometry it was diluted to different from those previously reported, this 7.5,°7o for the standard procedure. This reagent could reaction can be applied for small scale deter be used after storage in a refrigerator for about ten mination of cu-amino acids in general with days. satisfactory sensitivity, accuracy and repro Borate BuFer (pH 9.0)-This was prepared by mixing 50 ml of 0.2m boric acid and 59ml of 0.2M ducibility, and the determination is not affect sodium borate. ed by the presence of small amounts of most Standard Procedure-After a series of preliminary a-amino acids. The present paper reports experiments (10) and the experiments which are the procedure developed for measuring cu-amino described in detail below, the following method was acids, factors influencing the determination developed as the standard procedure for determina and its application in measuring the reaction tion of (o-amino acids. To 0.1-0.3 ml of a sample velocity of pumpkin glutamate decarboxylase solution in a test tube (18•~120mm) are added in [EC 4. 1.1. 15]. order 0.2 ml of borate buffer and 1.0 ml of phenol reagent. After mixing thoroughly and cooling in ice water, 0.4 ml of the sodium hypochlorite reagent is

* Present address: Department of Agricultural added. The tube is shaken vigorously in ice water Chemistry, College of Agriculture, University of and placed in a boiling water bath for 10 min. It Osaka Prefecture, Sakai, Osaka. is then immediately cooled by immersion in ice water 87 88 S. KITAOKA and Y. NAKANO

for 5 min. When the reaction mixture, 1.7-1.9 ml, is too little to fill the photometer cell, it is diluted to a suitable volume with 60%, ethanol. In some of

the experiments described below 1.0 ml of 60°b ethanol was added before estimating the optical density in a Hitachi Photo-Electric Spectrophotometer while in the

others the estimation was made in a Shimadzu-Bausch and Lomb Spectronic 20 photometer after adding 2.0 ml of 60% ethanol to the colored reaction mixture. The optical density was read at 630 mƒÊ against a blank containing reagents but no sample. The sample solution may contain ethanol, butanol or other similar solvents. As described later the addition of borate buffer augments the color intensity but has no buffering FIG. 1. Color intensities with different com action. In some experiments on individual factors binations of phenol and NaClO concentrations. affecting the reaction, borate buffer was omitted. In Reaction mixtures (1.7 ml) containing 100 ƒÊg the experiments described below leading to estab- ƒÀ-alanine (0 .3 ml), phenol solution (1.0 ml) and lishment of the standard procedure, ƒÀ-alanine was NaClO solution (0.4 ml) with different combina mainly used as the sample w-amino acid. tions of concentrations of the latter two reagents were subjected to the color reaction at room RESULTS temperature for 30 min. Effects of the Concentrations and Ratio of Phenol -•ü-: Phenol 1%, -•œ- Phenol 2%, Sodium Hypochlorite-The concentrations of these -ƒ¢-: Phenol 3%, -• - Phenol 4%, two reagents employed by previous workers x Phenol 5%, -•£- .. Phenol 6%. ( 2-8) differ considerably and relatively low

concentrations have usually been used under rating into the the reaction mixture, acetate,

strongly alkaline conditions with little or no citrate, phosphate, borate or carbonate buffer

heating, Fig. I summarizes the results of ex at various pH values.

periments to determine the optimum concen The results are shown in Table I. The trations and ratio of phenol and sodium hypo reaction mixtures consisted of 0.1 ml of A-

chlorite reagents for determination of (ƒÖ-amino alanine solution (100 ƒÊg), 0.2 ml of a buffer

acids. The color reactions were conducted solution (0.2 M), 1.0 ml of phenol reagent and

with 0.3 ml of sample solution containing 100ƒÊg 0.4 ml of sodium hypochlorite reagent.

of ƒÀ-alanine and different combinations of The results show that the color developed

1.0 ml phenol reagents containing 1 to 6 % is less in acidic buffers and generally more

phenol and 0.4 ml of sodium hypochlorite in alkaline buffers. Of the buffers examined, reagents containing 0.5 to 15% available chlo borate buffer (pH 9.0) had the most effect

rine. Color was developed at room tempera increasing the absorbance by 20 to 50% de ture for 30 min and after diluting the mixture pending on the reaction conditions. Table II with 2.0 ml of 60% ethanol optical densities indicates the effects of different concentrations were read at 630 mƒÊ. of borate buffer. Addition of 0.3 to 0.4 M buffer The results clearly indicate that 6%° phenol had the most effect. However , for convenience and 7.5%o sodium hypochlorite gave the highest and because of its stability, 0.2 tit borate buffer optical density. Accurate results were obtained was used in subsequent experiments and in with 0.1 to 0.3 ml of sample with and without the standard procedure. 0.2 ml of 0.2 ma borate buffer (PHI 9 .0) and at It was rioted that the borate buffer had higher temperatures also. no buffering action, for the reaction mixture

Effects of Buffers-Since the pH value has remained at pH 10-11 after addition of buffers been reported to influence the BERTHELOT re of different concentrations . Accordingly the action considerably (1), the effects of some effects of boric acid, sodium borate and borate representative buffers were studied by incorpo- buffer (pH 9.0) in augmenting the color devel- Determination of '_t;-Amino Acids 89

TABLE I TABLE II Effects of buffers on color intensity . Effeet of concentrationof borate buffer The reaction mixture contained 100 ƒÊg of on color intensity. ƒÀ-alanine (0 .1 ml;), test buffer (0 .2m, 0.2 mb, 6('b Reaction conditions were as for Table I ex phenol (1.0 ml) and 7.5% NaClO (0.4 m1). After ceptthat borate buffer (0.2 ml) of different con the reaction (100°C , 10min) 60% ethanol (2.0m1) centrations was used. was added.

TABLE III

Effects of boric acid, sodium borate and borate buffer on color intensity.

Reaction mixture contained 50 ƒÊg of ƒÀ-alanine

(0.2 ml), test borate compound (0.2 m, 0.2 ml), 6%,

phenol (1.0ml) and 7.5(e NaClO (0.4 ml). Before reading absorbance 60% ethanol (1.0 ml) was added.

opment were compared (Table III). The data show that all of these substances increase the color but that borate buffer has the most effect. Stability of the Reagents-The phenol rea gents employed by previous workers for deter mination of ammonia, containing different and so it appears that the reagents may be

concentrations of phenol and in most cases used reliably for considerably longer periods

sodium hydroxide and other substances, were if calibrations are made before determinations. reported to be stable for from at least one Effect of Temperature-The reaction temper

month to over one year at low temperatures ature was found to affect the velocity and

in the dark while sodium hypochlorite reagents intensity of color developed (Fig. 2). The data

were stable for longer periods ( 2-8 ). Experi shown were obtained on 2 samples containing

ments showed that the phenol and sodium 50 fig and 100 ƒÊg of N-alanine respectively. On

hypochlorite reagents used in the present heating at 100°C (in a boiling water bath)

method were stable for at least one month and both samples gave a more color after 10 min for about ten days, respectively, if stored in than after 15 min. But at lower temperatures a refrigerator. It was found that although reaction for 15 min yielded more color. The the absolute values of absorbance decreased reversal occurred at around 70°C with the gradually on storage, the linearity between 100 ƒÊg-sample and at around 55°C with the the concentration of the sample and the color 50 ƒÊg-sample. The decrease in color intensity intensity was maintained for longer periods, at higher temperature on prolonged heating 90 S. KITAOKA and Y. NAKANO

TABLE IV

stability of color.

Reaction mixture (1.7ml) contained 100 ƒÊg of

l3-alanine (0.3 ml) and no borate buffer. After the reaction (100°C, 10 min) the mixture was stood at room temperature, then 60% ethanol (2.0ml) was added at intervals and the optical density measured.

FIG. 2. Effect of reaction temperature and time on color intensity. Reaction mixtures (1.7 ml) containing 50 or 100 ƒÊg ƒÀ-alanine (0.3 ml), the phenol reagent

(1.0 ml) and the NaClO reagent (0.4 ml) were heated at different temperatures for different color developed was about 10% less than after

periods. Absorbances were read after adding cooling for 5 and 10 min. In the standard

60% ethanol (1.0 ml). procedure 5 min cooling is an essential step. -•ü- Heated for 5 min , Influence of Interval between Additions of Phenol -•œ- Heated for 10 min, Reagent and Sodium Hypochlorite Reagent-FAWCETT -•~- Heated for 15 min. and SCOTT (I) noted that the interval between the additions of the two reagents influenced is probably due to decomposition of the pig the final optical density. Under the present ment or its precursors. For the standard pro reaction conditions no significant difference cedure the reaction conditions were set at was found with intervals of 2 to 10 min, which

100°C for 10 min to obtain the maximum color are convenient even when handling a consid intensity. Should this method be modified erable number of samples. Too prompt ad for use at lower temperatures, it is advisable dition of hypochlorite reagent is not recom- to check the optimum reaction time at a given mended. temperature for the specific kind and concen Stability of the Color -The color obtained tration range of ƒÖ-amino acid to be deter by the present method was stable for at least mined. 5 hr at room temperature (Table IV).

Effect of Cooling before and after the Reac Dilution with 1.0 or 2.0 ml of 60% ethanol tion-In our standard procedure we prescribe did not influence the stability. mixing the sample solution and the reagents Sensitivities of Amino Acids, Amines and Am in ice water. If this is neglected and mixing monium Salts-Table V lists a number of re- is done at room temperature, there is slight presentative ƒ¿- and (o-amino acids, aliphatic spontaneous development of heat due to the and aromatic amines, ammonium salts and reaction between the phenol and sodium hy- micellaneous nitrogenous compounds together pochlorite, and the intensity of color developed with their molecular extinction coefficients at in the subsequent reaction is not reproducible. 630 m t when subjected to the standard pro

After the reaction at 100°C for 10 min, the cedure. reaction mixture must be cooled promptly in Simple w-amino acids show fairly high ice water. Color develops during the cooling. sensitivities in this reaction, 1 to 2 ƒÊg giving

When the mixture was not cooled (60%o ethanol a measurable color, while the co-amino acids being added immediately without cooling) the with an additional methyl, amino or hydroxy Determination of w-Amino Acids 91

TABLE V

Sensitivities of amino acids, amines and ammonium salts.

Molecular extinction coefficients (ƒÃ630) were calculated from the optical densities with sample solutions

(0.2 ml ; total volume 1.8 ml) containing 1 to 200 ug of the test compounds. The indicated values are mostly the means of values at 2 to 3 sample concentrations. A negative value was a value smaller than about 0.1.

1) Hydrochloride used. 2) Brownish color developed. 3) Sulfate used. 92 S. KITAOKA and Y. NAKANO

TABLE VI

Interferences by a-amino acids.

To 50ƒÊg of ƒÀ-alanine or ƒÁ-aminobutyric acid

(0.1 ml) were added serially varied amounts of ƒ¿- alanine or ƒ¿-aminobutyric acid (0.1 ml), respec

tively. Borate buffer (0.2 ml) and 60% ethanol

(2.0 m1) were used.

FIG. 3. Beer's plots of reactions of some ƒÖ- amino acids and amines. The standard procedure was followed with a total of 1 8 ml of each reaction mixture. 1 : Ammonium chloride, 2: ƒÁ-Aminobutyric acid,

3a: d-Aminovaleric acid, ƒÃ-aminocaproic acid, taurine, cadavine 2HC1, 3b: ƒÀ-Alanine, 4: ƒÀ-Aminoisobutyric acid, histamine 2HCl, 5: Ornithine, lysine, 6: ƒ¿-Hydroxy-ƒÁ-aminobutyric acid, tyramine

HCl, 7 : ƒ¿, ƒÁ-Diaminobutyric acid, 8: Glycine.

group at another position give less color. In contrast, ordinary a-amino acids did not give structure and the intensity of the color ob

positive reactions. Exceptions were ornithine tained in this reaction in reference to the re- and lysine which area, ƒÖ-diamino acids. Glycine action mechanism was not studied.

with an ƒ¿- and at the same time ƒÖ-amino This method is highly reproducible if the

group, histidine and serine gave slightly positive standard procedure is carefully followed, and

reactions. the optical density of w-amino acids and amines As in previously reported modifications was proportional to their concentration in the

of the reaction for determination of ammonia, range studied as shown in Fig. 3 with some

the present method revealed high sensitivity representative compounds. Exceptionally ƒÀ- with ammonium salts. It is noted, however, alanine showed rather a short measurable that in none of the previous modifications range. did w-amino acids give a positive color reac Interference by Various Substances-The in tion. Some amines, both aliphatic and aro terference of a-amino acids in the determina matic, gave positive reactions in different tion of w-amino acids by this method was degrees. An extremely high sensitivity was studied with two combinations of ƒ¿- and ƒÀ- found for p-methylaminophenol with an ƒÃ630 alanines, and ƒ¿- and ƒÁ-aminobutyric acids. value of 35,500. The data with methylamines Table VI shows the results of experiments suggest that the reaction is only positive with where different amounts of the a-amino acids primary amines and not with secondary or were added with 50 ƒÊg of ƒÀ-alanine or ƒÁ-ami tertiary amines. The correlation between the nobutyric acid. Interference by equivalent Determination of co-Amino Acids 93

TABLE VII termining the ƒÁ-aminobutyric acid liberated by the present method. The reaction mixture, Interference by acid and alkali. 0.2 ml in total volume, contained 0.1 ml of To the sample solution (0.1 ml) containing 0.1 si phosphate buffer (pH 5.8), 0.02 ml of 50 ƒÊg of ƒÀ-alanine and 0.2 M borate buffer (0 .2 ml) were added different concentrations of HCl or substrate solution (final concentration 10-3 --1.25 x 10-2 m) NaOH (0.1 ml). , and 0.08 ml of enzyme prepa ration. The observed optical densities were

corrected for interference by glutamic acid.

The Michaelis constant obtained from the

experiments was 4 x 10-3 M which is in fair

accordance with the reported value of 3.6±

0.4•~10-3 M, obtained manometrically (12).

The present method requires less sample and

time and is simpler than the manometric

method.

DISCUSSION

amounts of a-amino acids is relatively small A sensitive method for colorimetric deter

but it was increased gradually with the amount mination of u,-amino acids utilizing a specific

of the a-amino acids added. It is interesting modification of the Berthelot reaction is de

that the effects were reverse, i.e., ƒ¿-alanine scribed. The reaction conditions differ greatly

increased the color developed with ƒÀ-alanine from those reported for determination of am

whereas ƒ¿-aminobutyric acid decreased the monia. In recent modifications (2-8) sodium

color developed with ƒÁ-aminobutyric acid. The nitroprusside has heen used as an essential

reason for this is not clear but a preliminary catalyzer, but in the present method it did

examination should be made of the mode not improve the reaction and caused un-

and extent of interference by a-amino acids desirable precipitation. In none of the pre

if the present method is applied to a mixture viously reported methods for determination

containing significant amounts of a-amino of ammonia, did o)-amino acids give a positive

acids. color reaction. The reaction conditions em Absorbances of up to 100 ƒÊg of ƒÁ-amino ployed by SCHICK and DEGERING (9) for de butyric acid were not affected by the presence termination of taurine were not sufficiently

of 200 pg of ovalbumin. sensitive or accurate for micro-determination

The results shown in Table VII show the of a-amino acids in biochemical studies. In

strong inhibition of the color reaction by acid the present method ƒÀ-alanine gives an ƒÃ630

and alkali. These inhibitions can be removed value of 3500 and ƒÁ-aminobutyric acid a value

by preliminary neutralization of the sample of 5380. The corresponding values (8570) in

solution. the ninhydrin reaction (13) are 14,950 and

Determination of ƒÁ-Aminobutyric Acid Liberated 25,600, respectively.

by Pumpkin Glutamate Decaiboxvlase-To test the Although most neutral and acidic a-amino

application of this method for studies of en acids give no color, they may exert a con

zyme reactions, the reaction velocity of pump siderable influence on the color developed kin glutamate decarboxylase was estimated. with o)-amino acids if present in appreciable

Details of this experiment will be published amounts. A preliminary separation is therefore elsewhere. An enzyme preparation yielding recommended on application of this method

14.4 ƒÊmoles of 1•-aminobutyric acid per 1 mg to a mixture of ƒÖ-amino acids and a-amino of protein was obtained by the method of acids when the latter are present in sufficient

OKUNUKI and INAGAKI (11). The velocities amount to cause interference. In a rather of decarboxylation with different concentra simple system with a known a-amino acid, as tions of glutamic acid were estimated by de- is the case of the experiments on the glutamate 94 S. KITAOKA and Y. NAKANO

decarboxylase reported here, a correction of Dr. I. Chibata, Tanabe Seiyaku Co., Osaka, and

the observed values can be made. Dr. H. Kataoka, Nippon Shinyaku Co., Kyoto, for

Application of this reaction with slight generous supply of some amines. modification to paper or thin-layer chromato REFERENCES graphy provides an excellent technique for detecting cu-amino acids, some basic a-amino (1) M. Berthelot, Repert. Chim. Appl., 1, 284 (1859) acids and amines in the presence of other a- (2) B. Lubochinsky and J.P. Zalta, Bull. Ste. Chim. amino acids, as will be reported shortly. Biol., 36, 1363 (1954)

Ammonia and ammonium salts produce (3) J.L. Ternberg and F.B. Hershey, J. Lab. Clin. Med., 56, 766 (1960) intense colors in the present method as in

other modifications reported. Thus ammonia (4) J.K. Fawcett and J.E. Scott, J. Clin. Pathol., 13, 156 (1960) should not be used in experiments, as for ex- (5) R.L. Searcy and F.M. Cox, Clin. Chim. Acta, 8, ample, in eluting amino acids from a column. 810 (1963) The interference by acid and alkali is readily (6) A.L. Chaney and E.P. Marbach, Ctin. Chem., eliminated by preliminary neutralization of 8, 130 (1962) the sample. (7) M. Saito, K. Uchida, and E. Suzuki, Rinsho If sufficient care is paid to the precautions Kensa, 8, 878 (1964) discussed above, the present method has prom (8) R.G. Martinek, Clin. Chem., 9, 620 (1963) ising applications with its high sensitivity, (9) J.W. Schick and E.F. Degering, Ind. Eng. Chem.,

reproducibility and accuracy, and simplicity 39, 906 (1947)

and economy in handling, in studies of ƒÖ-amino (10) S. Kitaoka, K. Suzuki, and M. Ito, Seikagaku, 39, 580 (1967). Abstract of paper presented at acids, their distribution in nature and their the 40th Annual Meeting of the Japanese Bio metabolism. An example is included in this chemical Society, Sakai, Osaka, November 6, 1967 paper. The method may also be applied for (11) K. Okunuki and M. Inagaki, Proc. Japan. Acad., determination of some basic ƒ¿-amino acids 27, 658 (1951) and biochemically interesting amines. (12) O. Schales and S.S. Schales, Arch. Biochem., 11, 155 (1946) We thank Prof. M. Kandatsu, University of (13) E.W. Yemen and E.C. Cocking, Analyst, 80,209 Tokyo, for a gift of 2-aminoethylphosphonic acid and (1955)