a colorimetric method for the estimation · a colorimetric method for the estimation ......

A COLORIMETRIC METHOD FOR THE ESTIMATION OF AMINO-ACID a-NITROGEN.

BY VICTOR JOHN HARDING AND REGINALD &I. MAcLEAK.

(Prom the Biochemical Laboratory, McGill University, Afontreal.)

(Received for publication, January 14, 1915.)

Two methods are at present in use for the determination’ of amino-acid nitrogen. The first, that of Sorensen,’ is a rapid, easy method, but is neither very delicate nor accurate. The second, that of Van Slyke,2 is the method now usually accepted for the determination of nitrogen in amino groups. In it,s latest development, the micro chemical form: the method will estimate 0.5 mgm. of nitrogen with an accuracy of 1 per cent, and its application to the determination of amino-acid nitrogen in blood and tissues has yielded in its author’s hands very valuable and interesting results. It was felt, however, that a third and inde- pendent method for the estimation of the a-nitrogen of the amino: acids, especially if the sensitiveness of the reaction could be increased beyond that of the Van Slyke method without loss of accuracy, would not only be valuable but necessary as the time approached for a study of the chemistry of the single cell.

It soon became apparent that such a hope could be fulfilled only by the quantitative application of a color reaction of amino- acids, and of all such the most probable seemed the reaction with triketohydrindene hydrate.

This reaction was discovered by Ruhemann4 who found that all acids containing a free amino group in the LY position reacted with triketohydrindene hydrate with the production of an intense blue color. p-, y-, and &amino-acids only gave small amounts of color, and a-amino-acids substituted on the amino or carboxyl

1 S. P. L. Sijrensen: Biochem. Ztschr., vii, p. 45, 1908. 2 D. D. Van Slyke: this Journal, ix, p. 185, 1911; xii, p. 275, 1912. 3 Van Slyke: ibid., xvi,‘p. 121, 1913-14. 4 S. Ruhemann: Z’r. Chem. Sot., xcvii, pt. ii, p. 2025, 1910.

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218 Estimation of Amino-Acid a-Nitrogen

group did not react at all. This discovery was confirmed and extended by Abderhalden, who applied it to the det.ection of pregnancy and cancer, triketohydrindene hydrate now being a commercial product under the name of “ninhydrin.” The sensitiveness of the reaction is unquestioned; for, according to Abderhalden and Schmidt,6 it will detect one part of glycine in G5,OOO parts of water, though the other amino-acids will not react in quite so dilute a solution.

The constitution of the blue coloring matter was investigated by Ruhamann,6 who isolated from the interaction of alanine and triketohydrindene hydrate a body which was found to be identical with the ammonium salt of diketohydrindylidene-diketo- hy&indam.ine,

Thus the chemistry of the coloring matter was weil known and the problem became the determination of the conditions under which the grouping

RCH (NHZ) COZH

common t.o all a-amino-acids would react quantitatively with triketohydrindene hydrate to produce the blue colored ammonium salt of diketohydrindylidene-diketohydrindamine, which could then be compared calorimetrically with a definite amount of coloring matter as standard.

The reaction has already been made use of in a roughly quan- tit,ative way by Abderhalden and Lamp6’ in studying the fate of amino-acids during absorption, but they made no attempt to place their results on a strictly quantitative basis. Herzfeld,* however, devised a method for the estimation of the group

6 E. Abderhalden and H. Schmidt: Ztschr. f. physiol. Chem., lxxxv, p. 143, 1913.

6 Ruhemann: Tr. Chem. Sot., xcix, p. 1486, 1911. 7 E. Abderhalden and A. E. LampB: Ztschr. f. physiol. Chem., lxxx& p.

473, 1912. * E. Herzfeld:Biochem. Ztschr., lix, p. 249, 1914.


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V. J. Harding and R. M. MacLean

NH2 -CH

< COOH

using the ninhydrin reaction as a basis. The method was to evaporate the amino-acid and excess of triketohydrindene hydrate t’o dryness on a water bath, dissolve the purple colored residue in a little alcohol with a drop or two of ammonium hydroxide, make up to a known volume, and determine the amount of coloring matter by measuring its extinction coefficient in a spectrophotometer. The present authors have repeated this method of pre- paring the coloring matter in a quantitative way, estimating it, however, by a Duboscq calorimeter instead of measuring the extinction coefficient in a spectrophotometer. The former method is much more rapid, and at present is the only one which could be successfully applied in hospital laboratories to the study of amino-acid excretion in pathological conditions. Working in this way, the results were very unsatisfactory. It was found by heating varying amounts of an amino-acid with an excess of ninhydrin that the different amounts of the amino-acid could be estimated with moderate accuracy, using a fixed amount of the same amino-acid as standard. The following figures illustrate this point.

AMINO-ACID cc. OF 0.1 PER CENT COLORIXIETER READING SOLUTION ~- .-

Found:

I

Calculated: ~___~ ~~~____~_ __.--

Glycine.. . . . . . . . . .! 1.0 Standard 1 Set at 2.00 cm. 1.5 1.51

I 1.33

2.0 1.00 1.00 Alanine. . . . . . . . . . . . . . . 1 .O Standard Set at 2.00 cm.

2.0 0.99 1.00 4.0 1.0 Standard /

0.48 0.55 Aspartic acid.. . . Set at 3.00 cm.

/ 28 i 1.51 1.50 0.95 1.00

Glutaminic acid.. . . . . ./ 1 .O Standard Set at 2.00 cm. 1.5 1.33 1.33 3.0 0.69 / 066 I .

Thus it will be seen that varying amounts of glycine can be estimated by using the color produced by a known amount of


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glycine as standard. The same is t’rue for alanine, and aspartic and glutaminic acids.

When, however, we attempted to estimate alanine, or aspartic or glutaminic acids, using glycine as a standard, the method gave totally erroneous results.

, h‘t PER cc .4YINO-ACID

Found: Calculated: .-

mgm. nlgm.

Alanine............................I 0.016 0.036 Glutaminic acid.. . . . . . . . . . .’ 0.152 0.095 -. --__.

In the case of aspartic acid the color produced by the reaction was of such a pronounced reddish shade that it was found impossible to match it against the bluish violet color of the standard.

Thus it will be seen that this method of estimating amino-acid nit:ogen fails completely.

Reaction of amino-acids and triketohydrindene hydrate in slight excess.

In our earlier experiments on this subject, attempts were made to study the reaction between various amino-acids and the triketone, with strictly molecular amounts of the two substances, but, owing to causes at present unknown, these experiments failed to give any concordant results. Neither of us, working alone or together, could be certain of obtaining any two series of experiments which agreed within 5 per cent. What we desired to do was to heat a known amount of an amino-acid, usually glycine 01 alanine, with the molecular equivalent of triketohydrindene hydrate and determine the time at which the maximum development of the blue coloring matter took place. This could be accom- plished by immersing a series of test-tubes containing the equivn- lent amounts in a boiling constant-level water bath, removing them one by one at stated intervals (usually five minutes), diluting them to a known volume (100 cc.) with distilled.water, using the color produced in the test-tube first removed as standard, and comparing the others against that in a Duboscq calorimeter. At first the com- parisons were made by daylight, but it was soon found that much


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V. J. Harding.and R. M. MacLean 221

more consistent readings were obtained by. the use of a 25 watt tungsten lamp as a source of illumination in a dark room. The lamp was placed in a conical, semi-opaque shade, the outer end of which was covered with a sheet of tissue paper to diffuse the light; and the whole was placed about a foot away from the calorimeter. In this way a strongly and evenly illuminated field was obtained on the white reflector of the calorimeter only, and thus the eye was free from any other disturbing sources of light during the determinations. A small electric bulb on a convenient switch enabled one to take the vernier readings. In artificial light the bluish colored solution of the coloring matter changes to a red violet, resembling very much the appearance of, a dilute solution of potassium permanganate when viewed in daylight.

Even under these conditions, which greatly improved our ability to make concordant readings, the results continued to be very irregular and capricious, when using equimolecular amounts of the two reagents. The presence of traces of impurity, the rapid fading of the coloring matter, and the sensitiveness of the reaction to heat, probably were all factors, not under these conditions properly controlled, which contributed to the failure of our experiments from a quantitative point of view. However, under more stable and more accurately defined conditions, it is hoped at a later date to return to the study of the reaction in equimolecular solution and to endeavor to obtain from it some in- sight into the mechanism of what is without doubt a complicated series of reactions.

It was soon found, however, that a slight excess of triketohydrindene hydrate enabled us to approach a solution of our problem of the time of the maximum development of the coloring matter in dilute solution. The same method of procedure was adopted as that previously described. The coloring matter in the test-tube first removed was used as standard and the others were read against it. The series of curves in Figure 1 shows the relative amounts of coloring matter plotted against the time, each amino- acid being taken separately.

It will be noticed that the time of the development of the maximum amount of color is different for each amino-acid, thus precluding the use of this technique for a method of estimating amino-acid a-nitrogen in a mixture of amino-acids. No attempt

TRE JOcxiNAL OF BIOLOGIClL CHEMISTRY, VOL. xx. NO. 3


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222 Estimation of’ Amino-Acid a-Nitrogen

was made to compare one ,amino-acid against another, as such a series of experiments would have served no useful purpose at that time. Moreover, the actual amounts of color produced in the cases of aspartic and glutaminic acids and asparagine were so small that the reaction liquid was only diluted to 50 cc. instead of 100 cc. Also the color produced by aspartic acid and asparagine was of a pronounced yehowish tint and could not be compared with the reddish violet given by alanine or glycine.

’ i j h / I I I I I I

FIG. 1.

Reaction of amino-acids and triketohydrindene hydrate in large excess.

In our next series of experiments we determined the time of the maximum development of color between the different amino-acids and the triketone, using a large excess of the latter in high concentration. The necessity of a high concentration to produce quantitative amounts of color’in this reaction had been pointed out by Herzfeld,g and a series of experiments of our own had confirmed this conclusion. We heated together in a boiling constant- level water bath a series of 1 cc. of0.1 per cent solution of amino- acid and 0.5 cc. of 1 per cent solution of triketohydrindene hydrate

* Herzfeld: Zoc cil.


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V. J. Harding and R. M. MacLean 223

for periods of 5,10,15, and 20 minutes. The amount of color produced in the test-tube heated for five minutes was taken as standard, and the results were plotted as before. Each amino- acid was considered separately (Figure 2).

The effect of the higher concentration of the triketone was at once readily apparent. The amounts of color produced in the same time were much larger, and concordant experiments were

FIG. 2.

easily obtained except in the case of asparagine. It will also at once be noticed that the times of maximum development of color with the different amino-acids are much shorter than in the first series of experiments and lie closer together. Thus, in ihe first series, the times vary from twenty minutes (tyrosine) to seventy minutes (alanine), whereas in the second series the variation is only from ten minutes (glycine) to twenty minutes (aspartic acid and tyrosine). The other amino-acids possess the time of maxi-


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mum development of color at fifteen minutes. The colors with aspartic acid and asparagine, however, were still very weak and still possessed a strong yellowish tinge which rendered their comparison with the’ other acids extremely difficult. Other irregu- larities, too, were noticeable. The colors produced by aspartic and glutaminic acids faded very much more rapidly than those produced by the other amino-acids; that produced by aspart.ic acid being discharged completely in a dark room at the end of twelve hours. As these two acids differed from the others only in the presence of a second carboxyl group, thus rendering them more acidic in character, the disappearance of the coloring matter on standing was put down to this cause. Ruhemann had pointed out that long contact with acids causes a decomposition of the coloring matter, giving a colorless solution, and experiments by us had proved the strong inhibitory effect of small amounts of organic acids on the production of the ammonium salt of diketohydrindylidene-diketohydrindamine from the interaction of alanine and the triketone.

In order to t.est this assumption a third series of experiments was carried out in presence of a base which would neutralize the acidity of the second carboxyl group of aspartic and glutaminic acids. Such a base should not be strong enough to hydrolyze the triketohydrindene hydrate, as happens with the hydroxides of the alkali metals, and should not interfere with the reductions and condensations which take place in the reaction. The base chosen was pyridine.

Interaction of amino-acids and triketohydrindene hydwte in large excess, in presence of pyridine.

One cc. of a 0.1 per cent solution of the amino-acid was added to 0.5 cc. of a 1 per cent soIution of triketohydrindene hydrate, and 0.2 cc. of pure, freshly distilled pyridine was added, and the mixture heated in a boiling water bath for varying intervals of time, as in the two previous series of experimenbs. The estimations of the relative amounts of coloring matter were carried out in a manner similar to the former experiments, each acid being compared with itself onIy. The curves in Figure 3 show the results obtained. They show quite clearly that the addition of


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T(. J. Harding and R. M. MacLean 225

the pyridine had achieved the desired result. They show that the reaction between amino-acids and triketohydrindene hydrate in presence of pyridine takes place rapidly, and reaches a maximum amount of coloration which remains constant for some minutes, except in the case of aspartic acid. This time of maximum development of color is constant at about twenty minutes. Moreover, the addition of the pyridine had increased enormously the actual amounts of coloring matter produced, and the colors now did not fade appreciably in a short period of time. In the curves obtained with glycine and alanine 0.5 cc. of pyridine was used.

Time in Minutis

FIG. 3.

A determination was now made of the relative amounts of coloring matter produced by four of the amino-acids, of bvhose purity we were quite certain, by dissolving equivalent amounts of each of them in water and using one as a standard. The following table will illustrate the experiment and the result. Being in equivalent amounts, they should produce the same amount of coloring matter and thus give the same calorimeter reading.

It will be seen that aspartic acid was still a little low in amount of coloring matter produced when compared with the other acids.

As a result of further experiments, it was decided to reduce the amount of amino-acid nitrogen per cc. to 0.05 .mgm., to reduce


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226 Estimation of Amino-Acid Q-Nitrogen

nfmw-ncrD iv2 PEll cc.

~ mgm.

Alanine ............... .i Glycine ................ ; Aspartic acid I Glutaminic acid

................ ./

0.1 a.1

8::

cc.

0.2 0.2 0.2 0.2

TRIKETONE: 1.0 PER CENT

cc.

0.5 0.5 0.5 0.5

1.00 0.99 1.17 1.01

the pyridine to 1.0 cc. of a 10 per cent solution in water, and to increase the triketohydrindene hydrate to 1.0 cc. of a 2 per cent solution.

Method of estimation of amino-acid a-nitrogen by use of triketohydrindene hydrate.

One cc. of the solution to be estimated, containing not more than 0.05 mgm. of amino-acid a-nitrogen and neutral to phenolphtha- lein, is mixed with 1 cc. of a 10 per cent aqueous solution of pure pyridine and 1 cc. of a freshly prepared 2 per cent solution of t.ri- ketohydrindene hydrate and heated in a rapidly boiling constant- level water bath for twenty minutes. At the end of that time the test-tube is removed, cooled, and diluted to a suitable volume, usually 100 cc., but if the amino-acid a-nitrogen is very small in amount, a correspondingly smaller dilution can be used. The solution of coloring matter thus obtained is compared with the standard. color in the usual way, in a Duboscq calorimeter.

Preparation of a standard color.

The standard solution is prepared by dissolving 0.3178 of a gram of pure freshly crystallized alanine in a liter of distilled water. Such a solution contains 0.05 mgm. of nitrogen per cc. To prepare the standard color 1 cc. of the standard alanine solution is heated for twenty minutes in a boiling water bath with 0.5 cc. of a 10 per cent solution of pyridine and 1.0 cc. of a 1 per cent solution of triketohydrindene hydrate.‘O At the end of that time the contents of the tube are cooled and diluted to 100 cc. This solution of coloring matter is used as a standard and will keep for twenty-four hours. The standard solution of alanine is

lo The employment of a more concentrated solution of the ketone gives no further increase in the amount of coloring matter in the case of alanine.


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*MINO-AmD - Calorimeter T Van Slyke

~- _ ~~~ .~~~~- ~~. - - ~__ I- ~- .- mgm. mgm. mgm.

Glycine............................ 0.186 0.189 0.189 Alanine............................ 0.100 0.101 0.102 Valine............................. 0.201 0.201 0.196 Leucine........................... 0.104 0.102 cu-Amido-n-caproic acid.. . . . 0.160 0.164 0.161 Phenyl alanine.. . . . 0.128 0.126 0.126 i-Tyrosine......................... 0.084 0.087 Tryptophane. . . . . . . . . 0.090 0.092 Histidine.......................... 0.085 0.089 Bspartic acid.. . . . 0.105 0.103 0.104 Asparagine. . . . . . 0.17.5 0.178 Glutaminic acid.. . . . . .

i 0.15s 0.157 : 0.157

.-__-


st,able for three months. Attempts were made to prepare a per- manent standard color without success. A preparation of the pure coloring matter was made according to the directions of Ruhemann, and a dilute solution of it made in water. It was found, however, that when the dried coloring matter was used, it was not as freely soluble in water as the freshly prepared sub- stance, and that an aqueous solution of the pure coloring matter faded rapidly; it was much more stable in the presence of pyridine, but even then at the end of three months it had faded completely. There was thus no advantage to be gained in using a solution of the coloring matter prepared per se. The standard color prepared as directed is little or no trouble to prepare along with the determinations themselves.

Determination of amino-acid a-nitrogen in various amino-acids and comparison with the method of Van Slylce.

A series of determinations was then made of the amino-acid nitrogen in various amino-acids, and the results were compared with theory in the case of the pure acid. A parallel series of determinations was made by the method of Van Slyke and the results were compared with the calorimetric determinations. In the case of the acids whose purity was doubtful, the method of Van Slyke served as a standard. The results are expressed in mgm. of nitrogen per cc.


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The results show quite clearly the excell&t agreement of the calorimetric method with theory and with the method of Van Slyke. It must be pointed out that the solutions of the amino- acids were made of just sufficient concentration to be estimated in the Van Slyke micro apparatus. To determine the amount, calorimetrically, it was necessary to dilute those solutions from two to four times, so that our actual error of determination is less than appears in the table.

The estimation of cystine, however, by this method gives results which are much too low. Moreover, the coloration devel- oped has an intense red color, which makes it almost impossible to compare it with the standard alanine. As a consequence of this it, would be impossible to estimate the amino-acid a-nitrogen in a mixture of amino-acids containing a large proportion of cystine without a large error. Such cases, however, are not very common, and the authors do not think that the amount of cystine, occurring in the hydrolysis’mixture obtained from the majority of proteins, will introduce any serious error. To determine this point and to see if the method would estimate the a-nitrogen in a mixture of amino-acids in the proportions in which they occur in a native protein, an estimation of the amino-acid a-nitrogen in ereptone was made and compared with the Van Slyke method.

- . ..-------

Calorimeter....................... Van Slyke.. . . . . . . . . . . . . .

__~_

r-it PER cc. Ii* -

mym. per cent

0.132 8.5 0.139 9.0

It will be seen that the calorimetric method gives figures slightly lower than the Van Slyke method, but not, low enough to invalidate seriously the result. The difference, however, is capable of easy explanation. The coloritietric method gives too low a result in the presence of cystine. The Van Slyke method gives a result, a little too high in the case of glycine and cystine. These three facts are sufficient,, we think, to explain the difference of 0.5 per cent.

A series of determinations of the amino-acid a-nitrogen in peptones from various sources was next, carried out in order to


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test the efficacy of the method when dealing with partial hydrolysis products of proteins. The following table shows the results to be in good agreement with those obtained by the Van Slyke method.

7-72 PER cc. ORIOIIV OF PErnOHE I

I- ~-

--.--~-.__.

Calorimeter Van Slyke

Meat (Merck). . . . . . . . . . . . . . . . . . .I 0.062 0.061 “Ecarne” (Schuchardt). , . . . . . . ., 0.035 0.031 Precipitated by alcohol (Schu-l

chardt). . . . . . . . . . . . . . . . . . . . . . . 0.025 0.021 Witte.. . . . . . . . . . . . . . . . . . . . . . . . 0.067 0.067

Detemzination of the maximal and minimal amounts of amino-acid a-nitrogen capable of estimation by this method.

The early experimental results described in this paper (page 226) had shown that although some of the amino-acids could be accurately estimated when the solution contained 0.1 mgm. of nitrogen per cc., yet other amino-acids could be estimated only when the nitrogen was only 0.05 mgm. per cc. This value is taken as the maximal value. To determine the minimal value two series of experiments were performed, one on an aIanine solution, the other on an ereptone solution. A standard solution of alanine and a solution of ereptone were diluted by, known amounts of water and calorimetric estimations made of the amino- acid a-nitrogen present in the diluted solutions. The coloring matter produced was diluted to a suitable volume so that the calorimetric readings did not differ from the standard by large amounts. The theoretical amounts given against the results obtained for ereptone solutions were obtained by calculating from the mean result of the calorimetric and Van Slyke determinations given on page 228.

?ngm. 9n!pz. mpm. Alanine. . . . . . . . Found 0.050 0.025 0.012 ctJ5

Per cc. . . . . . . . Calculated 0.050 0.025 0.012 0.006 Ereptone. . . . . Found 0.065 0.032 0.016 0.008

Per cc.. . . . Calculated 0.067 0.033 0.016 0.008


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It will thus be seen that the method is accurate over a range of 0.05 mgm. to 0.005 mgm. per cc. Whether amounts less than 0.005 mgm. can be estimated, we have not determined. At that concentration the amount of coloring matter produced is so small that it can only be measured with difficulty. Qualitatively, however, it is possible to detect as little as 0.001 part of a mgm. of amino-acid a-nitrogen in 1 cc. of solution. This, in the case of alanine, means the detection of one part of the amino-acid in a little over 1,500,OOO parts of water.

The question naturally arises as to whether the reaction will proceed quantitatively in presence of other substances. From the results obtained with ereptone (page 228) and the various peptones (page 229), the method can be used for the determination of the amino-acid a-nitrogen set free in the hydrolysis of proteins. Its application to the analysis of urine and blood, however, is still attended with difficulties, ammonium salts and urea causing disturbances in the reaction. These points are being investigated, and the results will be reported shortly.

SUMMARY.

1. A method has been devised for the estimation of amino-acid a-nitrogen by the use of triketohydrindene hydrate and, pyridine.

2. The method possesses an accuracy equal to that of the Van Slyke method.

3. It will estimate within the ranges of 0.005 mgm. to 0.05 mgm. amino-acid a-nitrogen per cc.

4. The method is inaccurate, however, for cystine. 5. The method is applicable to the determination of amino-

acid a-nitrogen (in neutral solution) set free in protein hydrolysis.


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MacLeanVictor John Harding and Reginald M.

-NITROGENαESTIMATION OF AMINO-ACID

A COLORIMETRIC METHOD FOR THE

1915, 20:217-230.J. Biol. Chem.

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