The use of ninhydrine reaction in the quantitative determination of amino acids1

Transcription

1 The use of ninhydrine reaction in the quantitative determination of amino acids1 BY Artturi I. Virtanen and Tauno Laine (Biochemical Institute, Helsinki) The methods for the quantitative determination of amino acids are of utmost importance in biochemical investigations. For the determination of amino nitrogen the excellent method of van Slyke (1911) is employed everywhere. This method cannot, however, be employed when very small amounts of amino-nitrogen are to be determined, whereas the colour reaction of ninhydrine with amino acids ssems to be especially suitable for the determination of amino nitrogen in very dilute solutions, if the reaction is accomplished under suitable experimental conditions. We are presenting in the following our observations on the reaction and the determination method as we have developed it. The employment of ninhydrine reaction for the determination of carboxyl groups in amino acids has been recently introduced by van Slyke and Dillon (1938). The reaction seems to be very vigorous in this respect. On the basis of our observations, the ninhydrine reaction can be well applied also to the quantitative determination of certain individual amino acids, by determining the aldehyde formed in the reaction. The method for the determination of a-alanine is based upon this fact, and will be described below. In 1910 Ruhemann (1910, 1911) noted, that triketohydrindenhydrate or ninhydrine, discovered by him, reacted when heated with amino acids and formed a bluish-red colouring substance. The reaction with free amino acid occurs according to Ruhemann in the following manner. 1 Received for publication June Ist., 1938.

2 THE USE OF NINHYDRINE REACTION IN THE QUANTITATIVE ETC. 393 ninhydrine /\ / \, 1,-CO oc-- fi diketohy drindylidenedike tohydrindamine The mechanism of the reaction is not yet more accurately known. The resulting products of the reaction are acc. to Ruhemann, a colouring substance (diketohydrindylidenediketohydrindamine) and carbon dioxide, and an aldehyde, containing one carbon atom less than the amino acid, and water. Several investigators have employed the formation of the colouring substance in the reaction for the colorimetric determination of amino acids. As to the specificity of the ninhydrine reaction, Cherbuliez and Herzenstein (1934) have considered it to be specific only to the grouping - CO-CH (NH,) -. However, according to the earlier investigators, also the amino groups wich are at a greater distance from the carbonyl group, would react slowly with ninhydrine. E. Abderhalden and H. Schmidt (1911) employed the reaction qualitatively for detecting dialysable protein compounds formed in proteolysis. E. Herzfeld (1914) used the reaction for spectrophotometric determination of amino acids. Under the conditions he made his determinations, many other substances, however, interfered harmfully, as the reaction mixture was allowed to evaporate to dryness. H. Riffart (1922) worked out already a much better method. He accomplished the reaction with phosphates in a puffered solution at exactly ph 7. Only ammonium salts and amines interfered with the reaction. He investigated the behaviour of several amino acids as well as of their mixtures in the reaction and obtained fairly satisfactory results. M. Polonovski and Fr. Moreno-Martin (1935) published a colorimetric method distinctly superior to the former ones. They made the determination in a dilute acetic acid solution, saturated with (NH,),SO,. This acted as a condensing substance in the

3 394 ARTTURI I. VIRTANEN AKD TAUNO LAINE reaction, and the reaction occurred more completely than acc. to Riff art. The sensitiveness of the reaction was thereby considerably increased. Heating was accomplished on the water-bath for I 5 minutes. The precipitated colouring substance was extracted with cloroform and further with niroo soda solution, since in an acid solution the colouring substance is very susceptible to light, whereas in a slightly alkalinc solution it is very stable. The method is, however, deficient even in the form Polonovski and Moreno-Martin have used it, since it does not pay sufficiently attention to the effect of ph in the reaction, although this fact, according to our observations is of utmost importance to the quantitativeness of the result. The condensing substance (NH,),SO, namely reacts within a wide acidity range with ninhydrine. Our experiments with ammonium sulphate solutions, with varying ph values in different experiments, showed that the ninhydrine gives a distinct colour with ammonium sulphate at ph 2,5-7,0. If the ph of the solution is about 2-2,2, no colour is any longer formed with ammonium sulphate, whereas amina acids react fully quantitatively at this ph. As we worked out the method we desired to apply it to the Pulfrich-photometer. For this purpose we had to ascertain that the colouring substances formed in the reaction of amino acids with ninhydrine were really spectroscopically identical. We prepared the colouring substances with four different amino acids, viz. aspartic acid, alanine, tryptophane, and leusine acc. to the method described below. The absorption spectra in the visible range were photographed with Zeiss spectrograph and determined photometrically. The adsorption curves of the colouring substances were in each case identical, their maximum being around 567 mp. The determination of amino nitrogen by ninhydrine method 750 mg of (NH,),SO, for analysis was weighed into a test tube of dark glass. I ml of the amino acid solution under investigation (2-12 mg amino-n per I litre, 0.04ml of acetic acid and 0.2ml of I o/o ninhydrine solution were added (Ninhydrine Bayer ). If the amino acid is well puffered more acid must be added since the ph of the solution must not exceed 2,2. The heating was accomplished on a boiling water-bath for 15 minutes, whereupon the tube was rapidly cooled under a running tap. The precipitated colour was extracted with altogether I o ml of cloroform by repeating the extraction several times with small amounts of the solvent in a separatory funnel of dark glass. Chloroform was transferred into a 50ml funnel, n/ioo soda

4 THE USE OF NINNYDRINE REACTION IN THE QNANTITATIVE ETC. 395 solution was added and the funnel was shaken, whereby the colouring substance was dissolved with a beautifully blue colour. Appr. 45 ml of soda solution was employed. It was further filtered into the measuring flask and made up to exactly 50 ml. The colour remained very stable. Colorimetrical det'ermination was made with Pulfrich-photometer, using the solution of the blank test (reaction solution without amino-nitrogen) in the oth8er chamber. Extinctions with 1-aspartic acid Chamber 3 cm and filter S. 57. Amino-N, mg per litre s I0,O 1.50 Amino-N in the determination ZP v5 I0,O 15.0 Extinctions 0,06 0,IZ 0,18 0,24 0,39 The reaction of other amino acids proceeds in the same manner. We have obtained the same extinctions with glycine and alanine. Error limits heve bmeen in our determinations with pure amino acids within f 59'0. Determination of a-alanine by ninhydrine method As was already mentioned above, an aldehyde containing one carbon atom less than the corresponding amino acid is formed in the reaction of ninhydrine with amino acids. R. Abderhalden (1938) who has prepared several aldehydes from amino acids by ninhydrine reaction, has succeeded also in obtaining acetaldehyde from alanine. According to him acetaldehyde is not formed from othcer amino acids. No aldehyde at all was formed from glycine, serine, cystine, and histidine. We have now observed, that under suitable conetitions acetaldehyde is formed quantitatively from a-alanine through the effect of ninhydrine, and a-alanine can thus be determined in an easy and simple way. The determination was made with an apparatus designed by Lieb and Zacherl (1932) for the determination of lactic acid. ioml of amino acid solution was placed into the reaction flask, which contained mg alanine. 7.5 g of solid (NH,),SO, was add'ed as condensing substance and thereupon 0.5 g citric acid. After the solution had started to boil, 4 ml of IO/O ninhydrine solution was

5 396 ARTTURI I. VIRTANEN AND T.AUNO LAINE introduced through the side tube and the acetaldehyde which was formed was arrested in the receiver, containing ro/o Xa bisulphite. Heating was accomplished above a small burner for 30 mins. The titration was made according to Lieb and Zacherl iodometrically. - a- Alanine added, mg 085 1,o ! a-alanine found, mg % Recovery 0, ,OI I I,87 94

6 THE USE OF NINHYDRINE REACTION IN THE QUANTITATIVE ETC. 397 can be removed from the reaction solution by steam distillation, there are no greater difficulties in the determination of aldehydes. We shall continue our investigations on this line. Summary Modifications have been suggested to the ninhydrine reaction for the quantitative determination of amino nitrogen. These improvements reduce the experimental errors. The reaction must be accomplished at ph 2-2,2, as the ammonium sulphate, employed as a condensing substance, forms colour with ninhydrine already at ph 2,5 and above that. The ninhydrine reaction yields good values, if the solution to be investigated contains mg amino-nitrogen per I litre. a-alanine can be determined easily and with satifactory accuracy by determining the acetaldehyde formed in the reaction. From other amino acids investigated, aspartic acid and Palanine give some acetaldehyde. A method for determination of a-alanine has been developed. References Abderhalden, E. and Schmidt, H., 2. physiol. Chem Abderhalden, R., Ibidem Cherbuliez, E. and Herzenstein, A., Helo. Acta Herzfeld, E., Biochem Lieb, H. and Zacheri, M. K., 2. physiol. Chem Polonovski, M. and Moreno-Martin, Fr., C. r. SOC. Biol. Paris Riffart, H., Biochem Ruhemann, S., J. Chem. SOC s. Ruhemann, S., J. Chem. SOC a van Slyke, D. D., J. Biol. Chem van Slyke, D. D. and Dillon, R. T., C. R. Trav. Lab. Carlsberg Ser. chem Virtanen, A. I. and Laine, T., Enzymologia