A N A L Y T I C A L BIOCHEMISTRY 64, 2 8 4 - 2 8 8

(1975)

An Improved 2,4,6-Trinitrobenzenesulfonic Acid Method for the Determination of Amines The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1-3) since its introduction in 1960 by Satake et al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15-30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS. EXPERIMENTAL Materials. TNBS (Eastman) and a crystallized and lyophilized preparation of bovine serum albumin (Sigma) were used without further purification. All amino acids, amines, and salts were reagent grade. Amino compounds were dissolved in 0.10 M sodium tetraborate, pH 9.3. Procedure. Twenty-five microliters of aqueous 0.03 M TNBS is added to 1 ml of sample contained in a cuvette, agitated to ensure complete mixing, and allowed to stand for 30 rain at room temperature, 25 + 1° C. The reagent blank consists of 25 /~1 of 0.03 M TNBS in 1 ml of 0.10 M borate. Absorbance is read at 420 nm.

RESU LTS The absorption spectrum of the reaction product(s) of glycine and TNBS in pH 9.3, 0.1 M borate is shown in Fig. 1. This spectrum is nearly identical to that obtained by Mokrasch for glycine in 65% (v/v) methanolic borate solution (5). The time course of the trinitrophenylation of glycine at three different concentrations in the presence of a constant amount of TNBS in 0.1 M borate, pH 9.3, was measured using the 284 Copyright © 1975 by Academic Press, Inc. Printed in the United States. All rights of reproduction in any form reserved,

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FIG. l. Product Absorption Spectrum of Glycine + T N B S _ T N B S = 7.5 × 10 4 M, g l y c i n e = 5.5 X 1 0 - S M , p H 9.3 0 . 1 0 M borate.

Conditions:

appearance of the absorption peak at 420 nm (Fig. 2). These results show that the trinitrophenylation reaction is virtually complete within 30 rain. Furthermore, the first-order rate constants calculated from plots of In (,4® - A T ) versus time for the data shown in Fig. 2 were found to be insensitive to the initial glycine concentration. Each run gave a firstorder constant (kob,0 of 0.17 min ~. The reaction-progress curves (Fig. 2) show that under the conditions employed in our experiments it is unnecessary to quench the trinitrophenylation reaction, since at this time the color development is greater than 98% complete. Indeed, the effect of such a quenching step would be to dilute some of the color formed 0.g

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