The Biuret and Ninhydrin Tests for Proteins as Measured with Hardy's Spectrophotometer

Abstract

Color tests for proteins have long been in use, although in most cases the chemistry involved is not clearly understood. One method of attack upon this problem is to analyse the transmission spectra of the colored solutions with the aid of Hardy's recording photoelectric spectrophotometer. This has been done for the ninhydrin and biuret reactions.

The ninhydrin test was performed according to Koch 1 by adding 2.5 cc. 0.1 % ninhydrin to 10 cc. protein solution, and boiling for one minute. The cloudy blue to red solutions were filtered before measuring their transmission of light in the various regions of the spectrum. For the ninhydrin test on 10% bacto-peptone (Fig. 1, upper curve) the percent transmission of light by the colored solution increases continuously from 2% at a wavelength of 400 mμ to 88% at 700 mμ. A very similar curve is obtained for the ninhydrin test on 20% casein (Hammarsten). Digestion of these 2 proteins by 0.25% trypsin (Fairchild and Foster) at about pH 8 and at 50°C. caused very little change in the shape of the curve, with a slight decrease in percent transmission over the. middle of the spectrum. In all cases where digestion was studied, the course of the reaction was followed by means of the formal titration. Curves for the increase in carboxyl groups as a function of time were very similar to those obtained by Northrop and Kunitz. 2

The biuret reaction was performed according to Koch 1 by adding 5 cc. 10% NaOH to 5 cc. protein solution, and then adding 20 drops 1 % CuSO₄. The cloudy or clear red to purple solutions were filtered before analyzing with the spectrophotometer. The test was performed on 2 preparations of 20% casein, a 10% solution of bacto-peptone, 3.5 and 5% tgg albumen, and 5% edestin.