The Biuret Reaction of Proteins in the Presence of Ethylene Glycol

The biuret method offers some advantages as a simple, rapid photometric method for the determination of proteins. In the methods which have been proposed, precipitation of cupric hydroxide gives rise to certain difficulties (1, 2). It occurred to us that the precipitates might be avoided by the addition of a reagent which would form a compound with copper which was soluble in alkali, but of such a nature that the copper could still react with protein. Ethylene glycol has been examined and found suitable, and with the use of this substance it is possible to prepare a single, alkaline copper solution which may be added to the protein for the development of the biuret color. Absorption Spectra-The absorption spectrum of the complex of cupric ion and ethylene glycol is shown in Curve B of Fig. 1. The concentration of ethylene glycol was 8.0 ml. per 100 ml., and that of sodium hydroxide was 2.5 gm. per 100 ml. (hereafter concentrations expressed in these terms will be referred to as per cent). The values of E:?m. are calculated in terms1 of the total copper concentration, and were obtained with solutions in which the concentration ranged from 0.001 to 0.12 per cent. One maximum is observed at 630 rnp (@!& = 4.75) and another at 297 rnp (E:?,,,. = 64.~), with a minimum in the region from 380 to 430 rnp. The absorpt’ion spectrum of the egg albumin-copper complex is shown in Curve A of Fig. 1, and was determined by subtracting the blank due to the copper and ethylene glycol reagent and that due to the protein in the ultraviolet region. The values of I*: :‘%. are calculated in terms of the protein concentration, and maxima are found at 545 rnp (E:&. = 2.58) and at 3G0 rnp (E:3m. = ll.~), with a minimum at 430 mp. The position of the maximum reported by Sizer (3) is 552 m,u. The position of the maximum found for mixed serum proteins in the present study is