QUANTITATIVE BIURET TEST FOR MILK SERUM PROTEINS

Department of Dairy and Food Industry, University of Wisconsin, Madison The biuret reaction has been used for many years as a qualitative test for the presence of proteins in solution. It depends on the formation of a violet copperprotein complex in alkaline CuSQ solution. This reaction first was adapted as a quantitative test for protein by Autenrieth (1, 2), who determined the albumin and globulin in urine, ascitic fluid and blood serum.

A nmnber of modifications of the biuret test have been suggested, primarily to obtain solutions free of turbidity and to increase the sensitivity of the test.

Kingsley (7) recommended concentrations of 9 per cent NaOH and 0.1 per cent CuSQ in the test solution. He reported that the color complex was fully developed in 30 min. and remained stable until turbidity appeared in about 1 hr.

Later, Kingsley (8) recommended centrifugation to obtain clear tests. Robinson and Hodgen (13) altered the reagent to 3 per cent NaOH and 0.5 per cent CuSO4, and centrifuged and filtered to obtain a clear solution. Mehl (9) found that 0.05 per cent Cu was needed to give an excess for plasma protein concentrations up to 0.15 per cent.

Weiehselbaum (14) and later Wolfson et al. (]5) used a reagent similar to Fehling's solution and did not comment on trouble with turbidity. Trials in this laboratory have shown that the reagent used by Weichselbaum caused turbidity and was not sensitive to low concentrations of milk serum protein.

A quantitative biuret test has been reported by Molnar (12) for total protein in milk and in mixtures of milk and egg albumin. In this test the proteins are precipitated with triehloroacetic acid, centrifuged, the supernatant decanted and the proteins put into solution with alkali. Color is developed in this solution by addition of CuSQ. The method is satisfactory for protein concentration from 1 to 15 per cent. Below I per cent the color intensity is very low and the values range much below those obtained by the K.jeldahl method.

Although the biuret method is widely used in serum protein analysis, the method has not been standardized and difficulty with turbid solution still is experienced.

The purpose of this study was to develop a simple, sensitive biuret method for the measurement of milk serum proteins during fractionation.