A simple rapid biuret method for the estimation of protein in samples containing thiols

Interference in the Lowry protein determination by thiol compounds is now well known (1–3). We have found that the estimation of protein by the biuret reaction is also subject to interference when the protein sample contains various thiols. We wish to report that this interference can be prevented in most cases by using a biuret reagent which is chelated with ethylenediaminetetraacetate (EDTA). In samples containing dithiothreitol (DTT) it is also necessary to add iodoacetamide prior to the addition of the biuret reagent. The use of iodoacetate to eliminate thiol interference in the Lowry procedure has been reported previously (3). This report details the extent of interference of dithiothreitol, β-mercaptoethanol, β-mercaptoethylamine, and glutathione, and illustrates the extent of neutralization which is attained in each case. We have also introduced modifications which permit the development of a stable color in only 5 min.