Protein and Peptide Determination Based on the Modified Biuret Procedure: Implications for Various Biotechnologies

Abstract

Spectrophotometric methods for total protein analysis are generally simple, rapid and sensitive. Such sensitive protein assays may have applications in forensic science, in the detection of protein contaminants in drugs and in a number of other applications of research interest. Biuret reaction with proteins and peptides is widely used in clinical and biological laboratories. In this work, instead of copper sulphate, sodium hydroxide and Seignette salt, we used insoluble copper phosphate, potassium or sodium hydroxide and ethyl alcohol for the preparation of the biuret reagent. Absorbance of the biuret complex was recorded both at 219-230 nm (after dilution) and around 550 nm against a reagent blank. Amino acid interference was investigated around 550 nm at the same concentration as proteins. The sensitivity of the method at 226 nm was greater than those of other spectrophotometric assays (old biuret method, Lowry, and BCA) with a LOD of about 0.5 �g mL?1 BSA. The new variants of the biuret method for total protein analysis eliminate the need for precise reagent addition and vortexing inherent in the widely used Lowry method, providing flexibility of application. The method developed, which uses an alkaline-alcoholic reagent and insoluble copper phosphate, is simple, rapid, reproducible and sensitive; it is not influenced by detergents, solvents and buffers containing ammonium and is flexible enough to change the analytical protocol when necessary. A discussion was made on the applications of protein and peptide determination with the new biuret assay.