Membrane Filtration for determining Protein in the Presence of Interfering Substances

Abstract

SEVERAL substances that are present in biological materials and some compounds which are added during protein purification procedures interfere with protein determinations by the biuret and Folin-phenol procedures¹. For example, substances with two or more peptide bonds and ammonium salts give a positive biuret reaction and aromatic amino-acids, uric acid, guanine, and xanthine cause similar difficulties in the Folin-phenol method. Reducing agents such as 2-mercaptoethanol interfere with both procedures. The effects of these substances can be minimized if protein samples are analysed after being precipitated, centrifuged and further washed with trichloroacetic acid (TCA). It, however, the initial protein solution is very dilute and large portions must be taken for analysis, or if only a small amount of protein is to be assayed, problems arise in recovering and washing the precipitated protein free of interfering substances. Micromethods have been devised² to surmount these problems, but for routine analysis a rapid and effective procedure is needed for removing these undesirable substances and determining protein. These requirements appear to be fulfilled in the method reported here. This method involves the analysis of protein precipitated by TCA and then separated from interfering substances by membrane filtration on ‘Millipore’ filters.