Quantitative determination of serine proteinase inhibitor activity using a radial diffusion assay

Abstract

An improved, time efficient, visual assay for quantitative determination of proteinase inhibitor activity in protein extracts is reported. Proteinase inhibitor activity of mammalian, bacterial, and fungal serine proteinases can be quantified. The method relies on radial diffusion of proteinase inhibitor containing extracts from a central well through an agar gel containing a serine proteinase. After an incubation period the agar gel is stained via the diazo coupling of the beta-naphthol produced by the enzymatic hydrolysis of N-acetyl-DL-phenylalanine-beta-naphthyl-ester. Circular zones containing inhibitor-proteinase complexes remain colorless while the region containing only proteinase shows a bright pink-purple color. A reference curve relates the diameter of the colorless zone to the logarithm of the proteinase inhibitor concentration. The error in the estimation of a proteinase inhibitor quantity varying between 10 and 1000 pmol is 4-12%. The sensitivity of the assay is approximately 2-20 pmol of inhibited proteinase molecules depending on the inhibitor-proteinase complex assayed. The sensitivity of the assay can be enhanced 10-fold or more by dilution of the proteinase concentration in the agar and by a reduction of the agar thickness.