Natural protease inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis

An electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors is reported. This assay is particularly suited for investigations of crude biological materials when specific antisera are not available. The supporting medium consists of agarose into which denatured fibrinogen is incorporated as the substrate for proteases. The processing then is divided into two steps: (1) electrophoretic resolution of the inhibitor containing material and (2) detection of the inhibitor bands through their protease inhibiting activity. The inhibitor position is thus made visible as a colored band of denatured fibrinogen which has escaped digestion by protease. By electrophoretic separation of multiple copies of a sample of biological fluid followed by soaking each of them in the solution of a distinct protease, the enzyme specificity of a particular inhibitor band can easily be established. The bands can in selected cases be quantitated accurately by densitometry and the inhibitor activity thus determined using a reference serum calibrated with Trasylol as a standard. The activity of alpha-1-protease inhibitor in healthy horses is reported.