Gel diffusion--a simple and sensitive technique for the assay of proteinase inhibitors and its use for the determination of the ratio of proteinases in mixtures.

In casein-containing agarose gels, pepsin and chymosin form radial diffusion zones; the diameters of these zones show rectilinear correlations with the logarithm of the enzyme concentration at constant time. The sensitivity for both enzymes is below 1 microgram. Addition of the inhibitor pepstatin A to these enzymes causes a reduction of the diameters of the diffusion zones, with large differences for both the enzymes. With this procedure, the pepsin/chymosin ratio in rennet preparations was assayed with an accuracy of +/- 5%. Identification of the inhibitors allows the determination of amounts in the namomole range. This method is a simple technique for the evaluation of proteinases and their inhibitors in screening systems.