Identification of protease isozymes after analytical isoelectric focusing using fluorogenic substrates impregnated into cellulose membranes.

Abstract

A technique to characterize active isozymes of proteases is described. After isoelectric focusing in an ultrathin gel, a cellulose diacetate or hydrate membrane previously impregnated with media containing a fluorogenic substrate is applied to the surface of the gel. Low-molecular-weight peptide inhibitors can also be added to the membrane to enhance specificity. The technique shows excellent reproducibility, specificity, and sensitivity, and has promise for the study of isozymes of proteases in tissues and body fluids in normal and pathological states.