Electrophoretic detection of chitinase isoenzymes using the PhastSystem

Zymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter‐staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4‐methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4‐methylumbelliferone). The p I s of chitinase isoenzymes were estimated by using coloured p I markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii .