Enzyme visualization with partially dehydrated agarose substrate layers: detection of paraoxonase after thin-layer isoelectric focusing in agarose.

A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3-naphtyl acetate, paraoxon, 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA-stable. Immunofixation with anti-human serum albumin-antibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and/or cleavage products is the major problem.