The construction and testing of a simple, slow delivery-rapid quench apparatus

Abstract

A simple, inexpensive, slow delivery-rapid quench apparatus is described. The apparatus can be used to mix small volumes (about 50 .mu.l) of equilibrium mixtures of enzyme-substrate and enzyme-product complexes with a quenching solution, ideally to inactivate the enzyme more rapidly than such complexes can be interconverted. The efficiency of the apparatus is tested by injecting basic solutions of an indicator dye into acid and observing the length of the unbleached plume of dye produced at the delivery tip, and by forming an enzyme-substrate complex with 32P-labeled phosphoglucomutase and measuring the extent of label transfer prior to inactivation by the quenching solution. Problems that may be encountered in attempts to inactivate equilibrium mixtures of enzyme-substrate and enzyme-product complexes without producing quenching artifacts are considered.