Determination of trace amounts of a proteolytic enzyme by fia with a stopped-flow and an on-line preconcentration technique.
An automated method for the determination of small amounts of proteolytic enzymes has been developed, by combining a stopped-flow and an on-line preconcentration technique, using sorbent extraction. It is based on the hydrolysis of a synthetic chromogenic substrate during the stopped-flow period, yielding p-nitroaniline, which is then preconcentrated on a C-18 column. The p-nitroaniline is then eluted from the column to the spectrophotometer flow cell with methanol. In operation, the substrate solution (500 mul) is injected into 0.1M tris-HCl buffer carrier and merged with the sample stream. The mixed plug is stopped in a waterbath, kept at constant temperature, for reaction. The operation of the manifold, including data acquisition, is controlled by a personal computer. Optimal parameters were 40 degrees , pH 8.3, a substrate concentration of 5 mM and wavelength 400 nm. The detection limit, using a 30 min stopped-flow reaction time, is 0.1 muU protease/ml. The method is intended to be used in combination with portable low volume air samplers for monitoring workplace enzyme dusts in the detergent industry.
Gübeli
T
T
Department of Chemistry, BG-10, University of Washington, Seattle, Washington 98195, U.S.A.
Růzicka
J
J
Christian
G D
GD