PRINCIPLES OF STOPPED-FLOW SEQUENTIAL INJECTION-ANALYSIS AND ITS APPLICATION TO THE KINETIC DETERMINATION OF TRACES OF A PROTEOLYTIC-ENZYME

The concept of sequential injection analysis is described, and an application of this novel technique to an assay of traces of a proteolytic enzyme is demonstrated. The automated method is based on a stopped-flow procedure with fluorescence detection. Instead of using the conventional constant flow rate, a sinusoidal flow pattern, as generated by a robust cam-driven piston pump, has been applied. The validation of the novel flow injection system using a nonreactive fluorescent compound yielded highly reproducible results over a concentration range of 3 orders of magnitude. The calibration graph for the enzyme activities in the range 5-75-mu-u mL-1 could be fitted a second-order polynomial. The detection limit is 2.5-mu-u mL-1, which corresponds to 7.2 ng mL-1 pure active enzyme.