Abstract: Three methods are described for spectrophotometric reaction rate determination. The first was based on an optical biosensor, which integrated reaction and detection in the flow cell; the second was based on the reversal of flow direction and passing of the sample plug iteratively through the enzyme reactor and the detector; and the third was based on a cyclic system that allowed the sample plug to be trapped in a circuit, which contained the enzyme reactor and detector. Optimization of variables specific to each method is discussed. The reaction rate of the hydrolysis of glucuronides catalyzed by β-D-glucuronidase was studied, and a critical comparison of the three techniques was made. Based on reaction-rate measurements, a parameter was defined to compare the features of the methods; no significant differences were found for the biochemical system studied.

Abstract: The activity of β-glucuronidase (I) in serum was assayed by monitoring its catalysis of the conversion of a p-nitrophenyl derivative to p-nitrophenol (II) using an optical flow-cell. Sample solution in 100 mM dipotassium hydrogen phosphate buffer of pH 6.8 (buffer A) was injected into a carrier stream of buffer A which was merged with the stream of 0.32 mM p-nitrophenyl-β-D-glucuronide in buffer A. After passing through a 250 cm reaction coil and merging with a stream of 10 mM NaOH, the stream was passed through a 50 cm reaction coil, followed by a Dowex-1 anion-exchange resin support (chloride form; 2% cross-linked and 100-200 mesh) packed in the flow cell, where I was retained; elution was effected with 0.1 M ethylenedinitrilo tetra-acetic acid disodium salt using a switching valve. All flow rates were 1.7 ml/min. The retention-elution process was monitored continuously at 405 nm. The calibration graphs for I and II were linear from 0.1-20 iu/l and 0.1-5 µg/ml, respectively with corresponding RSD of 1.7-3.0% and 2.0-2.4%. The sampling rate was 20 samples/h with recoveries of 95-104%.