We describe an investigation of the action of hyaluronidase on hyaluronan using viscosimetry. A new viscosimetric approach was developed for determining the activity of the enzyme in katal units. This approach requires knowledge of several parameters (e.g. Mark-Houwink constants) which were determined by combining viscosimetric measurement and gel-permeation chromatography analysis. Using all the necessary parameters we determined the kinetic parameters of the enzyme and found that 250 i.u. correspond to 1 nkat. An empirical viscometric was used to estimate the activity of the enzyme, and the Km was determined using the kinetic dilution method. The estimates produced by the absolute and empirical approaches were in good agreement. We demonstrate that the empirical estimation of the reaction rate is related to the rate of reaction expressed in absolute units and thus provides a good estimate of enzyme activity. Furthermore, we have found an empirical relationship which enables investigation of the kinetics of the enzyme in a simple and sensitive way by viscosimetry.
In this paper we describe kinetic investigations of the action of testicular hyaluronidase on hyaluronan. We have compared the use of two spectrophotometric assays, the first based on the Morgan-Elson reaction and the second on the neocuproine reaction. With the neocuproine reaction Km was found to be 0.46 mg/ml and Vmax to be 126 nmol l-1 s-1. Because of a low sensitivity and the production of interfering precipitates, the Morgan-Elson assay cannot be used for kinetic investigation of the enzyme. Furthermore this assay is prone to interference from compounds such as disodium cromoglycate, (+)-catechine, penicillamine, CaCl2 and acetate buffer.
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