Absolute and empirical determination of the enzymic activity and kinetic investigation of the action of hyaluronidase an hyaluronan using viscosimetry

Abstract

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 K(m) 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.