Volume 3, Number 2, pp. 35-44
J.-C. Vincent, T. Asteriou and B. Deschrevel
Laboratoire “Polymères, Biopolymères, Membranes”, UMR 6522 Universitè de Rouen – CNRS, 76821 Mont-Saint-Aignan CEDEX, France
Kinetics of hyaluronan hydrolysis catalysed by hyaluronidase.
Determination of the initial reaction rate and the kinetic parameters
Hyaluronan (HA) hydrolysis catalysed by testicular hyaluronidase (HAase)
can be followed as a function of time by using a couple of techniques including
the N-acetyl-D-glucosamine reducing end assay. Many research groups have
reported hyaluronidase assays using end point measurements, although the
time course is generally not linear. By using our improved version of the
method of Reissig et al., we have shown the exponential type of evolution
of the kinetics. A mono-exponential model has been established by considering
the cleavable ß(1,4) bond as the substrate entity instead of the HA
chain molecule. This modelling is in good agreement with the experimental
points at the beginning of the reaction, but does not perfectly fit them
in the later period. The HA chain length seems to be a parameter that should
be taken into account in the kinetic equation. Thus, two different types
of equation have been tested to obtain a better fitting of the whole experimental
kinetics. It appeared that a bi-exponential equation gave the best fitting
for all the kinetics. Hypotheses involving the effect of the HA chain length
on the mode of action HAase have been suggested about that equation. These
hypotheses concern the stability of the enzyme-substrate complex, the proximity
of cleavable HA sites leading to possible processive processes and the possible
transglycosylation reaction instead of hydrolysis. The consequence is that
the apparent kinetic constant of the reaction is different at the beginning
and at the end of the overall process. The bi-exponential equation being
the most suitable model for determining the initial reaction rate, we used
it to study the substrate-dependence of the reaction and determine its kinetic
parameters.
Keywords: initial reaction rate, kinetic modelling, reducing
end assay, substrate-dependence, substrate chain length effect, testicular
hyaluronidase