Random coil scission rates determined by time-dependent total intensity light scattering: hyaluronate depolymerization by hyaluronidase.

ABSTRACT

A recently developed theory of the light scattering by random coils undergoing random scission is applied to the digestion of hyaluronate by hyaluronidase. The time dependence of the scattered light from solutions undergoing digestion was monitored. Working at a high angle with high molecular weight hyaluronate allowed the use of a powerful approximation for determining initial velocities and the Henri-Michaelis-menten coefficients, without explicit knowledge of the hyaluronate molecular weight, radius of gyration, second virial coefficient, or polydispersity. Effects due to a molecular weight dependent second virial coefficient and to non-Gaussian behavior are briefly considered. Assays were performed over nearly two orders of magnitude in substrate concentration. The initial velocities are compared with those obtained by a standard reducing sugar assay, which was performed on identical samples. The main advantages of the light scattering assay procedure over the more traditional assays are that many relatively high-precision data points can be quickly and automatically collected with simple apparatus, and that the technique is most sensitive for the initial period of digestion, where the other assays are least sensitive. The shapes of the scattering curves also provide evidence that hyaluronate in these solutions is not a stable double strand and that the hyaluronidase cleaves bond randomly. The curves also indicate that enzyme deactivation occurs, which accounts for the lower velocities yielded by the slower reductimetric assay, which is measured over longer initial periods.