pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins. Part III. The electrostatic non-specific hyaluronan-hyaluronidase complex

摘要

The existence of electrostatic non-specific complexes between hyaluronan (HA) and hyaluronidase (HAase) has been demonstrated over a very large pH domain ranging from 3 to 10. At 1 gL~(-1) HA and HAase concentrations and 6 °C, two types of electrostatic HA-HAase complexes have been pointed out: sedimentable slightly charged complexes at pH ranging from 3 to 5 and soluble highly charged complexes at pH ranging from 5 to 10. The HAase over HA mass stoichiometric ratio varies from 2 to 6 for the sedimentable complexes and is maximum at pH ranging from 5 to 6. This characterises the size of the non-specific HA-HAase binding site: one HAase molecule is associated with 23-71 HA carboxyl groups, depending on pH. The dissociation constant of the electrostatic HA-HAase complex has been estimated to 3.8 x 10~(-5) mol L~(-1). The pI value for HAase has been estimated to 5.3 by Zeta potential measurements and is consistent with the theoretical predictions based on the charge state of the HAase molecule as a function of pH. The numbers of basic and acidic amino acids of HAase are quite equal and constant over the 5-9 pH domain, including pH 7. This favours the existence of positive patches on the surface of the HAase molecule, consistent with the formation of electrostatic HA-HAase complexes over this pH domain. This supports that HAase may be present under an inactive bound form in the extracellular matrix together with HA.