Effect of Ionic Strength and pH on the Properties of Purified Bovine Testicular Hyaluronidase

Abstract

Studies on the effect of pH and ionic strength upon the activity of purified bovine testicular hyaluronidase have shown that the pH optimum for the hydrolysis of hyaluronic acid occurs at 5.2 in the presence of, and at 6.0 in the absence of NaCl. Hydrolytic activity towards various mucopolysaccharide and hyaluronate octasaccharide substrates was dependent upon the presence of strong electrolyte (LiCl, NaCl, KCl, CsCl, NaNO3 and Na2SO4), maximum activity being obtained at electrolyte strengths of 0.2. Identical weights of sulphated and unsulphated mucopolysaccharides were hydrolysed at similar rates under optimal conditions, except that double chains of chondroitin 4-sulphate were hydrolysed at twice the rate of the other polysaccharides. Hydrolytic activity towards hyaluronate hexasaccharide was favoured at pH values below 5.2 whereas transglycosylation activity was favoured at higher pH. Hyaluronate tetrasaccharide was neither a substrate for the hydrolytic or transglycosylation activity, nor was it an inhibitor of the enzymic hydrolysis of hyaluronic acid. No conformational change in hyaluronic acid was detected by CD-spectroscopy in the presence of varying concentrations of salt and the collective results suggest that the salt effect is exerted on the enzyme rather than on the substrate.