THE TRANSGLYCOSYLATIVE ACTION OF TESTICULAR HYALURONIDASE

Abstract

Recently, there was reported the resolution of -digests produced from hyaluronic acid by testicular hyaluronidase into a series of oligosaccharides (2): The sizes of the oligosaccharides isolated ranged from di-through tetradecasaccharide in regular increments of 1 disaccharide unit.As in hyaluronic acid itself (3), equal numbers of glucuronic acid and acetylglucosamine residues are linked in these oligosaccharides in an alternating structure.The free reducing group is on the terminal acetylglucosamine residue.The oligosaccharides represent well defined substrate models of hyaluronic acid, and availability of methods for their individual estimation in mixtures permits a more precise analysis of the mode of action of some mucolytic enzymes than was previously possible.This report is concerned with the application of these substrates and these methods to studies on the action of bovine testicular hyaluronidase.EXPERIMENTAL Meth& and Materials-The qualitative and quantitative paper and ion exchange chromatographic procedures used (2), the isolation and properties of the oligosaccharide substrates (2), the preparation of hyaluronic acid and of a solid oligosaccharide mixture (4), and the conditions used in the Macleod and Robison hypoiodite reducing sugar method (2) have been described previously.The enzyme used in this study, except when otherwise stated, was a bovine testicular hyaluronidase prepared by an alcohol fractionation method, lot No. W-179, kindly supplied by Dr.Joseph &sifter of Wyeth, Inc.It assayed (5) about 1200 turbidity reducing units (abbreviated elsewhere as t.r.u.) per mg.With exceptions as indicated, all incubations were performed at 37" under toluene at a substrate concentfation of 20 mg. per ml. in 0.1 M acetate buffer of pH 5, 0.15 M in sodium chloride, containing 1 mg.per ml. of gelatin.Where specified, cysteine hydrochloride and sodium pyrophosphate, both at 0.01 M concentration, were added.