GB1060513A - Process for the preparation of glucuronoglycosaminoglycan hyaluronate lyase - Google Patents

Abstract

Hyaluronidase (glucuronoglycosaminoglycan hyaluronate lyase) is prepared from animal organs or products thereof containing the said enzyme, by: i) macerating with aqueous acetic acid containing 2.5 to 10% by volume hydrochloric acid, and centrifuging off the solids, ii) treating the supernatant with 21 g. ammonium sulphate per 200 mls. solution and removing the precipitate, iii) treating the resulting supernatant with 56.5 g. ammonium sulphate per 200 mls. solution, collecting the precipitate, and dissolving it in the minimum amount of water, iv) ultra-filtering this solution to 1/10 of its original volume, and dialysing the concentrae against water, v) treating the dialysate with two volumes of chloroform, separating the aqueous layer, centrifuging off any precipitate, and adjusting to pH 2.3, and standing for 1 to 15 minutes, then adjusting to pH 4.8, and removing the precipitate, vi) treating the clarified solution with 10 g. ammonium sulphate per 100 mls, and collecting the precipitae, vii) dissolving the precipitate in water and dialysing against physiological saline, viii) chromatographing the dialysate through a column of cross-linked sulphonated polystyrene ion exchange resin and eluting with pH 1.5 KCl/HCl buffer, ix) adjusting the pH of the elurate to 6.0 and ultra-filtering to 1/20 of its original volume and x) lyophilising the concentrated solution, if required. The product may be further purified by preparative electrophoresis on starch gel or polyacrylamide gel. Suitable starting materials are bulls' testicles or liver lysozymes. The product is said to have an activity of at least 21,500 International units and to be almost free from contaminating enzymes.