Improvements relating to the purification of labile enzymes contaminated with hemolysiní¬o

摘要

Labile enzymes contaminated with haemolysin-O are purified by treating aqueous solutions thereof with a solution of cholesterol in an organic liquid protein-precipitant, removing the resulting precipitate, adding water and separating the insoluble cholesterol-haemolysin complex and free cholesterol from the enzyme solution; the amount of cholesterol should be sufficient to combine with all the haemolysin, i.e. at least 0.01 mg. (and preferably 0.2-2.0 mg.) per 10,000 Bernheimer units thereof; the temperature is kept below 5 DEG C. throughout. The process is especially applicable to enzymes obtained by bacterial fermentation, with or without preliminary purification. The organic liquid may be ethanol, methanol, acetone or ether; another liquid may be added to increase the solubility of the cholesterol. Sufficient liquid protein-precipitant should be added to give a concentration of 5-60 per cent by volume; when the concentration is greater than 40 per cent the temperature should be kept below 0 DEG C., and in general it is desirable to keep the temperature within 5 DEG C. of the freezing point of the mixture. By suitable control of concentration, ionic strength, temperature, pH and amount of organic solvent it is possible to retain some non-enzymatic protein material in solution when all the enzymes have been precipitated. To ensure that all the haemolysin is in its reduced form it is desirable to add a mercapto compound before the cholesterol. The cholesterol complex is often too fine to be readily separated from the reconstituted solution and it is therefore advantageous to add protamine (0.005-0.02 parts by weight of total N in the solution), thereby precipitating non-enzymatic proteins in addition; this method is described in Specification 715,572. Examples are given of the purification of a mixture of streptokinase and streptodornase, with and without protamine treatment.