Uricase and its action

Experimental methods.Preparation of uricase.Uricase was in some cases prepared according to the method described in Part vii [Truszkowski, 1934] (this will be termed Method I).The following procedure (Method II), which has the merits of economy of material, time and labour, was later adopted.The extracts are more highly coloured than are those of uricase prepared by the previous method, but for many purposes they serve equally well.Minced ox kidney was placed in a 10-litre bottle, and cold water, entering the bottle through a tube reaching to the bottom, was run through until the super- natant liquid became quite clear and colourless.The residual tissue, freed in this way of its soluble constituents as well as of smaller insoluble particles, was dried at 370 in a current of air, and the dry residue was ground to an impalpable powder in a mill.The yellow powder so obtained exhibits considerable uricolytic activity, and on extraction with aqueous Na2CO3 affords deep yellow, active extracts, often possessing a disagreeable odour.The quality of the product is greatly improved by repeated washing on a Buchner funnel with 96 % alcohol at room temperature, followed by ether; the washing is continued until the filtrate becomes colourless.The residue is then washed four times with anhydrous alcohol- free ether, spread in a thin layer and dried at 300 under reduced pressure.The dry uricase so obtained is a light yellow powder, which can be stored for many months without deterioration, provided that traces of solvent (water, alcohol, ether) are absent, and that undue exposure to the air is avoided.The yield is roughly 100 g. per kg. of kidneys.Solutions of uric acid were prepared by adding uric acid to boiling aqueous NaOH (65 ml. of 0.1 N NaOH per g. of uric acid).The solutions so obtained had pH 8-6-9-0.Uric acid was determined colorimetrically by the method of Folin and Denis.Standardisation of conditions of determination of activity of uricase.10 flat- bottomed test-tubes (of uniform calibre), containing various volumes of a mixture of uric acid and uricase solutions and 0 5 ml. each of toluene, were kept at 370 for 48 hours, the contents of the tubes were mixed once by inversion, and uric acid was determined.The results, given in Table I, indicate that the apparent activity of the uricase diminishes at first rapidly, and then more slowly, with increasing height of the column of the solution.Since the only difference between the systems consists in the supply of oxygen to the lower layers of solution, it may be concluded that dissolved oxygen in the body ofthe systems is rapidly consumed, and that further oxidation can take place only at the expense of fresh oxygen diffusing into the ( 2787 )