Obtención de catalasa cristalina

CONVENIENT

METHODS CATALASE
BY HENRY TAUBER

FOR PREPARING CRYSTALLINE FROM COW LIVER
AND EDWARD L. PETIT

(From

the Venereal Service, School

Disease of Public

Experimental Laboratory, United States Public Health Health, University of North Carolina, Chapel Hill, North Carolina) for publication, October 21, 1951)

(Received

The preparation of crystalline catalase from beefliver was first reported by Sumner and Dounce (1, 2). Their procedure, in which dioxane is employed as the fractionating solvent, is time-consuming. Some investigators have failed to obtain catalase crystals by the dioxane method, possibly because of the presence of harmful peroxides in the dioxane. Our present procedures are simple and are readily followed. Crystalline cow liver catalase may beobtained within 48 hours. Water is employed for extraction of the enzyme and acetone for its isolation in crystalline form. All steps of the procedure, with the exception of dialysis and crystallization, may be carried out at room temperature.
EXPERIMENTAL

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Procedure-Cow liver is put through an electric meat grinder four times, and 650gm. are mixed with 700 ml. of distilled water for 6 minutes with an electric stirrer. Usually 1150 ml. of the mixture are obtained. The mixture is placed in a 3 liter beaker and 460 ml. (0.4 volume) of acetone (A. C. S. quality) are added in small portions with stirring, which is continued for 1 minute. The mixture is filtered in two port,ions through separate folded filters (Whatman No. la), andusually 850 ml. of this fraction are obtained. The filtrate is kept overnight at 4” and a precipitate forms. Without a second filtration, 196 ml. (0.23 volume) of acetone are added with stirring which is continued for about 1 minute to precipitate the catalase, which is removed by filtration. The moist precipitate is collected wit.h a spatula and extracted with 60 ml. of distilled water. Theaqueous solution is centrifuged for 15 minutes at 4” and 1250 X g. Centrifuging may be carried out at room temperature and at lower speed if desired. The clear supernatant is divided into two portions, and each portion is placed in a cellophane tubing and dialyzed at 4” against 10 liters of distilled water, which is continuously stirred. Within 24 to 48 hours microscopic, yellow-colored, arrow-shapedprisms or needles appear in the cellophane tubings (see Fig. 1). Precipitation of the catalase crystals continues for a few days.
703

704

CRYSTALLIKE

CATALASE

FROM

LIVER

If dialysis is started the same day as extraction, crystals may not form during dialysis. In order to obt.ain crystals from t.he dialyzed solution, however, it is only necessary to add 0.3 volume of acetone,remove the amorphous impurities by centrifuging at 4’ and 1250 X y, and place the supernatant in the refrigerator at 4”. The catalase begins to crystallize wit.hin 6 hours in the form of a greenish black precipitate, which under the microscope appears to consist only of golden yellow octahedrons (see Fig. 2). Precipitation of crystals continues for a few days. Similar re-

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FIG. aqueous

1. Cow liver catalase solution on dialysis.

crystals 320 X.

obtained

by

fractionation

with

acetone

from

sults were obtained with bull liver, but frozen liver must be completely thawed before it can be used for the preparation of crystalline catalase. This method is not suitable for the preparation of crystalline catalasefrom rabbit liver. Recryslalli~ation-The catalase crystals which formed during dialysis had an average Kat. f. value of 20,000. Recrystallization was carried out by dissolving this material at 40” in the least possible volume of 0.05 M phosphate buffer of pH 7.3. The centrifuged, almost black solution was placed in a refrigerator at 4” for a few days. Crystallization from the dilute phosphate...