404 IX. HEMATIN ENZYMES, II 



Considerable progress in the purification of catalase was made by 

 von Euler and his co-workers {720J25,1239,12W)- These autiors 

 also studied the kinetics of the enzyme carefully and developed 

 methods for its estimation. It is now evident that some of von 

 Euler's preparations were not far from purity. 



In 1930, Zeile and Hellstrom {3166) prepared hemoglobin-free 

 catalase from horse liver, using the method of Tsuchihashi {2832). 

 The hemoglobin is removed by precipitation with alcohol and chloro- 

 form. The enzyme is then adsorbed to alumina C or calcium phos- 

 phate and elutriated with secondary phosphate. They showed that 

 the catalatic activity was proportional to the hematin content of the 

 preparation, and thus proved catalase to be a hemoprotein. Similar 

 methods were used by Keilin and Hartree {14-87). 



Agner {2^) showed that one of the impurities of liver catalase is 

 ferritin. Since ferritin contains a much larger percentage of iron 

 than catalase, even small amounts of this impurity greatly influence 

 the iron content of catalase preparations. Hence no correlation 

 between iron content and activity had been observed in earlier 

 preparations {1239,1240). Agner removed ferritin by fractional 

 precipitation with ammonium sulfate. 



For the preparation of catalase from horse liver, Lemberg and 

 Legge {1705) used the method of Zeile, followed by removal of phos- 

 phate by dialysis and then by removal of the ferritin according to 

 Agner. Still better is the use of ammonia instead of phosphate for 

 elution of the enzyme. Another method in which no adsorption is 

 used, has been described by Agner {27). 



Sumner and Dounce {2698) prepared ox liver catalase by a greatly 

 simplified procedure using fractional precipitation with dio.xane, diges- 

 tion of glycogen by salivary amylase, and crystallization by addition 

 of ammonium sulfate or dialysis against ammonium sulfate solution. 

 Dioxane can be replaced by acetone {612). 



Ox liver catalase and catalases from the livers of other animals 

 were obtained as well-formed crystals by Sumner and his collaborators 

 {2698-2700,612). Erythrocyte catalase was prepared by Agner {28) 

 and crystallized by Laskowski and Sumner {1657). The isolation 

 and chemistry of catalase has been reviewed by Sumner {2697).* 



Catalase is readily adsorbed on alumina, calcium j)hosphate, silica 



gel, and also on cellulose {2742). The catalase adsorbed on alumina 



or silica gel is still active {2333,2651). 



* Recently, Boiinichseti (ol'tii) crystallized human erythrocyte catalase; Herbert 

 and Pinsent {12.'t'f.a) crystallized the cataliuse of Micrococcus ly.sodeicticu.s. 



