Aiitoxidalion of Flaiocytoc/ironie b., 525 



saturated with ammonium sulphate. This solution caused the clulion of the 

 enzyme. The eluted material was treated with solid ammonium sulphate to 

 a final saturation of 90% and kept at 4°C for at least overnight. 



The precipitate was centrifuged and washed with 60% saturated ammonium 

 sulphate, and finally dialyzed for 3 hr in the cold against 0-05 m sodium 

 lactate under the exclusion of the air. The solution was then treated with 

 cold acetone, which was slowly added under agitation until the cytochrome 

 spectrum disappeared from the supernatant. The excess of acetone was 

 removed by centrifugation and vacuum suction. The precipitate was sus- 

 pended in 0-01 M lactate and dialysed as before. Insoluble material was 

 discarded. The enzyme solution was brought over a column of DEAE- 

 cellulose according to Nygaard (1958) and purified according to the same 

 author. The eluted material was finally adsorbed onto and eluted from 

 calcium phosphate gel as before, precipitated with 70 % saturation of am- 

 monium sulphate and dialysed (as before) to remove the salt. 



In summary, this procedure contains steps from the purification according 

 to Bach, Dixon and Zerfas (1946), Appleby and Morton (1954, 1959) and 

 Nygaard (1958). The yield is about 0-1 micromole flavocytoclirome bjkg of 

 dried yeast. 



We were unable to obtain the crystalline preparation described by Appleby 

 and Morton (1954, 1959; but see Note, p. 533). Our best preparation had a 

 content of one flavin monophosphate and one haematin residue for each 

 140,000 g of protein. The turnover number was 16,000 moles ferricyanide 

 reduced by one mole of enzyme/min in the presence of excess lactate. This 

 corresponds to 6,900 Morton units/mg of protein. 



Other Techniques 



Oxygen consumption was measured in a Warburg manometric apparatus 

 at 37°C. 



L(4-)-Lactate determinations were performed with an enzymic method, by 

 measuring the amount of ferricyanide reduced in the presence of excess 

 flavocytochrome b^. The lactate used was 90% in the l(-|-) form and 10% 

 in the d(— ) form. 



Pyruvate determinations were performed according to the method of 

 Friedmann and Haugen (1943). 



Catalase was extracted from horse liver according to Sarkar and Sumner 

 (1955). 



RESULTS 



Effect of the Enzyme Concentration 



Figure 1 shows the effect of the concentration of flavocytochrome b^ on the 

 oxygen consumption for the oxidation of L(+)-lactate as catalysed by this 

 enzyme. The effect is linear. 



