CONDITIONS FOR THE AUTOXIDATION OF 

 FLAVOCYTOCHROME h. 



By E. BoERi* AND M. RiPPAf 

 Institute of Human Physiology, University of Ferrara, Fetrara 



INTRODUCTION 



FLAVOCYTOCHROME b^ is the L^+)-lactate dehydrogenase of aerobic yeast. 

 Several acceptors can be used for the oxidation of lactate by this enzyme ; 

 most of these acceptors are artificial. Ferricytochrome c (which is also 

 present in aerobic yeast) is probably the physiological acceptor. Oxygen is 

 also an acceptor, the flavocytochrome Z?2 having been described as autoxidiz- 

 able by Appleby and Morton (1954), by Boeri and Tosi (1956) and as 'very 

 sHghtly autoxidizable' by Morton (1958). 



It is the purpose of the present investigation to gain information on how 

 efficient an electron acceptor oxygen can be for flavocytochrome b^,. This 

 information can tell us how likely is a direct by-pass of the usual cytochrome 

 route. Furthermore, a study of the conditions needed for the autoxidation 

 of a cytochrome can give useful general information, as the reasons making 

 certain cytochromes (like cytochrome a^ and b^ readily autoxidizable and 

 others (Uke cytochrome c) only autoxidizable under extreme conditions are 

 still poorly understood. 



METHODS 



Extraction of Flavocytochrome h^ 



Compressed aerobic bakers' yeast was obtained from the factory and dried. 

 Lots of 3 kg of dried yeast were used each time. The yeast was ground, 

 suspended in 3 x its weight 0- 1 m K2HPO4, blended in a Waring blendor, 

 and autolyzed for 45 min at 37°C. After centrifugation, the supernatant was 

 brought to pH5-6 with 0-5 m lactic acid and heated for 15 min at 52°C. 

 After cooling and centrifugation, the supernatant was diluted with an equal 

 amount of distilled water and brought to pH 5-4 with lactic acid. Calcium 

 phosphate gel (45 mg dry weight/ml) was added in a ratio 1 : 6 and the 

 mixture was slowly agitated for 30 min. The centrifuged gel was treated with 

 a solution which was 01 m in K2HPO4, 002 m in sodium lactate and 20% 



* Professor Enzo Boeri died in October, 1960 (Editors). 



t Investigator of the Comitato Nazionale per le Ricerche Nucleari, Divisione Biologica 

 Group 3 C. 



524 



