514 



R. K. Morton, J. McD. Armstrong and C. A. Appleby 



the enzyme with these acceptors is not changed during the purification pro- 

 cedure, and the enzyme is unhkely to have been modified during isolation. 



However, prolonged storage of cytoclirome b^, under aerobic conditions, 

 even in the presence of EDTA, causes partial or complete dissociation of the 

 flavin group and a corresponding decline in activity with ferricyanide (Apple- 

 by, 1957; Appleby and Morton, 1959b). After this type of modification, 

 some of the haem of the preparation is only slowly reducible by lactate 

 (Table 7). 



Table 7. Effect of aerobic storage on the enzymic 



ACTIVITY OF cytochrome b.. 



Crystalline cytochrome li< was suspended in water and held aerobically at CC. Portions of the 



suspension were diluted into 0-5 M sodium lactate containing 10~^ m EDTA at pH 6-8 and at 0°C at the 



times indicated. Activity with ferricyanide was measured immediately. The position and extinction of 



the Soret band was measured after 10 min at 0°. (From Appleby and Morton. 1959b.) 



The considerable autoxidizability of the flavin-free derivative of cyto- 

 chrome ^2) and the slow reduction of inactive cytochrome b^ by active enzyme 

 (Appleby and Morton, 1954; Yamashita et al., 1958), are complicating 

 features in dealing with mixtures of inactive and active cytochrome b.^. 

 Stepwise modification of crystalline cytochrome bo, by (a) removal of the 

 polynucleotide, and (b) subsequent dissociation of the flavin, is being carried 

 out and the physicochemical, chemical and enzymic properties of the 

 derivatives of the crystalline enzyme are being studied. However, intact 

 crystalline cytochrome b^ has been used throughout the kinetic studies 

 described below. 



KINETICS OF LACTATE OXIDATION CATALYSED BY 

 CYTOCHROME b. 

 Differences between the mechanisms of reduction of ferricyanide and of 

 cytochrome c by cytochrome bo appeared likely when it was found (Boeri 

 et al., 1956; Appleby, 1957; Appleby and Morton, 1959b) that reduction of 

 ferricyanide was apparently of zero order, and of cytochrome c of first order. 

 Further kinetic studies by Armstrong and Morton (1959) were carried out 

 with a double-beam recording spectrophotometer (Optica CF 4). Extinctions 

 (E) were measured at 420 m/^ (ferricyanide), 550 m^ (cytochrome c) or 600 m/j, 



