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



Kinetic values vary according to conditions of determination. The con- 

 ditions used by Appleby and Morton (1954) were suboptimal, as indicated by 

 subsequent kinetic studies (Appleby, 1957; Appleby and Morton, 1959a, b). 

 The procedure for purification and crystallization of cytochrome b^ has not 

 been altered since 1953, when the first crystalhne preparations were obtained. 

 The recent statement by Nygaard (1959), which suggests that the original 

 preparations of Appleby and Morton had low activity as compared with 

 preparations obtained subsequently, is misleading. The values given here 

 and by Appleby and Morton (1959a, b) are higher than those reported 

 initially (Appleby and Morton, 1954) only because of diff'erent methods of 

 determination of activity. All values are vahd and refer to the activities of 

 crystalline cytochrome b^ under the conditions specified. 



Further information concerning the mechanism of action of cytochrome 

 Z>2 with different acceptors has been obtained from kinetic studies in which 

 the concentration of lactate (S) was varied at several different concentrations 

 of hydrogen acceptor (A), in order to differentiate between several possible 

 enzymic mechanisms. 



Oxidations catalysed by flavin-linked enzymes may conform to any one of 

 at least three different types of mechanisms. Using a simplified notation (in 

 place of the detailed notation shown in the Appendix (p. 521)), these 

 mechanisms and the corresponding steady state rate equations are as follows. 



/. Oxidation of a binary complex by acceptor (cf. Chance, 1953). 



E + S ^ ES 



ES-f Aox--E-f P + Ared 



(where Aox and Areci represent the oxidized and reduced forms of the acceptor 

 respectively; other symbols follow usual conventions). 



Here K„, 



' + (1) 



Both V and K^ are functions of the acceptor concentration [A], and have no 

 theoretical finite limit; —^ is not generally constant (see Appendix). 



//. Formation of (a) two or more binary complexes or (b) a ternary 

 complex (see Alberty, 1956). 



(a) or (b) 



E-I-S^ES E + S^ES 



ES + A ^ EP + H2A ES + A ^ ESA 



EP ^ E + P ESA ^ E + P + HoA 



