THE CHEMICAL AND ENZYMIC PROPERTIES 

 OF CYTOCHROME b., OF BAKERS' YEAST 



By R. K. Morton,* J. McD. Armstrong* and C. A. AppLEBYf 



Department of Agricultural Chemistry, The Waite Agricultural Research 



Institute, University of Adelaide, and Division of Plant Industry, C.S.I. R.O., 



Canberra 



Dixon and colleagues (Bach, Dixon and Keilin, 1942; Bach, Dixon and 

 Zerfas, 1946) first described cytochrome ^2 as a haemoprotein associated with 

 highly purified preparations of lactate dehydrogenase of bakers' yeast. 

 Bach et al. (1946) concluded that 'cytochrome b.^ forms an essential part of 

 the enzyme system either as the dehydrogenase itself or as an essential inter- 

 mediate carrier between lactate and methylene blue.' Subsequently, Morton 

 and co-workers (Appleby and Morton, 1954, 1959a, b, 1960; Armstrong and 

 Morton, 1959) continued studies of cytochrome b,^, to determine the relation- 

 ship between the haemoprotein and lactate dehydrogenase activity of bakers' 

 yeast. The cytochrome b^ was isolated as a crystalline deoxyribonucleo- 

 protein which contained equimolecular proportions of protohaem and 

 riboflavin phosphate (Appleby and Morton, 1954, 1959a, b, 1960). Appleby 

 and Morton (1954, 1959b) showed that the flavin group was essential for 

 enzymic activity. Thus cytochrome b^ was identified as a flavohaemoprotein, 

 a unique type of haematin enzyme with dehydrogenase activity. Boeri and 

 colleagues (Boeri, Cutolo, Luzzati and Tosi, 1955; Boeri and Tosi, 1956), and 

 Nygaard (1958) have confirmed the stoichiometric occurrence of flavin and 

 haem groups in this enzyme. 



The isolation procedure (Appleby and Morton, 1954, 1959a; Morton, 

 1955a, b) involves few treatments and depends on the use of organic solvents 

 (n-butanol and acetone) rather than on ammonium sulphate fractionation 

 and adsorption and elution procedures (Bach et al., 1946; Boeri et al., 

 1955; Yamanaka, Horio and Okunuki, 1958). Lipid is extracted first from 

 finely-ground, air-dried bakers' yeast by treatment with n-butanol (Morton, 

 1950, 1955a, b). The cytochrome b.;, and cytochrome c are then extracted from 

 the yeast with lactate solution; the solution is fractionated with acetone at 

 about -5°C, and the cytochrome bo, is crystallized free of cytochrome c by 

 dialysis to low ionic strength against dilute lactate at about pH 6-8 (Appleby 



* University of Adelaide. 

 t C.S.I.R.O. 

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