The DNA component of cytochrome [formula omitted]: I. Isolation of [formula omitted] and the behaviour of cytochrome [formula omitted] during chromatography on DEAE-cellulose and sedimentation in a sucrose gradient

Abstract

1. 1. The DNA component of Type I cytochrome b 2 of baker's yeast was extracted, together with FMN, by shaking a solution of the enzyme with phenol or by heating it with 2 M NaCl. The DNA was separated from the FMN by stepwise chromatography on DEAE-cellulose. 2. 2. Type I cytochrome b 2 was subjected to chromatography on DEAE-cellulose using a linear gradient of NaCl (pH 7.0) for elution. In the presence of 3 M urea, the oxidised haemoprotein, FMN and DNA components were separated and eluted in that order. In the absence of urea, oxidation of the enzyme occurred but the separation of the haemoprotein and FMN was incomplete. In all cases, the DNA peak was eluted with maximum absorption at 0.7 M NaCl. 3. 3. Stepwise chromatography of Type I cytochrome b 2 on DEAE-cellulose in the presence of urea gave similar results to gradient elution. However, in the absence of urea, the enzyme remained reduced and active and it was possible to prepare both DNA-free enzyme (Type II) and b 2- DNA by a simple chromatographic procedure. Type II enzyme prepared in this way readily formed its typical hexagonal crystals on dialysis and it also re-combined with b 2- DNA to produce Type I crystals of high specific enzymic activity and in high yield. 4. 4. Sedimentation of Type I cytochrome b 2 in a sucrose gradient led to partial separation of the flavohaemoprotein and DNA components; this indicates that they are not firmly associated in solution under the conditions used. 5. 5. The ease with which these two components can be separated by sedimentation and by chromatography on DEAE-cellulose has been considered with all available data on the molecular weight of the DNA component. It was concluded that the DNA and flavohaemoprotein do not exist in a one to one ratio in solution and that all the evidence indicates that b 2- DNA has a molecular weight of approx. 10 5.