PHYSICOCHEMICAL STUDIES ON CYTOCHROME b2. SOME PROPERTIES OF MODIFIED FORMS OF THE ENZYME AND OF THE DEOXYRIBONUCLEIC ACID COMPONENT

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Abstract
The absorption spectrum, and the sedimentation and electrophoretic behavior of DNA-free cytochrome b2 (Type II cytochrome b2) are described. Only one protein component was found. The concentration-dependence of the sedimentation coefficient is described by the equation S[image] = S[image] (l-kc0), where c0 is the concentration ([mu]M) of cytochrome b2 (expressed in terms of hema), k is 0.534 x 10-3l./[mu]mole of cytochrome b2 (expressed in terms of hema), and S[image] (the intrinsic sedimentation coefficient) is 8.56s. The concentration-dependence of S20, w is about one-third of that found for DNA-containing cytochrome b2 (Type I cytochrome b2). Oxygen and p-chloromercuribenzenesulphonate cause dissociation of the enzyme-bound flavin. In the presence of lactate, exposure to air forms aggregates of the protein, apparently through formation of intermolecular disulphide bonds. p-Chloromercuribenzenesulphonate prevents the formation of these aggregates. These are 4-6 thiol groups/molecule of enzyme-bound heme, in Type I cytochrome b2 which react rapidly with p-chloromercuribenzenesulphonate. It is suggested that one of these groups is at the active centre of the enzyme and forms a labile bond with the riboflavin phosphate group. Treatment with urea causes displacement of the flavin group and inactivation of the enzyme; the inactivation can be partly reversed. No new sedimenting components were observed with cytochrome b2 in 2.7 [image]- or in 5 [image]-urea at pH 6.7, or after subsequent removal of the urea by dialysis. After removal of the urea, the sedimentation coefficient of the protein was substantially the same as that of untreated enzyme. The sedimentation coefficient of DNA isolated from Type I cytochrome b2 in 5.8 [plus or minus] 0.2s, and the diffusion coefficient is 3x10-7 [plus or minus] 1x10-7 cm.2/sec. From these values the molecular weight is calculated to be 1.2x105[plus or minus] -0.4x105. The DNA of cytochrome b2 appears to be aggregated when dissociated from the protein.