570 Discussion 



in the presence of oxygen or on addition of — SH binding reagents. Wfiere flavin dis- 

 sociation has occurred in the presence of oxygen, aggregation of the protein also 

 occurs, presumably due to the formation of intermolecular disulphide bonds. This 

 behaviour is not observed when flavin dissociation is brought about with /7-chloro- 

 mercuriphenylsulphonate (PCMS). 



Unless otherwise indicated, these studies were carried out with freshly prepared 

 twice crystallized cytochrome bo under anaerobic conditions; solutions were handled 

 in an atmosphere of nitrogen at 2°C. The buff'er used for most of the experiments had 

 the following composition: 0-3 m sodium lactate, 005 m tetra sodium pyrophosphate/ 

 HCl, 10-* M ethylenediamine tetra-acetate (EDTA), pH 6-85 ± 003, ionic strength 

 0-63. A few of the preliminary experiments were carried out in 04 m sodium lactate, 

 015 m sodium chloride, 10-« m EDTA, pH 6-80, ionic strength 0-65. EDTA and high 

 lactate concentrations protect cytochrome b^ (Morton et al., this volume, p. 501 ; 

 Boeri and Rippa, this volume, p. 531). Pyrophosphate was chosen as the buff'er ion 

 because of its protective eff"ect (Armstrong and Morton, unpublished). The rotor 

 temperature was usually 2-5°C. 



PROPERTIES OF INTACT CRYSTALLINE CYTOCHROME b^ (dEOXYRIBONUCLEOPROTEIN) 



In these buff"ers, cytochrome 63 sediments as a single component with a symmetrical 

 boundary, iao.w = 8-48 x 10~^^ sec. The concentration dependence of sedimentation, 

 over the concentration range 8-110/tmoles haem/I., is described by the relationship 

 ■^2o.w = ^20, w (1 — 1-54 X lO-^' c) where c is expressed in terms of /<moles of enzyme 

 haem/1. The pink colour of cytoclirome 62 ™^y be observed to sediment with the 

 boundary; in these experiments no detectable fluorescence of the solutions was observed 

 before or after centrifugation. The lactate reductase activities towards ferricyanide and 

 ferricytochrome c were typical of intact cytochrome 60. The spectral properties of 

 such preparations are: Amax at 2650, 3300, 4230, 527-5 and 556-5 m/<; and £'265 ,„^/ 

 £423 mni 0'89. 



PROPERTIES OF NUCLEOTIDE-FREE CYTOCHROME 63 



The polydeoxyribonucleotide was dissociated from intact cytochrome b^, by dialysis, 

 under nitrogen, against ammonium sulphate solutions containing lactate and EDTA 

 (see Appleby and Morton, Biochem. J. 75, 258, 1960). Here also a single sedimenting 

 component was observed, the pink colour again sedimenting with the boundary: 

 s%,,^ = 1-%1 X 10 13 sec. 



With adequate precautions, particularly with respect to oxygen and to possible 

 contamination with copper, no fluorescence of the solutions can be detected before or 

 after centrifugation. The concentration dependence (measured over the concentration 

 range 8-103 //moles haem/1.) may be described as follows j-jo.w = ^20. w (1 — 5-33 x 

 10"* c) where the value of c is estimated in //moles of haem/1. (based on the millimolar 

 extinction coefficients of intact cytochrome bo). Other properties of such preparations 

 are: Amax at 267-8, 328, 423-0, 528-0 and 556-5 m/<; and E^^^ mal^i-13 m/i- 0-6. 

 Enzymic activities of such preparations are comparable with those of intact 

 cytochrome b^. 



These results indicate that the polydeoxyribonucleotide has little influence on the 

 sedimentation behaviour of the reduced native flavohaemoprotein. The polynucleotide 

 contains approximately 15 nucleotide residues/protein-bound haem, corresponding 

 to a weight of 5,000 g/mole of protein-bound haem; intact cytochrome b.^ has a weight 

 of approximately 80,000 g/mole of protein-bound haem. 



Preliminary studies of the polydeoxyribonucleotide dissociated from the intact 

 crystalline enzyme by treatment with ammonium sulphate indicate that it behaves as 

 a single compound when chromatographed on Ecteola resin. Since only a small 

 quantity of material was available, the sedimentation behaviour is difliicult to interpret, 

 but no obvious heterogeneity was observed, and the slow rate of sedimentation 



