Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide.
The 1:1 covalently cross-linked complex between horse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184-5190]. This earlier study has been extended to show that efficient cross-linking of the two proteins can occur in a variety of buffers over a broad ionic strength range. The substitution of ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1:1 complex (approximately 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to the cytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1:1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.
Moench
S J
SJ
Department of Chemistry, University of New Mexico, Albuquerque 87131.
Satterlee
J D
JD
Erman
J E
JE
D000818:Animals; D002234:Carbodiimides / Q000493:pharmacokinetics*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D011522:Protons; D012967:Sodium Dodecyl Sulfate; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase