The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin.
A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90% and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.
D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D006736:Horses; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D010970:Plastocyanin / Q000378:metabolism*; D011485:Protein Binding
D002234:Carbodiimides; D003574:Cytochrome c Group; D010940:Plant Proteins; D010970:Plastocyanin; D005022:Ethyldimethylaminopropyl Carbodiimide