Identification of the Heme-Binding Cysteines in Cytochromes c Without Radioactive Labeling

Abstract

In spite of the recent improvements in manual and automatic Edman degradation technology, the identification of cysteine residues remains difficult, mainly because of the instability of its phenylthiohydantoin (PTH) derivative. A way to circumvent the problem is to modify the cysteine into a more stable derivative. Many products are known to react with sulfhydryl groups such as ethylenimine, 2-bromoethyltrimethylammonium bromide, methyl-p-nitrobenzenesulfonate, sodium sulfite, acrylonitrile and 2-nitro-5-cyanobenzoate (for references, see [1]). The most commonly used reagent, however, appears to be iodoacetic acid or its amide iodoacetamide, giving rise to carboxymethylcysteine (CMC) or carboxyamidomethylcysteine. The reagents are commercially available as C¹⁴ radiolabeled products, and those who have the facilities to deal with isotopes prefer to identify PTH-CMC by radioactive counting. It is also possible to identify nonlabeled PTH-CMC, either by thin-layer chromatography or by high pressure liquid chromatography (HPLC), the method mostly used in conjunction with automatic sequence analysis. Depending on the HPLC system used, PTH-CMC may not always be identifiable as a separately isolated peak. In the present contribution we present a simple method to recognize PTH-CMC even though it coelutes with another PTH residue. The method is illustrated for the two hemebinding sites of the diheme cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarinus.