title: A simple and rapid assay for heme attachment to apocytochrome c.
abstract: A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.
mesh_terms: D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism; D045304:Cytochromes c; D006418:Heme / Q000032:analysis*; D007457:Iodine Radioisotopes; D011485:Protein Binding; D011865:Radioisotope Dilution Technique; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D001059:Apoproteins; D003574:Cytochrome c Group; D007457:Iodine Radioisotopes; D006418:Heme; D045304:Cytochromes c