Nature of tryptic attack on cytochrome b5 and further evidence for the two-domain structure of the cytochrome molecule.
Rabbit cytochrome b5 was incorporated into single-walled phosphatidylcholine liposomes, and the cytochrome b5-liposome complex thus formed was digested with trypsin. Protein chemical characterization indicated that the main products formed were 1) a hydrophilic (heme-containing) fragment of the cytochrome corresponding to the sequence consisting of the masked NH2-terminus through residue 88, 2) a hydrophobic peptide which spans residue 91 to the COOH-terminus (residue 133), and 3) a dipeptide, seryl-lysine, derived from residues 89 and 90. The hydrophobic peptide was obtained in the form of its complex with liposomes. It was concluded that trypsin cleaved rather specifically the peptide bonds between residues 88 and 89 (Arg-Ser) and between residues 90 and 91 (Lys-Leu). Tryptic digestion of free, unbound cytochrome b5 also resulted in the cleavage of the same peptide bonds. These results are not consistent with the proposal of Visser et al. (Visser, L., Robinson, N.C., & Tanford, C. (1975) Biochemistry 14, 1194-1199) that the hydrophilic and hydrophobic domains of cytochrome b5 are connected to each other by a link peptide consisting of some 15 amino acid residues and that this link peptide  can be cut out by the action of trypsin. The circular dichroism spectrum of intact cytochrome b5 or its complex with liposomes in the far-ultraviolet region was closely similar to the sum of the spectra of the hydrophilic fragment and the hydrophobic peptide (or its complex with liposomes). This indicates that the tryptic cleavage of the cytochrome molecule does not induce any significant changes in the conformations of the hydrophilic and hydrophobic moieties of the molecule and thus provides further evidence that the three-dimensional structures of the two domains are independent of each other.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D003580:Cytochromes; D007770:L-Lactate Dehydrogenase; D008081:Liposomes; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011817:Rabbits; D014357:Trypsin
D000596:Amino Acids; D003580:Cytochromes; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D007770:L-Lactate Dehydrogenase; D014357:Trypsin