Cytochrome Tb5 from bovine liver mitochondria and its F35Y mutant, digested from cyt b5 membrane protein by trypsin, is a kind of electron transfer protein, which consists of 82 amino acid residues. The molecular weight of the F35Y mutant, 9 477.5, is obtained by ESI/TOFMS in external standard calibration method. Compared with molecular weight of the cyt Tb5, 9 461, this data is exactly coincided with the calculated value according to the mutant amino acid sequence. For studying the stability of heme binding to the F35Y mutant and cyt Tb5, a series of experimental conditions were selected such as nozzle potential, organic solvent, pH value, etc. It was found that the abundance of holoprotein content measured in the mass spectrum decreased with increase of the nozzle potential or/and increase of the composition of organic solvent of /and pH value deviated from neutral. Relatively, the holoprotein's mass abundance of cyt Tb5 is higher than that of F35Y mutant that indicates stronger binding of heme b to polypeptide chain in the wild type cyt Tb5. However, we found that if the nozzle potential was too low it would produce more metal plus ion peaks, suppressing the sample's signals of mass spectrum, and meanwhile increasing organic solvent would reduce stability of the holoprotein. So, the optimal conditions chosen are those: the composition of methanol is 10% and the nozzle potential is 70V. In addition, it is verified that the heme b dissociated from the F35Y mutant is a Fe(Ⅲ) porphyrin by means of internal standard method.

Key words: MASS SPECTROGRAPHY, CYTOCHROME, MUTANT, PROTEIN, HEME, INTERACTIONS, MITOCHONDRIA