Studies on the interaction of polypeptide of cytochrome Tb-5 and its F35Y mutant with heme b by electrospray ionization mass spectrometry

Citation
Ct. Yu et al., Studies on the interaction of polypeptide of cytochrome Tb-5 and its F35Y mutant with heme b by electrospray ionization mass spectrometry, ACT CHIM S, 59(6), 2001, pp. 956-960
Citations number
10
Language
CHINESE
art.tipo
Article
Categorie Soggetti
Chemistry
Journal title
ACTA CHIMICA SINICA
ISSN journal
0567-7351 → ACNP
Volume
59
Issue
6
Year of publication
2001
Pages
956 - 960
Database
ISI
SICI code
0567-7351(2001)59:6<956:SOTIOP>2.0.ZU;2-6
Abstract
Cytochrome Tb-5 from bovine liver mitochondria aid its F35Y mutant, digeste d from cyt b(5) 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, 9477.5, is obtained by ESI/TOFMS in external standard cal ibration method. Compared with molecular weight of the cyt Tb-5, 9461, this data is exactly coincided with the calculated value according to the mutan t amino acid sequence. For studying the stability of heme binding to the F3 5Y mutant and cyt Tb-5, a series of experimental conditions were selected s uch as nozzle potential, organic solvent, pH value, etc. It was found that the abundance of holoprotein content measured in the mass spectrum decrease d with increase of the nozzle potential or/and increase of the composition of organic solvent or/and pH value deviated from neutral. Relatively, the h oloprotein's mass abundance of cyt Tb-5 is higher than that of F35Y mutant that indicates stronger binding of heme b to polypeptide chain in the wild type cyt Tb-5. However, we found that if the nozzle potential was too low i t would produce more metal plus ion peaks, suppressing the sample's signals of mass spectrum, and meanwhile increasing organic solvent would reduce st ability of the holoprotein. So, the optimal conditions chosen are those: th e composition of methanol is 10% and the nozzle potential is 70 V. In addit ion, it is verified that the heme b dissociated from the F35Y mutant is a F e(III) porphyrin by means of internal standard method.