Secobarbital, an allyl barbiturate, is a sedative-hypnotic known to precipitate acute attacks of hepatic porphyria in genetically predisposed individuals. This we believe, is due to the fact that secobarbital not only induces the liver hemoproteins, cytochromes P450, but also results in their destruction, thereby causing an acute demand for hepatic heme. The overall objective of this project is to elucidate the mechanism of the suicidal inactivation of P450 2B1 by secobarbital. Ongoing studies have shown that this drug inactivates rat liver cytochrome P450 2B1 both by protein and heme alkylation. Using chemically synthesized 14C-labelled secobarbital and lysyl endopeptidase digestion coupled with HPLC peptide mapping, we have isolated a radiolabelled peptide, identified as an active site 2B1 peptide by micro Edman amino acid sequencing analysis. The mass of the native peptide w5420.8 Da and the N-terminal sequence is consistent with a Lys-C peptide corresponding to residues 277-323 of rat liver 2B1. Because this peptide is highly hydrophobic, and attempts to characterize the full peptide by electrospray mass spectrometry have not yet been successful at UCSF, it has been analyzed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analyses. This Lys-C peptide and the corresponding peptic peptide obtained from 14C-secobarbital-inactivated 2B1 are currently being isolated for MALDI-MS and subdigestions to identify the residue covalently modified.
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