Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl

Biochemistry. 1992 Nov 3;31(43):10556-63. doi: 10.1021/bi00158a019.


2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels*
  • Amino Acid Sequence
  • Animals
  • Azides*
  • Biphenyl Compounds*
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / metabolism
  • Humans
  • Microsomes, Liver / enzymology
  • Molecular Sequence Data
  • Naphthalenes*
  • Oxidoreductases / antagonists & inhibitors
  • Oxidoreductases / chemistry*
  • Oxidoreductases / metabolism
  • Peptide Mapping
  • Photochemistry
  • Rabbits
  • Rats
  • Sequence Alignment


  • Affinity Labels
  • Azides
  • Biphenyl Compounds
  • Cytochrome P-450 Enzyme Inhibitors
  • Naphthalenes
  • 2-ethynylnaphthalene
  • 4-azido-1,1'-biphenyl
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Cytochrome P-450 CYP1A2