IDENTIFICATION OF ACTIVE-SITE PEPTIDES FROM H-3 LABELED 2-ETHYNYLNAPHTHALENE-INACTIVATED P450 2B1 AND 2B4 USING AMINO-ACID SEQUENCING AND MASS-SPECTROMETRY

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P4 50 inactivated [Roberts, E. S., Hopkins, N. E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [H-3]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) almost-equal-to 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed tbe sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [H-3]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) almost-equal-to 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283. When the fraction containing these peptides was analyzed by MALDI-MS, peaks at m/z 4730.4 and 4897.6 were detected. The lower mass peak represents the MH+ for the peptide Glu273 to Met314 (theoretical 4729.3), while the higher mass peak corresponds to the MH+ of the modified peptide (theoretical 4897.5). Two additional peaks were identified from this fraction at m/z 8603.7 and 8435.6 which could be explained by the presence of a microheterogeneous form of 2B4 with Met3l4 replaced by Leu. Again, the difference in mass between the two peaks (approximately 168) would correspond to the addition of a 2-naphthylacetyl group to the unmodified peptide.