HIGH CONFORMATIONAL STABILITY OF CYTOCHROME-P-450 1A2 - EVIDENCE FROMUV ABSORPTION-SPECTRA

The fourth derivative of absorption spectra between 260 and 310 nm wer e used for monitoring the changes in exposure of tyrosine and tryptoph an side chains in cytochrome P-450 1A2 to solvent. Titration of the en zyme with a specific inhibitor, alpha-naphthoflavone (2-phenylazo[h]ch romen-4-one) to inhibitor concentration of 30 mu M resulted in small b ut pronounced changes in derivative spectra (decrease in the maximum a mplitude, downshift of the spectral maximum at about 293 nm) correspon ding to the exposure of tryptophans towards the solvent. Further addit ion of the inhibitor led to a decrease of the exposure of these aromat ic side-chains. Similar behaviour was also observed in this work for o ther cytochromes P-450 (2B4 and 11A1). The fourth derivative of absorp tion spectra was also used to examine the stability of the enzyme both in the presence and absence of alpha-naphthoflavone, with increasing pressure (up to 400 MPa) and temperature (up to 35 degrees C) as pertu bing factors. The results show that cytochrome P-450 1A2 has a stable conformation as all the conformational changes observed were (spectral ly) fully reversible.