Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniques

The acyl carbonyl group of [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin (5MeTA-chymotrypsin) has been investigated by using both resonance Raman (RR) and Fourier transform infrared (FTIR) spectroscopies. The spectrum of the acyl-enzyme carbonyl group has been obtained as a function of pH over the range 3.0-10.0 in the RR experiments and over the range 3.4-7.6 (p2H) in the FTIR experiments. The carbonyl spectral profiles obtained by using FTIR spectroscopy are substantially different from the carbonyl profiles obtained by using RR spectroscopy. The FTIR spectra were obtained by subtracting the spectrum of the free enzyme from that of the acyl-enzyme. Use of the active-site inhibitor phenylmethanesulfonyl fluoride demonstrates that part of the intensity observed in the FTIR spectra of 5MeTA-chymotrypsin is due to a subtraction artifact giving rise to enzyme-associated bands, probably from peptide groups perturbed by substrate binding. The enzyme bands can be removed by subtracting the FTIR spectrum of 13C=O acyl-enzyme from that of 12C=O acyl-enzyme. Additionally, this procedure reveals that one of the acyl-enzyme carbonyl bands observed at 1727 cm-1 using RR spectroscopy is absent in the FTIR acyl-enzyme spectrum. However, a feature near 1720 cm-1 can be induced in the FTIR spectrum by actinic light in the near-UV region. Thus, it is proposed that the 1727 cm-1 RR carbonyl band results from a population of acyl-enzymes which is generated by exposure to the laser beam during RR data collection. When both the RR and FTIR data are adjusted to remove artifacts, they provide essentially identical carbonyl stretching profiles.