Sato et al. 10.1073/pnas.0611560104. |
Fig. 7. Procedure for calculation of picosecond UVRR difference spectra showing pump-induced differences. The nearly identical curves (a) through (c) are UVRR spectra of horse skeletal Mb corresponding to the probe-only spectrum (a), pump + probe spectra with time delays of -5 (b) and +5 ps (c). In the spectra (a) through (c), the contribution of quartz cell has been subtracted. Spectra (a) through (c) have been divided by a factor of 30. The traces (d) and (e) are UVRR difference spectra that were generated by subtracting the probe-only spectrum from pump + probe spectra at a (b) -5 and (c) +5 ps delay, with an appropriate subtraction factor.
Fig. 8. Comparison of the intensity changes among the 1,620-cm-1, W3, and W16 bands around time 0. Note that there is a difference in the initial intensity decreases between the 1,620-cm-1 band and the other two Trp bands. A dashed line denotes the instrument response function.
Fig. 9. Fitting results for the Tyr bands around time 0. The filled circles correspond to the experimental data of the 1,620-cm-1 (A) and Y9a (B) bands, respectively. Dashed lines are fits using the function mentioned in the legend of Fig. 3 that is convoluted with the instrument response function. These lines correspond to the response signal when the band intensity of the Tyr residues undergoes instantaneous decay followed by the recovery. Solid lines are best fit by using the sum of two exponential functions mentioned in the legend of Fig. 4 that is convoluted with the instrument response function. The direct comparison of experimental data to the dashed lines clearly shows that the initial intensity changes of the Tyr bands are not fitted well without the decay component.
Fig. 10. Integrated intensity changes of W16 (A) and W18 (B) bands relative to the integrated intensity in the probe-only spectrum as a function of delay time. The circles plotted in each panel denote the intensities of the bands of wild-type horse skeletal Mb, which were already shown in Fig. 4, and the squares denote the intensities of the bands of wild-type sperm whale Mb.
Fig. 11. UVRR spectra of CO-bound Mb acquired with different excitation wavelengths and the corresponding deoxyMb-minus-MbCO difference spectra for the indicated four excitation wavelengths between 222 and 234 nm. The traces (a), (c), (e), and (g) are absolute UVRR spectra of MbCO at each excitation wavelength. The traces (b), (d), (f), and (h) are the difference spectra at each excitation wavelength.
SI Text
Procedure for Calculating UVRR Difference Spectra.
SI Fig. 7 shows the procedure for calculating picosecond UVRR difference spectra from unprocessed UVRR time-resolved spectra. The trace (a) is a probe-only spectrum, which represents the UVRR spectrum for horse MbCO. Traces (b) and (c) are pump + probe spectra with time delays of -5 ps and 5 ps, respectively. All distinct Raman bands in the raw spectra can be attributed to contributions from Trp and Tyr residues except for a strong OH stretching band of water around 3,300 cm-1. In traces (a)-(c), the quartz signal of the sample cell has been removed from the spectra. The probe-only spectrum was subtracted from the pump + probe spectrum, providing the difference spectra shown in traces (d) [ = (b) - (a)] and (e) [ = (c) - (a)]. The self-absorption effect in resonance Raman scattering was invariable in the entire observed spectral range (600-4,000 cm-1), because no difference features of perchlorate band at 934 cm-1 and of the OH stretching band of water around 3,300 cm-1 were observed in the difference spectra for the sample containing sodium perchlorate (data not shown). Thus, for all of the difference spectra, the Raman band of water was used as an internal standard of Raman intensity. In trace (d), no difference feature was observed for Trp and Tyr Raman bands at -5 ps; this corroborates the cross-correlation measurement of 3.3 ps. In contrast, trace (e) shows the pump-induced negative features observed for the Trp and Tyr Raman bands, indicating deligation-induced spectral changes in the Raman bands at + 5 ps. It should be noted that the water band was reasonably eliminated in traces (d) and (e).