In the absorbance spectroscopy, the spectrum of …show more content…
Samples were placed in a quartz and scattering was collected at a 90-degree geometry using a filter to reject Rayleigh scattering. A laser equipped with another filter provided a wavelength at 405 nm onto the samples with a power of 4 mW for 20-40 min for each spectrum. The Raman shifts were calibrated with toluene and analyzed using Origin software. At pH 7.5, the HRP/nitrite spectrum revealed there was no significant effect of nitrite on position and intensity of the bands. However, inspection of the trace differences in revealed existence of a nitrogen isotope sensitive band from a peak/trough at 1334/1309 cm-1 demonstrating that there is nitration of the vinyl group to some extent at pH 7.5 without nitrite coordination to the heme. This result was consistent in both the high and low frequency resonance spectra. At pH 5.5, the difference in traces of the high frequency RR spectra demonstrated formation of a 6cLS heme species and presence of two peak/trough features suggesting the nitrogen and oxygen isotope sensitivity of the bands. The low frequency RR spectra revealed an additional isotope sensitive band at 800-900 cm-1. Results suggested the detection of ν (Fe-NO2), δ(FeNO2) and νsym(NO2) which was used to characterize the structure of the heme Fe(III)-nitro adduct in HRP. At pH 7.5 the heme pocket allowed for partial nitration of the heme 4- vinyl