Effects of pH and chloride concentration on resonance Raman spectra of human myeloperoxidase and Raman microspectroscopic analysis of enzyme state in azurophilic granules
✍ Scribed by Kenji Araki; Hideo Takeuchi
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2000
- Tongue
- English
- Weight
- 246 KB
- Volume
- 57
- Category
- Article
- ISSN
- 0006-3525
No coin nor oath required. For personal study only.
✦ Synopsis
Resonance Raman spectra of human myeloperoxidase were examined at pH 3.3-10.5 in the absence and presence of chloride ions. Among the porphyrin vibrational bands, the core-size marker bands showed particularly large wavenumber downshifts on going from pH 8.7 to 5.3 with a transition midpoint at pH 6.5 in the absence of chloride ions. The chloride ions did not affect the spectrum at a pH below 5.3 and above 8.7 whereas an increase of chloride concentration at neutral pH caused spectral changes similar to those observed upon pH lowering. Analogous effects were also observed on the Raman intensity. In addition, the stretching mode of the bond between the heme Fe and proximal histidine shifted by Ϫ2 cm Ϫ1 on going from pH 8.7 to 5.3. Decomposition of the 3 band revealed the presence of two components, which was confirmed by an isosbestic point in the absorption spectra. The observed spectral changes indicated the existence of alkaline and acidic forms of the enzyme. The pK of interconversion was 6.5, and it was increased by binding of chloride ions. The porphyrin core was slightly expanded in the acidic form compared to that in the alkaline form. A molecular mechanism of the porphyrin core expansion was proposed on the basis of the X-ray crystal structure. The pH-spectrum relationships obtained for the isolated enzyme were applied to in situ analysis of the state of myeloperoxidase in azurophilic granules of living neutrophils. The enzyme was stored in the acidic form and kept inactive in catalyzing HOCl production.