W. Anthony Oertling

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiffs base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.

A Simple Mixer/Jet Cell for Raman Spectroscopic Studies

In biological applications of resonance Raman spectroscopy it is frequently desirable to reduce the instantaneous and long-term power density of the focused laser beam in order to preserve the sample. Both flowing sample methods and laser beam defocusing techniques have been used successfully by various groups to minimize damage to photolabile samples. The use of flowing sample cells under conditions in which sample recycling is practical has the additional advantage that long acquisition times can be achieved with fairly minimal sample consumption. Nonetheless, the flowing cells described to date are most useful when both fairly large volumes of sample are available and recirculation is feasible. Recirculation is often not possible if the sample of interest is an unstable reaction intermediate, and such an application requires rapid mixing of the precursor compounds. A further consideration with Raman flow cells involves the sample containment technique in the scattering volume. Quartz capillaries are often used, but with these, quartz scattering is severe in the low-frequency region, where it overlaps vibrational modes of the sample and makes their detection difficult. Several groups have avoided this problem by arranging their flowing cells so that the sample forms a free jet in air in the scattering volume. A notable example of this is the microdroplet mixing technique developed by Kincaid and co-workers that uses continuous wave excitation and both rapid mixing and Raman scattering in air.