Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes

Abstract

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.

Raman excitation wavelength investigation of single red blood cells in vivo

“…8 -10 Their studies primarily focused on lymphocytes 9,11 and granulocytes. 10,12,13 The group identified haem moieties including eosinophil peroxidase (EPO) and myeloperoxidase in eosinophils and neutrophils, respectively, 10 and confirmed the high-spin six-coordinated structure of these haems. Recently, Schuster and co-workers 14,15 analysed single Clostridium cells at 632.8 nm excitation and observed bands associated with the major macromolecules including lipids, proteins and carbohydrates.…”

“…The latter assignments are based on the work of Salmaso et al 12 for EPO. In the oxygenated cells a doublet dominates this region occurring at 1249-1248 and 1228-1223 cm 1 for all four excitation wavelengths.…”

Eosinophil Peroxidase Oxidation of Thiocyanate

“…extracellular destruction of metazoan parasites (6). In preliminary studies we find (55) that the ED 50 for killing of cultured human and mammalian cells, including cardiac myocytes and several types of endothelial cells, is approximately 10-fold higher (200 -300 M oxidant) for EPO/H 2 O 2 /SCN Ϫ system-derived products than for HOCl or HOBr; however, the ED 50 for killing of freshly transformed schistosomules, a model of parasitic disease, is nearly identical (20 -30 M oxidant). It is therefore conceivable that OSCN Ϫ and OCN Ϫ comprise a cytotoxic system that exploits subtle, sulfhydryl-based metabolic differences between host and parasites to effect selective killing of the latter.…”

Raman spectroscopy: techniques and applications in the life sciences

“…For example, Salmaso et al used resonance Raman spectroscopy to study the enzyme eosinophil peroxidase in living immune cells [197]. Sijtsema et al were able to distinguish redox states in single immune cells by measuring resonance Raman signals from cytochrome b 558 and myeloperoxidase [198].…”