Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.
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.
Salmaso
B L
BL
Department of Applied Physics, University of Twente, Enschede, The Netherlands.
Puppels
G J
GJ
Caspers
P J
PJ
Floris
R
R
Wever
R
R
Greve
J
J
D001665:Binding Sites; D004735:Energy Transfer; D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D010544:Peroxidases / Q000097:blood* / Q000737:chemistry*; D011524:Protoporphyrins / Q000032:analysis; D013059:Spectrum Analysis, Raman / Q000379:methods
D011524:Protoporphyrins; C028025:protoporphyrin IX; D047209:Eosinophil Peroxidase; D010544:Peroxidases