Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes

B. L. Salmaso*, G. J. Puppels, P. J. Caspers, R. Floris, R. Wever, J. Greve

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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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.

Original languageEnglish
Pages (from-to)436-446
Number of pages11
JournalBiophysical Journal
Issue number1
Publication statusPublished - 1994
Externally publishedYes

Bibliographical note

Funding Information:
The authors wish to thank Drs. T. Kuijpers and A. Tool of the Department of Blood Cell Chemistry of the Netherlands Red Cross Central Laboratory for a large sample of purified human eosinophilic granulocytes and Dr. J. Krijgsman of the department of Civil Engineering of the Technical University of Delft for the use of the integrating sphere absorption photospec- trometer. The technical assistance of Y. M. Kraan during the whole project is greatly appreciated. We are grateful to Dr. G. Smulevich for many helpful suggestions and to Dr. T. G. Spiro for allowing us to use results on the assignment of the propionate bending vibration before publication. R. Floris and R. Wever gratefully acknowledge financial support by the Netherlands Organization for Scientific Research (NWO).


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