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Mapping of amino acid residues at non-covalent complex prosthetic group site of apo-hemoproteins containing photosensitive porphyrins

Grant number: 05/03424-0
Support type:Scholarships in Brazil - Master
Effective date (Start): April 01, 2006
Effective date (End): February 29, 2008
Field of knowledge:Biological Sciences - Biophysics
Principal Investigator:Iseli Lourenço Nantes Cardoso
Grantee:Clemerson Fabricio Balbino Dias
Home Institution: Pró-Reitoria de Pesquisa, Pós-Graduação e Extensão. Universidade de Mogi das Cruzes (UMC). Mogi das Cruzes , SP, Brazil

Abstract

Phorphyrins are cyclic compounds formed by pyrrolic rings attached each other by methylenic bridges that present catalytic and photosensitizing properties. The porphyrins play also important roles in biological systems in which they are associated to transition metals such as iron, cobalt and magnesium. The Protoporphyrin IX (heme) is the prosthetic group of several globular proteins that belong to class of the hemeproteins encompassing hemoglobin, myoglobin, peroxidases and cytochroms. In the hemeproteins, the detachment of the prosthetic group leads to the loss of the native tridimensional structure with the formation of the apohemeprotein. The apohemeproteins exhibit the ability to fold around the Protoporphyrin IX and other porphyrins that are supplied to them and the result is the formation of a structured non-covalent complex. The structure of the non-covalent complex, specially concerning to the heme pocket is not well-known. In this project we intend to utilize the photosensitization ability of Protoporphyrin IX and other porphyrins such as Zn-Substituted Protoporphyrin IX and Photofrin® to identify the amino acid residues that are situated in the porphyrin pocket. To attain this objective we will incorporate different photosensitizing porphyrins in different apohemeproteins such as those derived from cytochrome c, myoglobin, hemoglobin and HRP in inert atmosphere and in the dark. The non-covalent complexes will be analyzed by mass spectrometry MALDI-ToF in order to identify the amino acid residues that were preferentially oxidized. The pattern of oxidative damage obtained in the two experimental conditions will be compared with that obtained for apohemoproteins irradiated in the presence of methylene blue in the presence and in the absence of oxygen. There is the possibility that in the non-covalent complexes the amino acid localized near to the porphyrin will be preferentially oxidized and the pattern of oxidative damage will be an indicator of the amino acid that are neighbor of the prosthetic group.