Structural lesion to ribonuclease A caused by reductive stress: Assessment by Raman spectroscopy

Armida Torreggiani

doi:10.1155/2008/878931

Abstract

The damages induced by reductive radical stress on bovine pancreatic ribonuclease A (RNAse A) were investigated by Raman spectroscopy. Gamma-irradiation was used to simulate the endogenous formation of reductive species, in particular•H atom that is a simple one-electron equivalent reducing agent. Specific damages occur at sensitive amino acid sites, selectively, rather than indiscriminately, leading to the structure modification of the protein. Sulfur-containing residues (Met and Cys) and aromatic residues are appreciably attacked. In particular, extensive changes in the disulfide bridge conformations are induced as well as conformational changes of the protein secondary structure; a gradual conversion ofα-helical to pleated-sheet geometry was evidenced, indicating a higher ability of reducing radicals in denaturing the protein structure compared with that of oxidizing radical species.

Highlights

Protein damage caused by free radicals makes part of the aetiology of several diseases and the aging processes
Since recently the reductive stress has been placed in the context of a biological damage, constituting the basis of tandem-lipid damage the separate contribution of a reducing species such as H to the changes produced in RNAse was investigated more in detail in the present work [5,6]
The degradation of ribonuclease A (RNAse A) due to radical exposure was followed by Raman spectroscopy and the conditions of an endogenous radical stress were mimicked by using γ-radiolysis as source of free radicals

Summary

Introduction

Protein damage caused by free radicals makes part of the aetiology of several diseases and the aging processes. Exposure of proteins to free radicals may cause structural modifications of primary, secondary and tertiary assembly; the activity of enzymes, receptors and membrane transporters can be greatly affected [1,2]. In this respect, the fate of radical species derived from protein damage has been subject of many investigations, a clear picture of the degradation paths and their influence on the disease aetiology is far from being achieved. The degradation of RNAse A due to radical exposure was followed by Raman spectroscopy and the conditions of an endogenous radical stress were mimicked by using γ-radiolysis as source of free radicals. To identify the action of reductive radicals, irradiations were performed under various environmental conditions to isolate the individual radicals

Experimental

Results and discussion

Conclusions