LAUSR

Abstract Hydrogen peroxide (H 2 O 2 ) is an important signalling molecule and a non-radical reactive oxygen species (ROS) which can convert into more potent oxygen radicals leading to oxidative stress. The correlation between oxidative stress and the cellular proteinase-antiproteinase balance has been a major component in the development of several pathologies such as emphysema and cystic fibrosis. The present studies attempt to explore the functional inactivation and structural alterations induced in one of the major antiproteinase, alpha-2-macroglobulin (α 2 M) on interaction with H 2 O 2. α 2 M, purified from sheep blood plasma is a highly abundant proteinase inhibitor which acts as a backup or reserve force of antiproteinase. Our studies provide biophysical insights into the interaction of H 2 O 2 with sheep α 2 M. Result demonstrates that antiproteolytic activity of α 2 M was significantly lowered upon interaction with increasing concentration of H 2 O 2. Analysis of fluorescence spectra reveals quenching of fluorescence intensity in a static manner which occurs due to ground state complex formation. Synchronous fluorescence illustrates alteration in microenvironment around tryptophan and tyrosine residues of α 2 M. Additionally, UV/visible spectroscopy, circular dichroism and fourier transform infrared spectroscopy results confirms the alteration in secondary structure and conformation of α 2 M due to H 2 O 2 induced modification. Moreover, polyacrylamide gel electrophoresis also confirms the structural change in α 2 M on H 2 O 2 interaction. Thermodynamic parameters of H 2 O 2 binding with α 2 M obtained by ITC indicate the reaction to be exothermic and spontaneous. Our studies suggest that H 2 O 2 induces functional inactivation and structural disruption of sheep α 2 M possibly due to perturbation of secondary structure, at concentration of oxidant achievable in patho-physiological conditions such as cancer.