| The objective of this research was to understand the effect of oxidative stress on metal binding proteins, especially zinc binding proteins. The importance of understanding the effects of oxidative stress on metal binding proteins stems from their influence on various pathological disorders, followed by their oxidative sensitivity to redox active transition metals and specifically the dual nature zinc to generate an apoptotic stimulus and also act as an inhibitor of oxidative stress in presence of other transition metals.;Chapter 1 gives a brief review about the importance of metals, metal binding sites in proteins and the effects of redox active transition metals on proteins. In chapter 2, the effect of various redox active transition metals on recombinant human insulin was addressed. It was concluded that the oxidative susceptibility of zinc-insulin, is specific only to Cu2+ and restricted to metal binding amino acids His5 and His 10. This chapter illustrates the importance of accessibility of metal binding domains in proteins to only certain metals and the inequality in generating redox modifications to the same extent by various transition metals.;In chapter 3, metal catalyzed oxidation was coupled with quantitative LC-MS/MS to characterize the metal binding site of human prolactin. In this chapter the site specific nature of metal-catalyzed oxidation was exploited and concluded that the His27, His30 and His173 form a putative metal binding site in human prolactin (hPRL). This result shows the similarity of hPRL to human growth hormone (hGH), a member of the same family as hPRL, where the mbs is composed of His 18, His21 and Glu174.;In chapter 4, we embarked on an ambitious project to understand and characterize the effect of oxidative stress on zinc transporter protein-1 (ZnT-1), during aging and under in vitro conditions in the presence of various oxidizing agents. A polyclonal antibody specific to ZnT-1 was made and characterized to study ZnT-1. It was concluded that ZnT-1 is prone to oxidative stress, evident by formation of disulfide bridged aggregates during aging and more importantly, this aggregation appears to be a metal catalyzed process. Mechanistic studies performed on recombinant His rich loop (rHR-Loop) of ZnT-1 confirmed the instability of this region of ZnT-1 to free radicals generated by redox active transition metals. |
