Date of Award

2011

Degree Type

Thesis

Department

Chemistry

First Advisor

Stuehr, Dennis

Subject Headings

Heme, Hemoproteins, Dehydrogenases

Abstract

The aim of my research is to understand the mechanism of heme transfer by carrier proteins into heme proteins. The mechanism of heme insertion into proteins is poorly understood. Previous experiments reported from our lab identified GAPDH as a possible heme carrier protein involved in heme transfer (Ritu Chakravarti et al PNAS). This work focuses on 2 aspects of heme transfer. a.) Rate of transfer of heme from GAPDH to apomyoglobin. We examined the rate of transfer of native proto-heme and the analogue meso-heme. The results showed that the rate of heme transfer was twice as fast for meso-heme compared to the native proto-heme, for both human and rabbit GAPDH's. This suggests that structural factors may be important for heme transfer from GAPDH to apo-myoglobin. b.) Nitrosylation of GAPDH and its effect on rate of heme transfer. We went on to examine different factors that could affect the extent of nitrosylation. The factors which we examined were dose of nitrosylation donor, pH, and stability of nitrosylated protein with temperature and presence of oxygen. 1.) Increasing the dose of nitrosylation donor increased the extent of nitrosylation in the case of human GAPDH, in contrary with rabbit GAPDH nitrosylation which is not dependent on dose 2.) Nitrosylated protein is more stable at 4oC than at room temperature. 3.) The extent of nitrosylation increased as the pH increases in case of rabbit GAPDH, however human GAPDH nitrosylation was favored at slightly acidic and alkaline compared to neutral pH. Reducing agents like DTT, tertiary amines like HEPES buffer and oxygen were found to have no effect on extent of nitrosylation. Previous cell biology work from our lab suggests that nitrosylation of Cys152 in vivo diminishes heme binding property of GAPDH. Cysteine residues that underwent nitrosylation were identified by mass spectrometry. The results from mass spectrometry suggest that the levels of nitrosylation on each individual cysteine are species-specific. In fact, rabbit and human GAPDH showed different leve

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