Graduate Thesis Or Dissertation
 

A proteomic approach to 1,2-dichloroethane bioactivation and reaction with redox-active protein disulfide isomerase

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  • Protein disulfide isomerase (PDI), a member of the thioredoxin superfamily, contains two domains with significant sequence homology to the active sites in thioredoxin. PDI facilitates the folding of nascent proteins in the endoplasmic reticulum (ER), binds hormones and Ca²⁺, catalyzes the glutathione dependent reduction of dehydroascorbate, serves as a major chaperone molecule in the ER and serves as a subunit for prolyl-4-hydroxylase and microsomal triglyceride transferase. Because of its abundance in the ER and association with disease and chemically induced toxicity, the goal of this research was to investigate the relative susceptibility of PDI thiols to alkylation. The sensitivity of PDI to 1-chloro-2,4-dinitrobenzene (CDNB), iodoacetamide (IAM) and biotinoylated iodoacetamide (BIAM) was explored. The relative susceptibility of the thiolate anions present in the two active sites of PDI each containing the -CGHC- sequence was investigated with mass spectrometric techniques. PDI was inactivated by CDNB but was not found as sensitive as thioredoxin reductase as shown by Amer and coworkers (1995). IAM and BIAM were used as model alkylating agents to explore the two active sites of PDI and determine the residues most susceptible to alkylation. Alkylation by IAM and BIAM was first detected at the N-terminal cysteine in each active site (-C*GHC-) followed by alkylation at the second cysteine residue (-C*GHC*-) as shown by tandem mass spectrometry. Mass spectroscopy showed that the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane, S-(2-chloroethyl)glutathione (CEG), decreased activity and protein thiols of PDI. CEG produced two protein adducts at very low excesses of CEG over PDI; however, higher concentrations resulted in several protein adducts. Only one modification in each active site at the N-terminal cysteine residue can be identified, indicating that while these thiolate anions of PDI are susceptible, it would appear that the episulfonium ion may present itself to other sites as well. This may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity and the role of PDI in the redox status of the cell.
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