Purification of Human CYPIA2 and Rat NADPH Reductase in Preparation for the Determination of Adduct Formed between 01tipraz and Human CYPIA2
Woodrow, Andrew Jon
A group of enzymes collectively called "Cytochrome P450's" play a very important role in the cellular process of natural detoxification. These enzymes detoxify environmental toxins and metabolize xenobiotics, among other things. At times, in the process of metabolization, the P450 enzymes can activate certain carcinogenic substances. CYPIA2, an isoform of the cytochrome P450's, activates Aflatoxin Bl, a hepatocarcinogen prevalent in Southeast Asia. Recently, research has been done with the drug Oltipraz, which has been shown to inhibit CYPIA2. The ultimate goal is to determine the nature of the adduct (chemical addition product) that Oltipraz forms with CYPIA2, so that new drugs can be designed to inhibit this enzyme more efficiently. To determine this adduct, assays must be done on a purified, reconstituted system of CYPIA2, NADPH-reductase, and other vital components necessary for a membrane-bound system. The purpose of this project was to purify CYPIA2 and NADPH-reductase in preparation for this reconstituted system. In the end, CYPIA2 was successfully purified to a final yield of 22.86 nmol and NADPH-reductase was not successfully purified.
vii, 33 p.
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