Functional reconstitution of cytochrome P-450scc with hemin activated with Woodward's reagent K. Formation of a hemeprotein cross-link. |
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Authors: | I A Pikuleva A G Lapko V L Chashchin |
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Institution: | Institute of Bioorganic Chemistry, Byelorussian Republic Academy of Sciences, Minsk, Union of Soviet Socialist Republics. |
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Abstract: | In order to evaluate structure-function relationships of heme moiety in cytochrome P-450scc, we carried out the reconstitution of apoprotein with Fe-protoporphyrin IX, one carboxyl group of which was converted to reactive enol ester by Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate). Woodward's reagent K can be used as a cross-linking reagent, since amino groups can apparently react with the enol ester. Treatment of cytochrome P-450scc with H2O2 was used to obtain the apoprotein. Functional reconstitution of the hemin derivative with apocytochrome P-450scc was achieved. The reconstituted hemeprotein was purified, and the resulting preparation contained no P-420 form and had the same cholesterol-hydroxylating activity as a control preparation. 30% of the reconstituted hemin was covalently bound to protein. Heme-linked peptide (Gly177-Phe194) was isolated. Its possible role in the active site formation of cytochrome P-450scc is discussed. |
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