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Novel Modifications of Job’s Method Applied to Determination of the Stoichiometry of Cytochrome P450 3A4 Interactions With Substrates
Author(s) -
Davydov Dmitri R,
Fernando Harshica,
Halpert James R
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a459
Subject(s) - cooperativity , allosteric regulation , chemistry , cooperative binding , substrate (aquarium) , allosteric enzyme , binding site , enzyme , biophysics , analytical chemistry (journal) , biochemistry , chromatography , oceanography , biology , geology
The prevailing concept of cooperativity in cytochrome P450 3A4 (CYP3A4) is based on the presumption of multiple substrate binding sites in the enzyme. Thus, it is vital to determine the stoichiometry of interactions of the CYP3A4 with allosteric and non‐allosteric substrates. For this purpose we developed two novel modifications of the continuous variation (Job’s titration) method for the studies of enzyme‐substrate interactions by absorbance spectroscopy. Both methods are based on gradual mixing of the enzyme and substrate solutions of similar concentration. In the first method the experiment is carried out in a cell with a vertical optical beam, so that the path length increases during the experiment. In the second approach the experiment is carried out by progressive mixing of the enzyme and substrate solutions placed into two compartments of a tandem cell, so that the recorded signal represents the sum of the ascending and descending branches of the Job’s plot. We used these methods to study the interactions of CYP3A4 with bromocriptine (BCT), 1‐pyrenebutanol (1‐PB), 1‐pyrenemethylamine (PMA) and coumarin‐6 (C6). In the case of 1‐PB, which exhibits prominent cooperativity with CYP3A4, both approaches reveal two binding sites, while BCT, PMA and C6, which show no cooperativity, exhibit only one binding site. This novel methodology is proposed as a universal approach for studies of the interactions of cytochromes P450 with substrates. (Supported by GM54995 and Center Grant ES06676 and by H‐1458 from the Robert A. Welch Foundation).