[8] Purification of cytochrome b5
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Cited by (129)
Structural and kinetic basis of steroid 17α,20-lyase activity in Teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2
2015, Journal of Biological ChemistryCitation Excerpt :The fractions eluted in a peak at ∼0.3 m NaCl (based on A413 measurements) were pooled and dialyzed twice against 50 volumes of 20 mm potassium phosphate buffer (pH 7.6) containing 20% glycerol (v/v) and 0.1 mm EDTA, aliquoted, and stored at −20 °C. The final amount of recovered protein was 515 nmol from a 6-liter culture, based on spectral quantitation (37). To generate complexes, inhibitor (abiraterone, in (CH3)2SO) or substrate (progesterone, in C2H5OH (P450 17A2)) was added to purified protein.
Interaction of modified tail-anchored proteins with liposomes: Effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b<inf>5</inf>
2012, Journal of Bioscience and BioengineeringCitation Excerpt :For the purification of native Hcytb5 and other mutants, essentially identical purification procedures were conducted, resulting in similar results both in quality and contents. Cytochrome b5 content was estimated using an extinction coefficient of 117 mM− 1 cm− 1 at 412 nm for the oxididzed form or 163 mM− 1 cm− 1 at 423 nm for the dithionite-reduced form (24). Since the purified forms of Hcytb5 and its mutants contained substantial amounts of non-ionic detergent, Triton X-100, which would interfere the stability of the proteoliposomes because of its strong dissolving ability of the lipid bilayer and a low CMC value (0.23 mM (0.015%)), we removed the most of them by a following procedure.
A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles
2008, Protein Expression and PurificationCitation Excerpt :Over a ∼2 min period, the Soret peak was shifted from 413 nm to 423 nm and the peak at 555 nm increased in intensity. The resulting spectrum matched that of the reduced state of detergent-solubilized fl-cytb5 [15,37]. In control experiments, free heme was not reduced by His8-cytb5R in the time-scale and NADH concentration used for the cytb5 reduction.
Metmyoglobin reducing activity
2005, Meat ScienceReduction of cytochrome b<inf>5</inf> by NADPH-cytochrome P450 reductase
2005, Archives of Biochemistry and BiophysicsCitation Excerpt :Tergitol NP-10 (from Sigma) was dissolved in H2O to prepare a 2.5% stock solution (w/v), which was diluted for use. Concentrations of b5[34] and NPR [35] were measured using UV–visible spectroscopy. Steady-state measurements of rates of NPR reduction were made in 0.3 M potassium phosphate buffer (pH 7.7) at 23 °C with 2.0 nM concentrations of NPR, using an OLIS/Cary 14 instrument (On-Line Instrument Systems, Bogart, GA).
The cationic charges on Arg <sup>347</sup>, Arg <sup>358</sup> and Arg <sup>449</sup> of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b <inf>5</inf>-dependent acyl-carbon cleavage activities
2004, Journal of Steroid Biochemistry and Molecular Biology