Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of den...

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Bibliographic Details
Published in:The Journal of biological chemistry 1981-06, Vol.256 (12), p.6262-6265
Main Authors: G C Wagner, M Perez, W A Toscano, Jr, I C Gunsalus
Format: Article
Language:English
Subjects:
Apoproteins
Chemical Phenomena
Chemistry
Cytochrome P-450 Enzyme System
Electron Spin Resonance Spectroscopy
Heme
Pseudomonas - enzymology
Spectrum Analysis
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Summary:Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(19)69157-x