Immunodetection and quantification of cytochromes P450 using epitope tagging: immunological, spectroscopic, and kinetic analysis of cinnamate 4-hydroxylase.
Cytochrome P450-dependent monooxygenases (P450s) are integral membrane proteins typically expressed at low levels both in vivo and by heterologous expression systems, often making quantification of these enzymes challenging. Since the time of their discovery, P450s have typically been quantified by their carbon monoxide (CO) difference spectra. Although this technique is reliable, it requires quantities of enzyme that are sometimes difficult to obtain, and spectroscopic instruments and expertise frequently unavailable in laboratories whose primary focus is genetics or molecular biology. We have developed a method for quantifying recombinant FLAG epitope-tagged proteins using fluorescence detection of a chromophore-labeled anti-FLAG monoclonal antibody and well-established immunoblot technology. The utility of this technique was tested using cinnamate 4-hydroxylase (C4H), one of the best-studied plant P450s. No substantial differences in the stability or kinetic properties were observed between the native and FLAG-tagged enzymes. Immunochemical quantification of epitope-tagged C4H reported slightly lower P450 concentrations than conventional methods but has a limit of quantification 400-fold lower than carbon monoxide difference spectroscopy.
Humphreys
John M
JM
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, 47907-2063, Indiana.
Chapple
Clint
C
D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000276:immunology; D013057:Spectrum Analysis; D050564:Trans-Cinnamate 4-Monooxygenase
D000939:Epitopes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase