High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450

Cytochromes P450 are a superfamily of enzymes that catalyze the monoxygenation of a variety of substrates, including aliphatic and aromatic compounds (1 ). They contain a noncovalently bound protoporphyrin IX, giving these enzymes characteristic spectral properties. This heme has an available sixth coordination ligand that is able to bind carbon monoxide. Difference spectroscopy yields a spectral peak at approx 450 nm when comparing bound and unbound forms (2 ). This difference demonstrates the presence of a correctly-folded cytochrome P450. Binding of carbon monoxide in the presence of a biologically inactive form of a cytochrome P450 yields a spectral peak at 420 nm (3 ). Thus CO binding effectively assays for the presence of a correctly-folded cytochrome P450, an incorrectly-folded P420, or a lack of either. Carbon monoxide binding assays are typically done in single-cuvet format (4 ) but are easily modified for high-throughput in microtiter plates. This method is useful in quickly assaying a library of cytochrome P450s for folded and possibly functional proteins while eliminating misfolded or low expressing variants. It can also be used to rapidly determine the P450 concentration of multiple samples or the relative expression levels of individual clones in a high-throughput screen of a mutant library.