A MICROASSAY FOR HEME OXYGENASE ACTIVITY USING THIN-LAYER CHROMATOGRAPHY

A sensitive and facile assay for heme oxygenase (HO) has been developed. The basis of the assay is the detection of [14C]bilirubin formation in a coupled enzyme assay involving HO and biliverdin reductase actions, respectively. Separation of substrate from product is accomplished by thin-layer chromatography with subsequent quantitation by liquid scintillation counting of radioactive material present on chromatograms. As little as 20 μg of total cellular protein derived from cells growing in a standard 25-cm2 culture flask is sufficient for detection of HO enzyme activity using this assay. The reaction is inhibited by tin-protoporphyrin (10 μm final concentration), a specific inhibitor of HO. The linearity of the enzyme reaction with respect to incubation time and amount of protein used was established. Comparison of the new HO assay with a spectrophotometric assay was made, and good agreement of the results from both methods was found. The assay described here should facilitate measurements of this important hemedegrading enzyme in tissue culture studies and cases where limited amounts of material are available. © 1992.