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 micrograms 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 microM 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 heme-degrading enzyme in tissue culture studies and cases where limited amounts of material are available.
Sierra
E E
EE
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Nutter
L M
LM
D001345:Autoradiography; D001663:Bilirubin / Q000302:isolation & purification; D001943:Breast Neoplasms / Q000201:enzymology; D002855:Chromatography, Thin Layer; D006418:Heme / Q000302:isolation & purification / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008665:Metalloporphyrins / Q000494:pharmacology; D011524:Protoporphyrins / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D008665:Metalloporphyrins; D011524:Protoporphyrins; D006418:Heme; C032628:tin protoporphyrin IX; D006419:Heme Oxygenase (Decyclizing); D001663:Bilirubin