A rapid fluorescence-based assay for detecting soluble methane monooxygenase.
A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be Km(app)=262 microM and Vmax(app)=821 nmol 7-hydroxycoumarin min(-1) mg protein(-1). The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.
Miller
A R
AR
Idaho National Engineering and Environmental Laboratory, Idaho Falls 83415-2203, USA. millar@inel.gov
Keener
W K
WK
Watwood
M E
ME
Roberto
F F
FF
D003374:Coumarins / Q000378:metabolism*; D005453:Fluorescence; D009682:Magnetic Resonance Spectroscopy; D020602:Methylosinus trichosporium / Q000201:enzymology* / Q000254:growth & development; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013997:Time Factors; D014468:Umbelliferones / Q000378:metabolism
D003374:Coumarins; D014468:Umbelliferones; C031477:7-hydroxycoumarin; C030123:coumarin; D010105:Oxygenases; C021555:methane monooxygenase