Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.
A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme.
Ping
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Department of Chemistry, University of Kentucky, Lexington 40506-0055.
Butterfiel
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D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010206:Papain / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013113:Spin Labels; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D005456:Fluorescent Dyes; D013113:Spin Labels; D020245:p-Chloromercuribenzoic Acid; C038518:acrylodan; D015081:2-Naphthylamine; D010206:Papain