Membrane protein proteolysis assayed by fluorescence quenching: assay of O-sialoglycoprotein endopeptidase.
The assay of the O-sialoglycoprotein endopeptidase of Pasteurella haemolytica has previously used the cleavage of 125I-labeled glycophorin A, measured by SDS-PAGE, autoradiography, gel-slicing, and scintillation counting. A new assay is based on the increased fluorescence which results from proteolytic cleavage of a fluorescence-quenched micellar substrate, 4,4-difluor-5,7-dimethyl-4-bora-3 alpha, 4 alpha-diaza-s-indacene-3-propionic acid conjugated to glycophorin A (BODIPY-FL-glycophorin A). Micellar association of glycophorin A molecules results in 97% fluorescence quenching despite a low molar ratio of BODIPY-FL-glycophorin A. Proteolysis of the membrane protein causes greatly enhanced fluorescence which is used for a rapid one-step proteolysis assay. Direct monitoring of proteolysis in microcuvettes, or routine assay in microtiter plates can be used. Reproducibility is higher than with the radiolabeled substrate and the K(m) values for the two substrates are similar. The assay is suitable for the O-sialoglycoprotein endopeptidase activity of chromatographically purified enzyme or unpurified bacterial culture supernatants and can be used to monitor inhibition of the O-sialoglycoprotein endopeptidase by neutralizing antibodies. The O-sialoglycoprotein endopeptidase assay employing BODIPY-FL-glycophorin A provides a rapid and nonradioactive method for the assay of this highly specific enzyme.
Jiang
P
P
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Ontario, Canada.
Mellors
A
A
D001896:Boron Compounds; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005456:Fluorescent Dyes; D006021:Glycophorins; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D016978:Mannheimia haemolytica / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000032:analysis* / Q000097:blood; D008823:Micelles; D012588:Scintillation Counting / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D006021:Glycophorins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D008823:Micelles; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase