A fluorogenic substrate for the continuous assaying of aryl sulfatases.
The most common fluorogenic substrate for assaying aryl sulfatases (ARSs) is 4-methylumbelliferyl sulfate (MUS). However, ARSs operate optimally at pH values that are less than the pK(a) (7.8) of the reaction product of MUS, 4-methylumbelliferone (4-MU). Thus, a major disadvantage of this assay is that it is usually run in a discontinuous mode due to the need for basification of the reaction mixture to achieve complete ionization of the phenolic products and maximum fluorescence. To circumvent this problem, 6,8-difluoro-4-methylumbelliferyl sulfate (DiFMUS) was prepared and examined as a substrate for ARSs. The product of the reaction is 6,8-difluoro-4-methylumbelliferone, a known coumarin with fluorescent properties equal to those of 4-MU, and has a pK(a) of 4.9. This allowed for the continuous assaying of human placental ARSs A, B, and C, which operate optimally between pH 5.0 and pH 7.0. Furthermore, DiFMUS exhibited a lower K(m) (up to 20-fold) for the ARSs than did MUS; for ARSA and ARSB, it exhibited a greater V(max) than did MUS. This substrate should have considerable utility for the continuous assay of ARS activity.
Ahmed
Vanessa
V
Department of Chemistry, University of Waterloo, 200 University Ave. West, Waterloo, Ont., Canada N2L 3G1.
Ispahany
Mehdi
M
Ruttgaizer
Scott
S
Guillemette
Guy
G
Taylor
Scott D
SD
D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D010920:Placenta / Q000201:enzymology; D013429:Sulfatases / Q000032:analysis* / Q000378:metabolism*
C504401:6,8-difluoro-4-methylumbelliferyl sulfate; D006923:Hymecromone; D013429:Sulfatases