Fluorogenic substrates for the screening assay of transketolase through beta-elimination of umbelliferone--Development, scope and limitations.
5-O-Coumarinyl-d-xylulose was studied as a fluorogenic substrate for the stereospecific assay of transketolase enzyme. Enzymatic C2-C3 cleavage released an alpha-hydroxyl, beta-coumarinyl substituted aldehyde. Although the subsequent beta-elimination step was rate limiting under chemical or enzymatic catalysis, we detected a TK activity as low as 0.7mIU. To improve the fluorescence signal release, kinetic and product distribution analyses of this reaction were performed by LC/UV/MS coupling.
Charmantray
F
F
Clermont Université, Université Blaise Pascal, Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand, France. Franck.CHARMANTRAY@univ-bpclermont.fr
Légeret
B
B
Hélaine
V
V
Hecquet
L
L
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D057230:Limit of Detection; D013058:Mass Spectrometry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013237:Stereoisomerism; D014153:Transaldolase / Q000378:metabolism; D014174:Transketolase / Q000378:metabolism*; D014468:Umbelliferones / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D014468:Umbelliferones; D012710:Serum Albumin, Bovine; C031477:7-hydroxycoumarin; D014174:Transketolase; D014153:Transaldolase