A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.
A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII, NIV-tetra-acetyl-chitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphtalene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo-chitinase and chitobiosidase activities could be specifically assayed by monitoring the variation of fluorescence after enzymatic hydrolysis of this substrate. Chitobiases and N-acetyl-beta-glucosaminidases are not active on the compound, the presence of a bulky chromogenic group at the 2 position of the nonreducing end of the subtrate preventing the binding and thus hydrolysis by these two exo-enzymes. The observation that chitobiosidases are able to hydrolyse a chitooligosaccharide functionalized on both extremities demonstrates the possibility of an endo-action for this class of chitinases, which are generally classified as exo-enzymes. This fluorogenic chitooligosaccharide should prove to be very useful for the detection and the convenient assay of chitinolytic activities at nanomolar concentrations.
Cottaz
S
S
Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, Grenoble, France. cottaz@cermav.cnrs.fr
Brasme
B
B
Driguez
H
H
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002688:Chitinases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D005453:Fluorescence; D006596:Hexosaminidases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013379:Substrate Specificity; D013890:Thiourea / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
C414756:N(I),N(II),N(III),N(IV)-tetraacetyl-NV-dimethylaminophenylazophenylthioureido-beta-chitopentaosyl-pyroglutamyl amidoethylnaphthalenesulfonic acid; D009844:Oligosaccharides; C079639:1,4-beta-chitobiosidase; D006596:Hexosaminidases; D002688:Chitinases; D013890:Thiourea; D011761:Pyrrolidonecarboxylic Acid