Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C.
An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.
Zaikova
T O
TO
Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Rukavishnikov
A V
AV
Birrell
G B
GB
Griffith
O H
OH
Keana
J F
JF
D019793:Fluorescein / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010738:Type C Phospholipases / Q000378:metabolism*
D005456:Fluorescent Dyes; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D019793:Fluorescein