Methods for measuring protein kinase and phosphatase activity

A method for determining dephosphorylating activity of an enzyme acting on a substrate molecule, said substrate comprising at least one phosphorylated moiety that is de-phosphorylated by said enzyme to yield a product and wherein said substrate is labelled with a fluorescent dye selected from the group consisting of acridone and quinacridone classes of dyes, said method comprising the steps of:

• i) measuring fluorescence intensity of the fluorescently labelled substrate;
• ii) combining said enzyme with said substrate molecule to yield a product;
• and
• iii) measuring a decrease in fluorescence intensity following step ii);
• wherein said decrease in fluorescence intensity of the fluorescent label is used to determine the dephosphorylating activity of said enzyme.

A method of simultaneously measuring kinase activities of two or more different enzymes each enzyme specific for a different substrate, wherein each substrate comprises at least one moiety that phosphorylated by an enzyme to yield a product, and wherein each said substrate is labelled with a different one of a set of fluorescent dyes, said method comprising the steps of:

• i) measuring fluorescence intensity and fluorescence lifetime of each of the fluorescently labelled substrates;
• ii) combining a mixture of said enzymes with each of said substrates in the presence of a phosphate source;
• and
• iii) measuring an increase in fluorescence intensity and in fluorescence lifetime of each of the fluorescent labels following step ii);
• wherein the fluorescent dyes in said set are selected from the group consisting of acridone and guinacridone classes of dyes;
• and wherein said increase in fluorescence intensity and in fluorescence lifetime of each of the fluorescent labels is used to determine the phosphorylating activity of each of said enzymes.