Fluorescence procedure used to determine activity of phosphatases, phosphodiesterases or kinases, employs trivalent lanthanide ion with organic ligand

摘要

The enzyme-catalyzed reaction is allowed to proceed using any natural or synthetic enzyme substrate. The following stages determine reaction conversion. Before, during or after reaction, the solution is mixed with a reagent comprising a complex, i.e. a trivalent lanthanide ion with an organic ligand. The ligand is not the enzyme substrate. The reagent is excited to photoluminescence by irradiation with light. Intensity or decay time of this photoluminescence is determined. Alteration of the fluorescence characteristics of the reagent resulting from the reaction, is used for the detection and determination of the respective enzyme. The enzyme is a phosphatase and the enzyme substrate a phosphate ester of glycerin, glucose, ribose or deoxyribose, a nucleoside, of serins, threonin, farnesol, creatinin, or of any other synthetic or bio-organic alcohol or phenol. The enzyme is a phosphatase and the phosphate ester a diphosphate or a triphosphate. The enzyme is a phosphodiesterase and the phosphate ester is a cyclic mono nucleotide e.g. the cyclic adenosin monophosphate or in the case of nucleases, a polynucleotide. The enzyme is a kinase and the enzyme substrate, if appropriate, is protein-bound serin, threonin or tyrosin, creatinin, glycerin, glucose, ribose or deoxyribose, a nucleoside or another synthetic or organic mono- or polyalcohol, or a phenol. The reagent general chemical structure contains the trivalent ion of europium, terbium, samarium, lanthanum, dysoprosium or holmium, with a chelator of the central ion. This has an absorption maximum in the waveband 300-450 nm. The luminescence of the ion is not extinguished. Neither substrate nor product are involved in the reaction. The ratio of ion to chelate is in the range 10:1 to 1:3. The lanthanide ion is a europium (III) or terbium (III) ion. The chelator is a hydroxylized heterocyclic or a chelatizing beta diketone (or its enol). The chelator is optionally a cumarin, chromon or chinolon-3-carbonic acid or tetracyclin or one of its derivatives. It is used to determine the effectiveness of enzyme inhibitors. It is used to determine the activity of an enzyme which is bound to a fixed substrate, on a biological membrane, another protein, e.g. another antibody, or on a natural or synthetic polynucleotide.