Methods for measuring enzyme activity
AssigneeGe Healthcare UK Limited
InventorJohn G. Whateley
Life of this patent
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Abstract
The invention relates to fluorescence methods for measuring enzyme activity, in particular enzyme cleaving and joining activities. The invention also relates to fluorogenic substrates which are useful for measuring enzyme activity and as in vitro and in vivo imaging probes.
Claims28
In a method of measuring the activity of an enzyme in cleaving a substrate, said substrate including at least one fluorescent label bound to a polymer which includes one or more tyrosine, tryptophan, phenoxy, indolyl or nitro-phenylalanine moieties, said moieties being separated from the said at least one fluorescent label by a linkage group cleavable by said enzyme, the improvement comprising the steps of:
- i) measuring the fluorescence lifetime of the at least one label of the substrate in a reaction mixture which facilitates enzyme activity;
- ii) adding the enzyme to said reaction mixture, and
- iii) measuring any increase in fluorescence lifetime of the at least one fluorescent label following step ii);
- wherein said increase in fluorescence lifetime indicates substrate cleavage and can be used to determine enzyme activity.
A method for measuring cellular location and distribution of a substrate, wherein the substrate is capable of being taken up by a living cell, the method comprising the steps of:
- i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a cell-free environment;
- ii) adding the substrate to one or more cells, and
- iii) measuring the fluorescence intensity and/or the lifetime of the fluorescent label following step ii);
- wherein an increase in fluorescence intensity and/or fluorescence lifetime indicates substrate cleavage and can be used to determine both enzyme activity and localisation.
A method of measuring the activity of an enzyme in joining a substrate to a reactant, said substrate including at least one fluorescent label and said reactant including one or more tyrosine, tryptophan, phenoxy, indolyl or nitro-phenylalanine, moieties, the method comprising the steps of:
- i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a reaction mixture which facilitates enzyme activity;
- ii) adding the enzyme to said reaction mixture, and
- iii) measuring any decrease in fluorescence intensity and/or lifetime of the fluorescent label following step ii);
- wherein said decrease in fluorescence intensity and/or lifetime indicates joining of the substrate to the reactant and can be used to determine enzyme activity.
A method for measuring cellular location and/or distribution of the substrate and/or reactant, wherein the substrate and the reactant are capable of being taken up by a living cell, the method comprising the steps of:
- i) measuring the fluorescence intensity and/or the fluorescence lifetime of the label in a cell-free environment;
- ii) adding the substrate and the reactant to one or more cells, and
- iii) measuring the fluorescence intensity and/or the lifetime of the fluorescent label following step ii);
- wherein a decrease in fluorescence intensity and/or fluorescence lifetime indicates substrate joining to reactant and can be used to determine both enzyme activity and localisation.
Dependencies are inferred from the claim text.