ASSAY FOR PHYTOL-FREE CHLOROPHYLL DERIVATIVES
The present invention provides a method for detecting a phytol-free chlorophyll derivative in a sample, comprising a step of detecting a fluorescent signal associated with the phytol-free chlorophyll derivative, wherein a fluorescent signal associated with chlorophyll or a phytol-containing chlorophyll derivative in the sample is quenched. The method may be used for quantifying activity of chlorophyllases and related enzymes in a sample without solvent fractionation of substrate and product.
1 . A method for detecting a phytol-free chlorophyll derivative in a sample, comprising a step of detecting a fluorescent signal associated with the phytol-free chlorophyll derivative, wherein a fluorescent signal associated with chlorophyll or a phytol-containing chlorophyll derivative in the sample is quenched, and wherein chlorophyll or the phytol-containing chlorophyll derivative is present in the form of an aqueous solution of dimers or other non-colloidal multimers.
2 . The method of claim 1 , wherein the fluorescent signal associated with chlorophyll or the phytol-containing chlorophyll derivative is quenched by dimerization of chlorophyll or the phytol-containing chlorophyll derivative.
3 . The method of claim 1 , wherein chlorophyll or the phytol-containing chlorophyll derivative dimerizes preferentially compared to the phytol-free chlorophyll derivative.
4 . The method of claim 1 , wherein the phytol-free chlorophyll derivative comprises pheophorbide or pyropheophorbide, or the phytol-containing chlorophyll derivative comprises pheophytin or pyropheophytin.
5 . The method of claim 1 , wherein the detecting step is performed in a detection solution comprising a surfactant, an alcohol or an alkali.
6 . The method of claim 5 , wherein the alcohol comprises isopropanol or ethanol.
7 . The method of claim 6 , wherein the detection solution comprises 12 to 20% by weight alcohol.
8 . The method of claim 5 , wherein the surfactant comprises 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol.
9 . The method of claim 5 , wherein the detection solution comprises 0.01 to 0.03% by weight surfactant.
10 . The method of claim 1 , wherein the detecting step is performed at 20 to 25° C.
11 . The method of claim 1 , wherein the detecting step is performed at a pH of greater than 10.0.
12 . The method of claim 1 , wherein a fluorescent signal of wavelength about 670 nm is detected.
13 . An assay method for quantifying enzyme activity in a sample, wherein the enzyme is capable of hydrolyzing chlorophyll or a phytol-containing chlorophyll derivative, the assay method comprising:
a.) contacting the sample with chlorophyll or a phytol-containing chlorophyll derivative; and
b.) detecting production of a phytol-free chlorophyll derivative by detecting a fluorescent signal associated with the phytol-free chlorophyll derivative, wherein a fluorescent signal associated with chlorophyll or a phytol-containing chlorophyll derivative in the sample is quenched, and wherein chlorophyll or the phytol-containing chlorophyll derivative is present in the form of an aqueous solution of dimers or other non-colloidal multimers.
14 . (canceled)
15 . (canceled)
16 . A kit for quantifying enzyme activity in a sample, wherein the enzyme is capable of hydrolyzing chlorophyll or a phytol-containing chlorophyll derivative, comprising:
a.) a reaction solution in which the enzyme is active;
b.) a substrate comprising chlorophyll or a phytol-containing chlorophyll derivative; and
c.) a detection solution in which the substrate dimerizes preferentially compared to a phytol-free chlorophyll derivative produced by the enzyme, and in which the substrate is present in the form of an aqueous solution of dimers or other non-colloidal multimers.
17 . The kit of claim 16 , wherein
(i) the reaction solution comprises a surfactant, acetone and a buffer, and
(ii) the substrate comprises pheophytin or pyropheophytin.
18 . (canceled)
19 . (canceled)
20 . (canceled)
21 . (canceled)
22 . (canceled)
23 . The method of claim 1 , wherein the detection step is performed in the absence of liposomes.
24 . The method of claim 5 , wherein the surfactant comprises 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, the alcohol comprises isopropanol and the alkali comprises sodium hydroxide.
25 . The assay method of claim 13 , wherein the enzyme is active during step (a) and the enzyme is inactive during step (b).
26 . The method of claim 1 , wherein the phytol-free chlorophyll derivative comprises pheophorbide or pyropheophorbide, and the phytol-containing chlorophyll derivative comprises pheophytin or pyropheophytin.
27 . The method of claim 1 , wherein the detecting step is performed in a detection solution comprising a surfactant, an alcohol and an alkali.