Method for detecting pyrophosphate by means of bioluminescence detection

Abstract

The invention relates to methods for detecting pyrophosphate by means of bioluminescence detection. In addition, methods for measuring chemical, especially enzyme-catalyzed, reactions in which pyrophosphate is formed or consumed are described. Such reactions are catalyzed for example by guanylyl cyclases, adenylyl cyclases, DNA polymerases or RNA polymerases. The novel methods are distinguished by high sensitivity and low susceptibility to interference, can easily be automated and miniaturized and are additionally suitable for carrying out continuous measurements. The methods can be employed particularly advantageously in the area of medical diagnosis and biomedical research, including pharmaceutical active ingredient research.

1 . A method for detecting pyrophosphate, characterized in that it includes the following stages:
 (a) contacting the components of a composition including dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate with the test sample (b) measuring luminescence.   
     
     
         2 . The method as claimed in  claim 1 , where the luciferase is selected from the group consisting of
 non-recombinant or recombinant luciferases,   non-recombinant or recombinant luciferases from insects of the superfamilies Elateroidea and Cantharoidea,   non-recombinant or recombinant luciferases from the insects  Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis  or  Luciola mingrelica,      luciferases derived or mutated therefrom,   or mixtures of luciferases derived therefrom.   
     
     
         3 . The method as claimed in  claim 1 , characterized in that the test sample originates from a natural source including the animate and inanimate environment. 
     
     
         4 . The method as claimed in  claim 1 , characterized in that the test sample originates from a vegetable or animal organism. 
     
     
         5 . The method as claimed in  claim 1 , characterized in that the test sample originates from the human body. 
     
     
         6 . The method as claimed in  claim 5 , characterized in that the test sample comprises samples or preparations of tissue or body fluids. 
     
     
         7 . The method as claimed in  claim 1 , characterized in that the test sample is a pyrophosphate-forming or -consuming chemical reaction. 
     
     
         8 . The method as claimed in  claim 1 , characterized in that the test sample is a pyrophosphate-forming or -consuming enzyme reaction. 
     
     
         9 . A method for measuring the time course of a pyrophosphate-forming or -consuming enzyme reaction, which includes the following stages:
 (a) contacting the components of the pyrophosphate-forming or -consuming enzyme reaction with the components of a composition including dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate;   (b) continuously measuring luminescence.   
     
     
         10 . The method as claimed in  claim 9 , where the luciferase is selected from the group consisting of
 non-recombinant or recombinant luciferases,   non-recombinant or recombinant luciferases from insects of the superfamilies Elateroidea and Cantharoidea,   non-recombinant or recombinant luciferases from the insects  Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis  or  Luciola mingrelica,      luciferases derived or mutated therefrom,   or mixtures of luciferases derived therefrom.   
     
     
         11 . A method for measuring the time course of a pyrophosphate-forming or -consuming enzyme reaction, characterized in that it includes the following stages:
 (a) contacting one or more components of the pyrophosphate-forming or -consuming enzyme reaction with a composition including dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate;   (b) contacting the combination from (a) with the remaining components of the pyrophosphate-forming or -consuming enzyme reaction; and   (c) continuously measuring luminescence.   
     
     
         12 . The method as claimed in  claim 11 , where the luciferase is selected from the group consisting of
 non-recombinant or recombinant luciferases,   non-recombinant or recombinant luciferases from insects of the superfamilies Elateroidea and Cantharoidea,   non-recombinant or recombinant luciferases from the insects  Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis  or  Luciola mingrelica,      luciferases derived or mutated therefrom,   or mixtures of luciferases derived therefrom.   
     
     
         13 . The method as claimed in  claim 8 , where the enzyme reaction is selected from the group consisting of
 pyrophosphate-forming or pyrophosphate-consuming enzyme reactions, guanylyl cyclase-catalyzed reactions,   adenylyl cyclase-catalyzed reactions,   squalene synthase-catalyzed reactions,   pyrophosphatase-catalyzed reactions,   DNA polymerase-catalyzed reactions or   RNA polymerase-catalyzed reactions.   
     
     
         14 . The method as claimed in  claim 8 , characterized in that the enzyme reaction is catalyzed by soluble vascular guanylyl cyclase. 
     
     
         15 . A chemical composition including
 dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate.   
     
     
         16 . The composition as claimed in  claim 15 , where the luciferase is selected from the group consisting of
 non-recombinant or recombinant luciferases,   non-recombinant or recombinant luciferases from insects of the superfamilies Elateroidea and Cantharoidea,   non-recombinant or recombinant luciferases from the insects  Photinus pyralis, Pyrophorus plagiophthalamus, Luciola cruciata, Luciola lateralis  or  Luciola mingrelica,      luciferases derived or mutated therefrom,   or mixtures of luciferases derived therefrom.   
     
     
         17 . An assay kit for carrying out the method of  claim 1  including at least
 a suitable first container which comprises at least dehydroluciferin, 
 a suitable second container which comprises at least luciferin, 
 a suitable third container which comprises at least ATP, and 
 a suitable fourth container which comprises at least luciferase which can be activated by pyrophosphate. 
 
     
     
         18 . An assay kit for carrying out the method of  claim 1  including at least
 one or more suitable containers which in each case comprise at least one or more components selected from the list consisting of dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate, with each listed component being present in at least one of the suitable containers. 
 
     
     
         19 . An assay kit for carrying out the method of  claim 1  including at least one suitable container which comprises at least dehydroluciferin, luciferin, ATP and luciferase which can be activated by pyrophosphate.