ADP detection using an enzyme-coupled reaction
View Patent ↗Methods and compositions are provided for determining ADP in the presence of ATP. These comprise including among the assay reagents at least one of the correcting components creatine phosphokinase and phosphocreatine, pyruvate kinase and phosphoenolpyruvate, peroxidase and a non-interfering peroxidase substrate, and catalase. One aspect of the method employs formation of hydrogen peroxide from the ADP by pyruvate kinase, phosphoenolpyruvate and pyruvate oxidase. The hydrogen peroxide is then determined. A combined reagent having all of the reagents may optionally include a peroxidase when the hydrogen peroxide is to be enzymatically determined. A peroxidase substrate is added to the sample in conjunction with the peroxidase substrate reagent, the mixture incubated and depending on whether the peroxidase substrate is a fluorescer or chemiluminescer, the mixture may be illuminated with excitation light and the emitted light determined as a measure of the ADP in the sample.
1. In a method for determining ATPase activity in a sample by measuring ADP to estimate the extent of reaction of ATP, where the method comprises the steps of: combining the sample, with reagents comprising ATP, pyruvate kinase, phosphoenolpyruvate, pyruvate oxidase and agents for determining the production of hydrogen peroxide from the reaction of pyruvate catalyzed by pyruvate oxidase, said agents producing a detectable product; and determining said detectable product;
the improvement which comprises:
prior to adding the ATP and said sample to said reagents, combining catalase or peroxidase and a non-interfering substrate with said reagents other than ATP and at least one of:
1) with said ATP, correcting components selected from the group consisting of (a) creatine phosphokinase and phosphocreatine and (b) pyruvate kinase and phosphoenolpyruvate, or
(2) with said phosphoenolpyruvate, correcting components selected from the group consisting of (a) lactate dehydrogenase (LDH) and reduced nicotinamide adenine dinucleotide (NADH), (b) glutamic pyruvic transaminase and glutamic acid and (c) pyruvate oxidase and wherein the amounts employed of said correcting components are substantially non-interfering in said determining of ATPase activity.
2. A method according to claim 1 wherein said phosphoenolpyruvate is combined with a member of the group consisting of
(1) lactate dehydrogenase and reduced nicotinamide adenine dinucleotide,
(2) glutamic pyruvate transaminase and glutamic acid and
(3) pyruvate oxidase.
3. A method according to claim 1 , wherein substantially non-interfering is that the difference in the observed results in the presence of the correcting components and in their absence with non-contaminated reagents is less than about 10%.
4. A method according to claim 3 , wherein said reagents further comprise peroxidase, where said reagents and correcting components are at a concentration in the range of:
Component
Range
Pyruvate kinase
5-500
U/ml
Pyruvate oxidase
1-100
U/ml
Peroxidase
1-100
U/ml
Catalase
0.01-10
U/ml
Phosphoenolpyruvate
10-1000
μM.
5. A method according to claim 3 , wherein said reagents and correcting components further comprise peroxidase, FAD, TPP, buffer, peroxidase substrate, and activating salt, where said reagents are at the concentration ranges indicated:
Component
Range
Pyruvate kinase
5-500
U/ml
Pyruvate oxidase
1-100
U/ml
Peroxidase
1-100
U/ml
Catalase
0.01-10
U/ml
Flavin adenine dinucleotide (FAD)
1-100
μM
Thiamine pyrophosphate (TPP)
10-1000
μM
Phosphoenolpyruvate
10-1000
μM
Buffer
10-100
mM
Peroxidase substrate
10-100
μM
Activatin salt (e.g., MgCl 2 )
0.2-40
mM.
6. A method according to claim 3 , wherein said reagents and correcting components further comprise peroxidase, FAD, TPP, buffer, peroxidase substrate, activating salt, and enzyme stabilizer, where said reagents are combined in a single reagent comprising the composition:
Component
Range
Pyruvate kinase
10-500
U/ml
Pyruvate oxidase
2-50
U/ml
Peroxidase
1-50
U/ml
Catalase
0.02-10
U/ml
Flavin adenine dinucleotide (FAD)
2-80
μM
Thiamine pyrophosphate (TPP)
20-500
μM
Phosphoenolpyruvate
20-800
μM
Buffer
10-100
mM
Activatin salt (e.g., MgCl 2 )
0.2-30
mM.
Enzyme stabilizer
2-40
wt %.