Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-07-28
1999-06-29
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 15, 435 17, 435 18, 435 19, 435 23, 435 24, C12Q 142, C12Q 148, C12Q 134
Patent
active
059167610
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method for determining adenosine 5' diphosphate ADP contained in a liquid sample by means of an enzymatic reaction, which comprises reacting the sample at 15 to 45.degree. C. at least in the presence of glucose, ADP-dependent hexokinase (ADP-HK), oxidized NAD(P) glucose-6-phosphate dehydrogenase (G6PDH), and one or more ion releasing salt selected from the group consisting of magnesium, cobalt and manganese ions and determining the ADP contained in the sample together with the AMP resulting from the reaction based on the amount of the reduced NAD (P) present invention further relates to a method for determining an enzyme for generating ADP or substrate thereof in a liquid sample by means of an enzymatic reaction, which comprises reacting the sample containing the enzyme for generating ADP or substrate thereof in the liquid sample at 15 to 45.degree. C. at least in the presence of a reaction reagent involving in the reaction based on the enzyme for generating ADP and the substrate thereof,ATP, glucose,ADP-HK, oxidized NAD (P),G6PDH and one or more ion releasing salt of magnesium ion, cobalt ion or manganese ion, and determining the enzyme for generating ADP or the substrate thereof contained in the sample together with the AMP resulting from the reaction based on the amount of the reduced NAD (P) yielded. The invention is applicable in the clinical chemistry. The method has advantageous for sample and exact assay of the ADP in the sample such as serum, plasma, urine and cerebrospinal fluid, by means of an amount of generation of or increase in the reduced NAD (P).
PRIOR ARTS
Methods for asssaying ADP in biological sample or enzyme, which generate ADP, or substrate thereof have known. These include a method using combination of the enzyme and other dehydrogenase or oxidase for the reaction product from substrate and the enzyme (for example, in case of enzymatic reaction with glycerol and glycerokinase, a method wherein glycerokinase is reacted with glycerol in the presence of ATP to generate ADP and glycerol phosphate, which is assayed by an action of glycerol phosphate dehydrogenase or glycerol phosphate oxidase) and a method assyaing ADP which is generated by enzymatic action.
However, in the method of assay by combining with other dehydrogenase or oxidase, a combination of each enzyme should be changed depending on the enzyme for generating ADP. This is not suitable for general use, furthermore there are possibilities of no enzymes with preferable combination for substrate or enzyme.
A method for assaying ADP,which is generated by enzymatic reaction, includes an assay by means of liquid chromatography. This has disadvantage due to complicated chromatographic operation.
Enzymatic assay methods of ADP which has good operability have known (Japan. Pta. Unexam. Publ. 7-8297). These include : a method measuring Reaction 1); a method using pyruvate kinase and pyruvate oxidase (oxidasemethod, Reaction 2); and a method measuring increase reduced NAD aldehyde dehydrogenase (increased method, Reaction 3).
The reactions of these methods are shown below.
In the equation ; PEP: phosphoenolpyruvate. Pi: phosphate, PDC: pyruvate decarboxylase, A1DH: aldehyde dehydrogenase, ans TPP: thiamine pyrophosphate. ##STR1##
In the decreased method of reduced NAD (NADH+H.sup.30) shown in Reaction 1, previously alliquot amount of reduced NAD should be added in the reaction mixture, and after termination of the reaction, residual amount of reduced NAD in the reaction mixture is measured. Consequently, this decreased method has number of problems as follows. assay. amount of added reduced NAD in the reaction mixture. assay should be controlled depending on the type of spectrophotometer for measurement of reduced NAD.
Quantitative assay of pyruvate by means of coloring indicator reagent of hydrogen peroxide, which is generated from the reaction by using pyruvate kinase and pyruvate as shown in Reaction 2, has known. (such as uric acid and ascorbate) or color substance (bilirubin,
REFERENCES:
patent: 4584272 (1986-04-01), Imahori et al.
patent: 5250416 (1993-10-01), Ohno et al.
by W.M. Kengen et al., "Purification and Characterization of a Novel ADP-dependent Glucokinase from the Hyperthermophilic Archaeon Pyrococcus feriosus", The Journal of Biological Chemistry, vol. 270, No. 51, Dec. 22, 1995, pp. 30453-30457.
by W.M. Kengen et al., "Evidence for the Operation of a Novel Embden-Meyerhof Pathway That Involves ADP-dependent Kinases during Sugar Fermentation by Pyrococcus furiosus", The Journal of Biological Chemistry, vol. 269, No. 26, Jul. 1, 1994, pp. 17537-17541.
Koga Shinji
Sakasegawa Shinichi
Asahi Kasei Kogyo Kabushiki Kaisha
Leary Louise N.
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