Enzymatic cycling method for detecting homocysteine

A homocysteine ​​and homocysteine ​​technology, which is applied in the preparation of test samples, measurement devices, color/spectral characteristics measurement, etc., can solve the problems of difficult to master production technology, difficult market application, and high cost

Active Publication Date: 2017-03-15
HUNAN YONGHE YANGGUANG SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In addition to the traditional high performance liquid chromatography, enzyme immunoassay, and fluorescence polarization methods, methods for the determination of homocysteine ​​have also emerged in recent years based on methionine and adenosylhomocysteine. Cyclic enzyme method (see Chinese patent CN200480026009.4), but because the production technology of key raw materials is difficult to master and the cost is very high, it is difficult to form market application

Method used

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  • Enzymatic cycling method for detecting homocysteine
  • Enzymatic cycling method for detecting homocysteine
  • Enzymatic cycling method for detecting homocysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] S1: Prepare a kit, the kit reagents include the following components: Tris buffer with a concentration of 20mM; calcium 5-methyltetrahydrofolate with a concentration of 5mM; glycine with a concentration of 10mM; NAD + ; a concentration of 2 mM MgCl 2 ; methionine synthase at a concentration of 5kU / L; aminomethyltransferase at a concentration of 1kU / L; glycine hydroxymethyltransferase at a concentration of 2kU / L.

[0037] S2: Weigh 1.211g of the Tris buffer in step S1, dissolve it in 300ml of purified water, and adjust the pH to 8.00±0.05 with concentrated hydrochloric acid;

[0038] S3: Weigh 1.244 g of calcium 5-methyltetrahydrofolate, 0.375 g of glycine, and NAD in step S1 + 0.331g and MgCl 2 0.203g was added to the buffer solution in step S2, and stirred until completely dissolved;

[0039] S4: Weigh 2.5kU of methionine synthase, 0.5kU of aminomethyltransferase, and 1kU of glycine hydroxymethyltransferase in step S1, dissolve them separately, add to the solution ...

Embodiment 2

[0044] S1: Prepare a kit, the kit reagents include the following components: Tris buffer with a concentration of 35mM; calcium 5-methyltetrahydrofolate with a concentration of 7.5mM; glycine with a concentration of 15mM; and a concentration of 3mM NAD + ; a concentration of 3.5 mM MgCl 2 ; Methionine synthase at a concentration of 7.5kU / L; Aminomethyltransferase at a concentration of 3kU / L; Glycine hydroxymethyltransferase at a concentration of 4kU / L.

[0045] S2: Weigh 2.120 g of the Tris buffer in step S1, dissolve it in 300 ml of purified water, and adjust the pH to 8.00±0.05 with concentrated hydrochloric acid;

[0046] S3: Weigh 1.866 g of calcium 5-methyltetrahydrofolate, 0.563 g of glycine, and NAD in step S1 + 0.994g and MgCl 2 0.356g was added into the buffer solution of step S2, and stirred until completely dissolved;

[0047] S4: Weigh 3.75kU of methionine synthase, 1.5kU of aminomethyltransferase, and 2kU of glycine hydroxymethyltransferase in step S1, dissolve...

Embodiment 3

[0052] S1: Prepare a kit, the kit reagents include the following components: Tris buffer with a concentration of 50 mM; calcium 5-methyltetrahydrofolate with a concentration of 10 mM; glycine with a concentration of 20 mM; NAD + ; a concentration of 5 mM MgCl 2 ; Methionine synthase at a concentration of 10kU / L; Aminomethyltransferase at a concentration of 5kU / L; Glycine hydroxymethyltransferase at a concentration of 6kU / L.

[0053] S2: Weigh 3.029g of the Tris buffer in step S1, dissolve it in 300ml of purified water, and adjust the pH to 8.00±0.05 with concentrated hydrochloric acid;

[0054] S3: Weigh 2.488 g of calcium 5-methyltetrahydrofolate, 0.751 g of glycine, and NAD in step S1 + 1.656g and MgCl 2 0.508g was added into the buffer solution of step S2, and stirred until completely dissolved;

[0055] S4: Weigh 5 kU of methionine synthase, 2.5 kU of aminomethyltransferase, and 3 kU of glycine hydroxymethyltransferase in step S1, dissolve them separately, add to the s...

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Abstract

The invention discloses an enzymatic cycling method for detecting homocysteine. The enzymatic cycling method comprises circular reaction of two reactants, namely a circular reaction of tetrahydrofolic acid and a circular reaction of 5,10-methyl tetrahydrofolic acid respectively; the circular reaction of tetrahydrofolic acid comprises the following steps: enabling 5-methyl tetrahydrofolic acid and L-homocysteine to react to generate tetrahydrofolic acid and L-methionine, and further enabling the reactant substrate tetrahydrofolic acid to react with glycine and NAD<+> to generate 5,10-methyl tetrahydrofolic acid and NADH (Reduced Coenzyme); the circular reaction of 5,10-methyl tetrahydrofolic acid comprises the following steps: enabling the reaction substrate 5,10-methyl tetrahydrofolic acid generated in the cycling reaction of tetrahydrofolic acid to react with glycine to generate tetrahydrofolic acid and serine, detecting the absorbancy increase degree of the NADH at the wavelength of 340nm, and calculating the concentration testing result of the L-methionine. According to the enzymatic cycling method, the cycling reactions are implemented on the basis of the tetrahydrofolic acid, NADH amplification detection signals can be accumulated and generated, the signals are tested at the wavelength of 340nm, and the NADH is in direct proportion to the concentration of the homocysteine participating in the reactions.

Description

technical field [0001] The invention relates to the technical field of detection methods for homocysteine, a small biological molecule, in particular to an enzyme cycle method for detecting homocysteine. Background technique [0002] Homocysteine ​​(Hcy), also known as homocysteine, is a heterogeneous amino acid cysteine, which contains a sulfhydryl group (-SH) in its side chain. Hcy is mainly derived from dietary methionine. It is an important intermediate product in the metabolic process of methionine and cysteine, and it does not participate in protein synthesis. In 1969, McCully found from the autopsy of children with hereditary homocystinuria that there were extensive arterial thrombosis and pathological manifestations of atherosclerosis (AS) in the systemic circulation, thus proposing hyperhomocysteinemia Hyperhomocysteinemia (HHCY) can lead to the hypothesis of atherosclerotic vascular disease. Hcy can directly or indirectly cause damage to vascular endothelial cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N1/28G01N1/38G01N1/34
CPCG01N1/28G01N1/34G01N1/38G01N21/31G01N2001/386
Inventor 沈林肖长河常萍杨小舟余琼林陈帅邓成刚钟玉霞辜振华
Owner HUNAN YONGHE YANGGUANG SCI & TECH
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