Oxidoreductase activity testing method

A test method and reductase technology, applied in the field of medical biological detection, can solve the problems of light color of the solution, easy fading, long reaction time, etc., and achieve the effect of wide application range and convenient operation

Inactive Publication Date: 2017-11-21
HANGZHOU JENNIFER BIOTECH CO LTD
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The TTC-dehydrogenase activity detection method needs to add organic reagents during the operation process, and the solution is light in color, easy to fade, and the reaction time is long

Method used

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  • Oxidoreductase activity testing method
  • Oxidoreductase activity testing method
  • Oxidoreductase activity testing method

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Experimental program
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Embodiment 1

[0066] Such as Figure 1 to Figure 3 As shown, the cDNA gene fragment encoding and expressing glutamate dehydrogenase (GDH) protein has a wide range of sources and exists in various living organisms.

[0067] Add 0-8nM GDH, appropriate amount of NADP+, and 5-30mM glutamic acid to 100ul reaction Tris buffer solution (pH7-9) at a final concentration, and shake and react at room temperature for 10-60min. Then, add monosulfonic acid tetrazolium salt detection reagent, after mixing, measure 450nm absorbance value (reference wavelength 620nm), obtain the Escherichia coli source glutamate dehydrogenase linear equation is y=0.21x+0.075 ( 2 =0.999), x is glutamate dehydrogenase activity (nM).

[0068] In the screening test of glutamic acid inhibitors, HJ7 and HJ8 were selected as positive controls. Negative control HJ1. Specific operation: add 50 ul of 0-30 μM inhibitor and 4 nM GDH Tris-NaCl buffer solution (pH 7-9) into a 96-well plate, shake and react for 1-5 hours at room temper...

Embodiment 2

[0070] 1~50mM lactate dehydrogenase corresponding to the base lactate and NAD + , Monosulfonic acid tetrazolium salt detection reagent is mixed in Tris-NaCl buffer or phosphate buffer (pH8~10); the solution to be tested containing lactate dehydrogenase is according to the volume ratio of 1:(0.01~1) Add buffer solution, mix evenly, place at 25-38°C to react for 0.5-4 hours, detect the absorbance at 450nm, and obtain the enzyme activity of the dehydrogenase in the solution to be tested. For details, see Figure 4 .

[0071] The content of lactate dehydrogenase in serum can be used as a detection index for certain diseases. Dilute the serum of different serum samples to an appropriate multiple, and then add 1-50mM lactic acid, NAD + , monosulfonic acid tetrazolium salt detection reagent mixed with Tris-NaCl buffer or phosphate buffer (pH 8 ~ 10); the test solution containing lactate dehydrogenase is added to the buffer according to the volume ratio of 1: (0.01 ~ 1) solution, mi...

Embodiment 3

[0074] 1~50mM Glucose-6-phosphate, NADP + , monosulfonic acid tetrazolium salt detection reagent mixed with Tris-NaCl buffer or phosphate buffer (pH7~8); The volume ratio is added to the buffer solution, mixed evenly, placed at 25-38°C for 0.5-4 hours, and the absorbance at 450nm is detected to obtain the enzyme activity of the dehydrogenase in the solution to be tested. For details, see Figure 5 .

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Abstract

The invention relates to the technical field of medicine biological detection, and provides a dehydrogenase activity detection method which can monitor oxidoreductase activity in real time in accordance with color change of a novel tetrazole monosulfonate detection reagent. A tetrazole monosulfonate detection system is adopted, and a good linear dose relationship is presented in detection of dehydrogenase activity; and an EC50 curve is obtained in accordance with an absorbance value of a reactant at 450nm, and dehydrogenase activity evaluation is implemented. The dehydrogenase activity detection method provided by the invention is highly sensitive and is convenient to operate; and the detection method is broad in application range, covering dehydrogenase of various organisms, including archaebacteria (extreme thermophilic bacteria and extreme halophilic bacteria), bacteria (lactobacillus, nitrifying bacteria, escherichia coli, diplococcus pneumoniae and the like), various cellular structure bio-actinomycetes, cyanobacteria (oscillatoria, chroococcus, nostoc and the like), most unicellular algae (chlamydomonas, green algae and the like), fungi (edible fungi, saccharomycetes, mould and the like) as well as animals and plants.

Description

technical field [0001] The invention relates to the technical field of medical biological detection, in particular to real-time monitoring of NAD(P)-dependent oxidoreductase activity by using the color change of a monosulfonic acid tetrazolium salt detection reagent, and provides an activity detection method of this type of enzyme. Background technique [0002] Oxidoreductase is the largest class of known enzymes, among which oxidase (oxydase) can catalyze the oxidation of substances by oxygen; dehydrogenase (dehydrogenase) can catalyze the removal of hydrogen from substance molecules, It mostly catalyzes the redox of alcohol group (-CHOH), aldehyde, ketone group (-HCO or -RCO) and alkyl gene (-CH2-CH2-) in the donor. As a kind of protein, it can activate some special hydrogen atoms, so that these hydrogen atoms can be transferred by appropriate hydrogen acceptors to oxidize the original substance. The main natural receptors are nicotinamide adenine dinucleotide (NAD + ) a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12Q1/32
CPCC12Q1/26C12Q1/32
Inventor 阮奔放阮健昵福
Owner HANGZHOU JENNIFER BIOTECH CO LTD
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