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Urease michaelis constant determination method

A Michaelis constant, urease meter technology, applied in the fields of biochemistry and applied enzymology, can solve the problems of long test time and many steps, and achieve the effect of simple test method

Inactive Publication Date: 2015-06-10
SHANDONG UNIV OF TECH
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  • Summary
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the urease-indophenol method is generally used to determine the Michaelis constant of urease, which has many steps and a long test time.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] (1) Preparation of urease solution (10kU / L): According to the number of enzyme activity units, weigh a certain amount of enzyme powder, dissolve it in deionized water, and prepare a 10kU / L urease solution.

[0012] (2) Preparation of phosphate buffer solution with a pH value of 6.8: Take 510ml of 0.2mol / L sodium dihydrogen phosphate, mix with 490ml of 0.2mol / L disodium hydrogen phosphate, add 0.02g of phenol red, and stir to dissolve.

[0013] (3) Configuration of urea buffer solution (pH=6.8) with different concentrations: Take 100mL of the phosphate buffer solution with a pH value of 6.8 in (2) above, add 0.12g of urea to obtain a buffer solution containing 20mmol / L of urea, and use this solution as The mother liquor was diluted with the above (2) phosphate buffer solution with a pH value of 6.8 to obtain phosphate buffer solutions with urea concentrations of 20, 15, 10, 6.67, 5 and 4 mmol / L respectively.

[0014] (4) Adding samples: Take a 96-well plate, add 25 μL of...

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Abstract

The invention relates to a urease michaelis constant determination method, namely a urease-phenol red method, belonging to the technical field of biochemistry and application enzymology. The determination method provided by the invention is characterized in that a series of urea solutions with different concentrations act as a substrate, and phenol red acts as an indicating agent, urea is decomposed to generate ammonia under the action of urease michaelis, and pH value is increased; the color of a system becomes dark under the action of the phenol red indicating agent, the absorbancy is increased, and is determined by a spectrophotometer or a microplate reader. The enzymatic reaction velocity is in direct proportion to the increase of the absorbancy value, so that the reciprocal 1 / delta A of the absorbancy increase value can replace 1 / V in a linewaver-Burk equation 1 / V=Km / (Vmax[S])+1 / Vmax, and the reciprocal 1 / [S] of the concentration of the substrate urea is plotted; the intercept -1 / Km of a straight line on the X axis can be obtained via the linewaver-Burk equation, and the Km value is resolved.

Description

technical field [0001] The invention relates to a method for measuring the Michaelis constant of urease, which belongs to the technical field of biochemistry and applied enzymology. Background technique [0002] Urease, also known as amidohydrolase, is widely found in various bacteria, fungi, plants, animals and humans, and can rapidly catalyze the hydrolysis of urea to generate ammonia and carbon dioxide. The catalytic properties of urease have been widely studied in various fields: in medicine, urease is closely related to the pathogenesis of nephritis, hepatic coma and peptic ulcer; damage; the non-productivity of urea has also caused some pollution to the environment, so it is of great significance to study urease. [0003] The Michaelis-Menten equation V=Vmax[S] / (Km+[S]) is extremely important in enzymology and can describe a variety of non-alteromeric enzyme kinetic phenomena, indicating the initial velocity V of an enzymatic reaction (some data Also known as the equ...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 盛桂华陈相飞胡晓明徐英韩艳伟陈杰王峰朱海亮
Owner SHANDONG UNIV OF TECH
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