Method for measuring enzymatic activity by integration method and initial rate method

A technology of initial velocity and integral method, which is applied in the field of determination of enzyme activity by combining integral method and initial velocity method, can solve problems such as error, random error system, and not reaction time

Inactive Publication Date: 2008-05-07
CHONGQING MEDICAL UNIVERSITY
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Problems solved by technology

This strategy has been tried (see Biochem J, 1975, 149:305-312; Biochem J, 1982; 203:117-123; Biochem J, 1985; 228:55-60), but in these attempts, when analyzed In the enzyme reaction curve, the substrate consumption ratio exceeds 50%, but the deviation is getting bigger and bigger, especially sensitive to the error of the reaction starting point and the error of the initial substrate concentration, so it cannot be used routinely
[0004] The independent variable of the integral velocity equation used in the previously attempted joint method is not the reaction time, and all contain random errors and systematic errors, which may be the reason for its low reliability

Method used

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  • Method for measuring enzymatic activity by integration method and initial rate method
  • Method for measuring enzymatic activity by integration method and initial rate method
  • Method for measuring enzymatic activity by integration method and initial rate method

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: this combined method can measure human serum butyrylcholinesterase activity, it is characterized in that carry out as follows:

[0035] (1), add 50mmol / L sodium phosphate buffer solution (pH 7.4) totally 0.90ml in test tube, and the thiobis(2-nitrobenzoic acid, Dithiobis-(2-nitrobenzoic acid) dissolved in this phosphate buffer solution acid), DTNB) in total 0.10ml (final concentration 0.50mmol / L), 0.10ml of an appropriately diluted serum sample, keep the temperature in a water bath at 30°C for 5 minutes;

[0036] (2) Add 100 μl of the substrate thiobutyrylcholine dissolved in the above-mentioned phosphate buffer solution to a final concentration of 54 μmol / L, and vortex to mix thoroughly;

[0037] (3), transfer the reaction mixture into the cuvette quickly, delay for 15 seconds, continuously monitor the 410nm absorption change at intervals of 5 seconds, and monitor for 5.0 minutes in total to obtain the reaction curve of the reaction product absorption inc...

Embodiment 2

[0045] Embodiment 2: This combined method can measure the gamma-glutamyl transferase activity in mouse kidney or human serum, it is characterized in that carry out as follows:

[0046] (1) Add 110 μmol / L of L-γ-glutamyl p-nitroaniline, 15.0 mmol / L of diglycine, and MgCl to the test tube. 2 10.0mmol / L Tris-HCl buffer solution (100mmol / L, pH 8.1) 1.10ml in total; keep the temperature in a water bath at 30°C for 5 minutes;

[0047] (2) Take 100 μl of an appropriate diluted sample and add it to the above-mentioned test tube, mix well so that the final concentration of the initial substrate is 100 μmol / L;

[0048] (3), transfer the reaction mixture into the cuvette rapidly, delay for 15 seconds, continuously monitor the 405nm absorption change at an interval of 5 seconds, and monitor for 10.0 minutes in total to obtain the reaction curve (accompanying drawing 3) of product p-nitroaniline absorption increase;

[0049] (4) Delete the data whose light absorption increases by less tha...

Embodiment 3

[0056] Embodiment 3: the characteristic steps of this combination method measuring mouse kidney, human serum alanine aminotransferase (ALT) activity are as follows:

[0057] (1), add 54 μmol / L of L-γ-glutamyl p-nitroaniline containing 600mmol / L alanine, 0.22mmol / L NADH, 1400U / L LDH, 100mmol / L Tris-HCl, bis Glycine 15.0mmol / L, MgCl2 10.0mmol / L, buffer solution (pH 7.3) containing 100mmol / L Tris-HCl 1.00ml in total; then add 100μl of properly diluted sample, mix quickly and in 30 ℃ constant temperature for 5 minutes;

[0058] (2) Add 100 μl of 16.3 mmol / L α-ketoglutarate solution into the above-mentioned test tube, shake and mix quickly;

[0059] (3), transfer the reaction mixture into the cuvette rapidly, delay for 30 seconds, continuously monitor the 340nm absorption change at 1 second intervals, and monitor for 5.0 minutes in total to obtain a reaction curve indicating that the instantaneous absorption (Ai) of the substrate (NADH) drops ( Accompanying drawing 5);

[0060] ...

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Abstract

The invention relates to an enzymatic activity test method using combining integration and initial velocity method, which is characterized in that the classical initial velocity is measured when the concentration of initial substrate is 70% higher than that of the preset substrate, and the classical initial velocity is still within linearity range; when the substrate consumption proportion in 80% record period is higher than the lower limit required in integration, the fitting reaction curve of integral velocity equation is used to determine the maximum reaction velocity using reaction time as independent variable, then the maximum reaction velocity is calculated as the initial velocity with preset substrate concentration is 93%; meanwhile, the slope of classical initial velocity and computation initial velocity to the enzyme amount reaction curve; when the substrate consumption proportion of the coupling enzyme reaction system in 80% record period is higher than the lower limit required in integration, the fitting enzyme reaction curve is used to determine the initial velocity using corresponding numerical integral velocity equation, otherwise the classical initial velocity is directly measured; total time for the optimization monitoring reaction guarantees the combination of the two methods.

Description

technical field [0001] The present invention relates to the method for measuring enzyme activity in fields such as clinical inspection, hygienic inspection, it is characterized in that the initial velocity of low-activity enzyme reaction system is measured by classical initial velocity method, and the maximum velocity of enzyme reaction of high-activity reaction system is measured by integral method, By converting to the initial velocity at the optimized substrate concentration, the response curves of the integral method and the initial velocity method become the same straight line, so that a higher linear response upper limit and a wider linear response range can be obtained with a lower substrate concentration , and guarantee the analysis efficiency. Background technique [0002] Determination of enzyme activity is routine work in the fields of clinical testing and environmental sanitation testing. At present, the initial velocity method is commonly used in the determinat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/17G06F17/10C12Q1/00
Inventor 廖飞赵运胜赵利娜陆巍杨晓兰廖红于明安桑宇
Owner CHONGQING MEDICAL UNIVERSITY
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