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Determination method of enzyme activity of collagenase

A collagenase and determination method technology, applied in the measurement of color/spectral properties, etc., can solve the problems of substrate difference, inconsistent enzyme activity data, inconsistent detection standards of collagenase enzyme activity, etc., achieving simple instrument, convenient operation, The effect of high measurement accuracy

Active Publication Date: 2012-12-05
ZHEJIANG MARINE DEV RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to overcome the deficiencies of the prior art, such as inconsistent detection standards for collagenase enzyme activity, substrate variability, and inconsistent enzyme activity data, and provides a measurement method with unified standards, overcoming substrate variability, and consistent enzyme activity data. Determination method of collagenase enzyme activity with high precision

Method used

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  • Determination method of enzyme activity of collagenase
  • Determination method of enzyme activity of collagenase
  • Determination method of enzyme activity of collagenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] ①Reagent preparation

[0037] (1) 0.1mol / L Tris·Cl buffer (containing 50mmol CaCl 2, pH7.5): Weigh 3.15g Tris·Cl and dissolve it in 200mL distilled water, add 1.11g calcium chloride, fully dissolve and adjust the pH to 7.5 with 3mol / L NaOH;

[0038] (2) 10% trichloroacetic acid (TCA) solution: mix 50mL of trichloroacetic acid with 450mL of distilled water;

[0039] (3) 0.6mol / L o-phthalaldehyde solution: dilute 0.6mol o-phthalaldehyde to 1L with distilled water;

[0040] (4) 0.3mmol / L hydroxyproline standard solution: weigh 39.4mg of hydroxyproline, dissolve it in distilled water, and dilute to 1L;

[0041] (5) 2mol / L acetic acid buffer solution: dissolve 2mol of sodium acetate with distilled water, and dilute to 1L, dilute 2mol of acetic acid with distilled water to 1L, and then mix the above sodium acetate solution and acetic acid solution at a volume ratio of 43:7 , the pH is 5.4;

[0042] (6) Ninhydrin chromogenic solution: mix 85mg ninhydrin hydrate and 15mg re...

Embodiment 2

[0057] 1. reagent preparation: with embodiment 1, difference is that the concentration of o-phthalaldehyde is 0.8mol / l;

[0058] ② Hydroxyproline gradient sample preparation: pipette 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0mL of hydroxyproline standard solution into 25ml colorimetric tubes, make up to 1mL with distilled water, and dilute to the concentration For 7 hydroxyproline gradient samples of 0.03, 0.06, 0.12, 0.15, 0.18, 0.24, and 0.30 μmol / mL, take 1.0ml of distilled water in a 25ml colorimetric tube as a reference sample;

[0059] ③ Hydroxyproline Gradient Sample Determination: The operating conditions are the same as in Example 1, except that the temperature of the water bath is 105° C., and the time is 15 minutes. The standard curve formula is fitted: C=2.7483A-0.2328.

[0060] ④Preparation of enzymatic hydrolysis solution to be tested: Weigh 1.2mg insoluble collagen, add 0.6mL 0.1mol / L Tris Cl buffer solution, preheat at 40°C for 12min, then add 0.1mL enzyme solution, rea...

Embodiment 3

[0069] 1. reagent preparation: with embodiment 1, difference is that the concentration of o-phthalaldehyde is 0.7mol / l;

[0070] ② Hydroxyproline gradient sample preparation: pipette 0.1, 0.2, 0.3, 0.4, 0.6, 0.7, 0.8, 1.0mL of hydroxyproline standard solution into 25ml colorimetric tubes, make up to 1mL with distilled water, dilute 8 hydroxyproline gradient samples with concentrations of 0.03, 0.06, 0.12, 0.15, 0.18, 0.21, 0.24, and 0.30 μmol / mL were prepared, and 1.0ml of distilled water was taken in a 25ml colorimetric tube as a reference;

[0071] ③ Hydroxyproline Gradient Sample Determination: The operating conditions are the same as in Example 1, except that the temperature of the water bath is 102° C. and the time is 18 minutes; the formula of the standard curve is obtained by fitting: C=2.7488A-0.2432.

[0072] ④Preparation of enzymatic solution to be tested: Weigh 1.0mg of insoluble collagen, add 0.5mL 0.1mol / L Tris Cl buffer solution, preheat at 37°C for 10min, then a...

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Abstract

The invention discloses a determination method of enzyme activity of collagenase, and aims to solve the problems of non-uniform enzyme activity detection standard and poor data consistency existing in the prior art. The determination method comprises the following steps of: (1) preparing a reagent; (2) preparing hydroxyproline gradient samples; (3) determining the hydroxyproline gradient samples, drawing a C-A standard curve according to a determined absorbance A and hydroxyproline gradient sample concentration C and fitting a standard curve formula; (4) preparing enzymatic hydrolysate to be determined: adding insoluble collagen or gelatin serving as a collagen substrate into an enzyme solution to be determined for enzymatic hydrolysis, centrifuging to obtain supernatant, and diluting the supernatant to prepare the enzymatic hydrolysate to be determined; (5) preparing a blank solution; (6) determining the enzyme activity and determining the absorbance A of the enzymatic hydrolysate to be determined according to the operation conditions of the step (3); (7) calculating the hydroxyproline concentration in the enzymatic hydrolysate to be determined; and (8) calculating the enzyme activity of the collagenase. The determination method has the characteristics of uniform enzyme activity detection standard, capability of solving the problem of substrate difference, high measuring accuracy and consistent enzyme activity data.

Description

technical field [0001] The invention relates to a method for measuring protease activity, in particular to a method for measuring collagenase activity. Background technique [0002] Collagen is a biopolymer synthesized by animal cells with a relative molecular weight of more than 300,000. It is widely found in animal bones, tendons, cartilage, skin and other connective tissues, accounting for about 1 / 3 of the total protein in mammals. 3. More than 90% of the total protein in the bone and muscle bonds, and more than 50% of the total protein in the skin. Collagen has a unique triple helical structure, which is difficult to be hydrolyzed by general proteases, and can only be specifically hydrolyzed by collagenase. Collagenase is a kind of proteolytic enzyme characterized by hydrolyzing insoluble collagen protein. It exists in many protease families, such as metalloprotease family and serine protease family. At present, collagenase has been widely used in medicine , food and ot...

Claims

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Application Information

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IPC IPC(8): G01N21/31
Inventor 周宇芳朱鹏杨会成廖妙飞肖金星钟明杰付万冬
Owner ZHEJIANG MARINE DEV RES INST
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