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Detection method for PEG modification capability of protein alkylation modifier, and application thereof

A technology of modification ability and detection method, which is applied in the direction of material analysis by observing the influence on chemical indicators, and analysis by making materials undergo chemical reactions, etc., which can solve the problem of unquantitative detection of activated PEG-modified proteins, unsatisfactory results, Long operation time and other issues, to achieve important promotion and application value, avoid waste, clear mechanism effect

Inactive Publication Date: 2014-02-05
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chang Yuan et al. (1996) used bovine serum albumin (BSA) as a model protein, and used gel electrophoresis to determine the modification ability of various activated PEGs. However, this method is cumbersome to operate, takes a long time to operate, and the results are not ideal, and it cannot be quantified. Ability to detect activated PEGylated proteins

Method used

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  • Detection method for PEG modification capability of protein alkylation modifier, and application thereof
  • Detection method for PEG modification capability of protein alkylation modifier, and application thereof
  • Detection method for PEG modification capability of protein alkylation modifier, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Preparation of reagents

[0049] (1) 1.0mmol / L GSH solution:

[0050] GSH 3.07mg was dissolved in 0.1mol / L barbiturate sodium-hydrochloride buffer solution with pH9.0, diluted to 10mL, and prepared immediately.

[0051] (2) 0.32mol / L Na 2 HPO 4 Solution:

[0052] Na 2 HPO 4 Dissolve 29.92g of double distilled water and dilute to 200mL.

[0053] (3) 1.0mmol / L DTNB (5,5'-dithio-p-dinitrobenzoic acid) chromogenic solution:

[0054] Weigh 20mg of DTNB and 500mg of trisodium citrate, dissolve in double distilled water and dilute to 50mL (the brown bottle should be kept in a refrigerator at 4°C in the dark).

[0055] (4) 5mmol / L activated mPEG5000 solution:

[0056] A. Activation of modifiers

[0057] Refer to the method of Abuchowski (1977) and improve and optimize: cyanuric chloride is recrystallized twice with anhydrous benzene (3A molecular sieve to remove water) before use; take 5.5g of secondary recrystallized cyanuric chloride and dissolve it in 400mL con...

Embodiment 2

[0066] 1. Preparation of reagents

[0067] (1) 1.0mmol / L GSH solution, 0.32mol / L Na 2 HPO 4 The preparation method of the solution and 1.0mmol / L DTNB (5,5'-dithio-p-dinitrobenzoic acid) chromogenic solution is the same as in Example 1.

[0068] (2) 5mmol / L activated mPEG10000 solution:

[0069] A. Activation of modifiers

[0070] Refer to the method of Abuchowski (1977) and improve and optimize: cyanuric chloride is recrystallized twice with anhydrous benzene (3A molecular sieve to remove water) before use; take 5.5g of secondary recrystallized cyanuric chloride and dissolve it in 400mL containing Add 100g of mPEG10000 to 10g of anhydrous sodium carbonate in anhydrous benzene, stir overnight at room temperature, filter, take about 400mL of the filtrate and slowly add 600mL of diethyl ether while stirring, and continue stirring for 20min after the white product precipitates, then filter with suction, and then use 400mL of Water and benzene are dissolved, and such precipit...

Embodiment 3

[0078] Example 3 The Determination Test of the Optimum Measuring Conditions of the Method of the Invention

[0079] In order to determine the best reaction conditions, the present invention has carried out a large number of long-term experimental explorations including single factor experiments and comparative analysis. The final experimental results are described as follows:

[0080] (1) Selection of the optimal molar ratio of modifier to GSH reaction mass

[0081] According to the analysis of the reaction mechanism, the inventors determined that the mass ratio of mPEG to GSH reaction in the method of the present invention is carried out in a molar ratio of 2:1. GSH has two reactive groups, amino and thiol, so one molecule of GSH can react with two molecules of mPEG, so the mass ratio of mPEG to GSH is 2:1, which is convenient for the calculation of the modification ability of modifiers. The experiment proves that the modification ability of the modifier can be accura...

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Abstract

The invention discloses a detection method for PEG modification capability of a protein alkylation modifier, and an application thereof. In the method, two groups of one GSH modified by the modifier and the other unmodified by the modifier are set. In the two groups, 5,5-dithio p-bis-nitrobenzoic acid (DNTB) is used as a color developing agent; light absorption values of the two groups of reaction systems are measured at a wavelength of 412 nm by using a spectrophotometer; and the modification capacity of the protein aminated PEG modifier is detected according to difference of the light absorption values of the two groups. The detection method is used for detection of the modification capacity of the modifier, and can realize both qualitative detection and quantitative detection. The whole detection process can be finished within 60 min; and the method has the beneficial effects of being rapid, simple and reliable.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a method and application for detecting the ability of a protein alkylation PEG modifier. Background technique [0002] In the late 1950s, using chemical modification methods to study the relationship between the structure and function of protein molecules became a hot spot in the fields of biochemistry and molecular biology. The purpose of chemical modification of proteins is in biomedicine and biotechnology. In biomedicine, chemical modification can reduce immunogenic immunoreactivity, inhibit the production of immunoglobulin E, etc.; in the field of biotechnology, enzymes can efficiently catalyze in organic solvents after chemical modification, and show Novel catalytic properties. [0003] The nature of the reaction between protein modifiers and proteins can be mainly divided into four types: (1) acylation and related reactions, such modifiers can undergo acylation reactions...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 陈芳艳冯定远王林川
Owner SOUTH CHINA AGRI UNIV
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