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Method for degrading heparan sulfate and detecting heparan sulfate disaccharide composition

A technology of heparan sulfate and deacetylation, applied in the field of medicine, can solve the problems of poor repeatability of analysis results, cumbersome detection steps, unstable properties, etc., and achieves the effects of low requirements for experimental instruments, short detection time, and small sample consumption.

Active Publication Date: 2015-07-08
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymes, strong anion exchange columns and disaccharide standards in the traditional method are very expensive, the enzyme is easily inactivated, the property is unstable, the detection steps are cumbersome, and the reproducibility of the analytical results of this method is poor

Method used

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  • Method for degrading heparan sulfate and detecting heparan sulfate disaccharide composition
  • Method for degrading heparan sulfate and detecting heparan sulfate disaccharide composition
  • Method for degrading heparan sulfate and detecting heparan sulfate disaccharide composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The chemical degradation of embodiment 1HS

[0036] (1) Dissolve 1mg HS in 500uL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 98 ° C, reacted for 8 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained.

[0037] (2) Concentrate the product obtained in step (1) to dryness, dissolve it in 100uL water, add 100uL sodium nitrite aqueous solution with a pH of 1.5, adjust the pH to 4.0 after reacting at 0-5°C for 10 minutes, and add 100uL sodium nitrite with a pH of 4.0 Nitric acid, react at 0-5°C for 10 min, add 60 uL of ammonia water to terminate the reaction, and obtain heparan sulfate disaccharide.

Embodiment 2

[0038] The chemical degradation of embodiment 2HS

[0039] (1) Dissolve 1 mgHS in 1 mL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 90 ° C, reacted for 4 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained.

[0040] 2) Concentrate the product obtained in step (1) to dryness, dissolve it in 100uL water, add 100uL sodium nitrite aqueous solution with a pH of 1.5, adjust the pH to 4.0 after reacting at 0-5°C for 10 minutes, and add 100uL sodium nitrite with a pH of 4.0 Nitric acid, react at 0-5°C for 10 minutes, add 60uL of ammonia water to terminate the reaction, and obtain heparan sulfate disaccharide.

Embodiment 3

[0041] Embodiment 3HS chemical degradation

[0042] 1) Dissolve 1mgHS in 1mL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 111 ° C, reacted for 20 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained.

[0043] 2) Concentrate the product obtained in step (1) to dryness, dissolve in 100uL of water, add 100uL of sodium nitrite aqueous solution with a pH of 1.5, react at 0-5°C, adjust the pH to 4.0, add 100uL of nitrous acid with a pH of 4.0, React at 0-5°C for 10 minutes, add 60 uL of ammonia water to terminate the reaction, and obtain heparan sulfate disaccharide.

[0044] The degradation products of Examples 1-3 were detected, and the results are shown in Table 1.

[0045] Table 1 Analysis of chemical degradation products

[0046]

[0047]The results show that Examples 1 and 3 can completely chemically degrade HS, and the time used in Example 1 is 60% le...

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Abstract

The invention belongs to the field of medicine, and relates to a method for degrading heparan sulfate and detecting heparan sulfate disaccharide composition. The method comprises the following steps of (1) degrading heparan sulfate into disaccharide by utilizing a chemical degradation method; (2) deriving the heparan sulfate disaccharide by utilizing a derivation reagent; (3) detecting the derived heparan sulfate disaccharide obtained in the step (2) by utilizing the liquid chromatography (LC) or liquid chromatography-mass spectrography (LC-MS) method. The heparan sulfate can be completely degraded into the disaccharide through the two chemical degradation methods, so that the structural information of the heparan sulfate disaccharide can be maximally obtained. The degradation condition is moderate, the cost is low, simplicity in operation can be realized, the requirement on the experimental instruments is low, the application range is wide, the detection time is short, the consumption of samples is small, the sensitivity is high, and the repeatability of the analysis result is good.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a method for degrading heparan sulfate and detecting its disaccharide composition. Background technique [0002] Heparan sulfate (HS) is a kind of glycosaminoglycan (GAG) widely present in animal tissue matrix and cell membrane. Proteoglycan, glycosylphosphatidylinositol glycan, β2 proteoglycan and CD44, etc. They have important biological functions in signal transduction, proliferation and differentiation of animal cells, immune regulation, cell adhesion, and information transmission of nerve cells. The HS sugar chain is composed of glucosamine (GlcN) connected with glucuronic acid (GlcA) or iduronic acid (IdoA) through α (1→4) glycosidic bonds to form a disaccharide repeating unit. The main difference between HS and heparin is that The degree of sulfation modification and the degree of acetylation of the disaccharide repeat unit are different. HS is one of the most complex biomacromole...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02C07H5/06C07H1/00C12P19/14C12P19/12
Inventor 张丽娟韩章润曾洋洋兰莹邱培菊
Owner OCEAN UNIV OF CHINA
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