Preparation method of oligosaccharide containing N-acetylated structure heparin

A technology of heparin oligosaccharide and acetylation, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve problems such as difficult preparation and structure determination.

Inactive Publication Date: 2015-07-08
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The invention solves the problem of difficult preparation and structure determination of N-acetylated heparan oligosacchar...

Method used

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  • Preparation method of oligosaccharide containing N-acetylated structure heparin
  • Preparation method of oligosaccharide containing N-acetylated structure heparin
  • Preparation method of oligosaccharide containing N-acetylated structure heparin

Examples

Experimental program
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Embodiment 1

[0033]1) Dissolve 200 mg low molecular weight heparin in 2.0ml, 0.1 M, pH 7.0 sodium acetate buffer solution (containing 0.1mM calcium acetate and 100μg / ml BSA), add 50 mIU heparinase Ⅰ, and place in a 37°C water bath After 12 hours of enzymatic hydrolysis, add 50mIU heparanase I to continue enzymatic hydrolysis for 12 hours; then heat to 100°C and boil for 3 minutes to terminate the reaction, centrifuge at 10,000 rpm for 10 minutes, take the supernatant, and freeze-dry;

[0034] 2) Take the freeze-dried sample and dissolve it with 1ml 0.2M ammonium bicarbonate, load the sample to a series Bio-Gel chromatography column, elute with 0.2M ammonium bicarbonate, and collect it with an automatic collector at a flow rate of 0.2ml / min; the separated oligosaccharides Detect with a UV spectrophotometer at a detection wavelength of 232 nm; sequentially collect the sugar fragments corresponding to each chromatographic peak, heat at 55°C for 24h to volatilize ammonium bicarbonate, and freez...

Embodiment 2

[0048] 1) Dissolve 100 mg low molecular weight heparin in 1.0ml, 0.1 M, pH 7.0 sodium acetate buffer solution (containing 0.1mM calcium acetate and 100μg / ml BSA), add 50 mIU heparanase Ⅰ, and place in a 37°C water bath After 12 hours of enzymatic hydrolysis, add 50mIU heparanase I to continue enzymatic hydrolysis for 12 hours; then heat to 100°C and boil for 3 minutes to terminate the reaction, centrifuge at 10,000 rpm for 10 minutes, take the supernatant, and freeze-dry;

[0049] 2) Take the freeze-dried sample and dissolve it with 1ml 0.2M ammonium bicarbonate, load the sample to a series Bio-Gel chromatography column, elute with 0.2M ammonium bicarbonate, and collect it with an automatic collector at a flow rate of 0.2ml / min; the separated oligosaccharides Detect with a UV spectrophotometer at a detection wavelength of 232 nm; sequentially collect the sugar fragments corresponding to each chromatographic peak, heat at 55°C for 24h to volatilize ammonium bicarbonate, and free...

Embodiment 3

[0063] 1) Dissolve 300 mg low molecular weight heparin in 1.0ml, 0.1 M, pH 7.0 sodium acetate buffer solution (containing 0.1mM calcium acetate and 100μg / ml BSA), add 50 mIU heparanase Ⅰ, and place in a 37°C water bath After 12 hours of enzymatic hydrolysis, add 50mIU heparanase I to continue enzymatic hydrolysis for 12 hours; then heat to 100°C and boil for 3 minutes to terminate the reaction, centrifuge at 10,000 rpm for 10 minutes, take the supernatant, and freeze-dry;

[0064] 2) Take the freeze-dried sample and dissolve it with 1ml 0.2M ammonium bicarbonate, load the sample to a series Bio-Gel chromatography column, elute with 0.2M ammonium bicarbonate, and collect it with an automatic collector at a flow rate of 0.2ml / min; the separated oligosaccharides Detect with a UV spectrophotometer at a detection wavelength of 232 nm; sequentially collect the sugar fragments corresponding to each chromatographic peak, heat at 55°C for 24h to volatilize ammonium bicarbonate, and freeze...

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Abstract

The invention relates to a preparation and purification method of a carbohydrate library containing N-acetylated heparin. The method comprises the following steps: enriching oligosaccharide containing an N-acetylated structure through deep enzymolysis of low molecular weight heparin with heparinase I, then, preparing a series of heparin oligosaccharide crude samples ranging from disaccharide to tetradecasaccharide by Bio-Gel P10 gel chromatography, further separating the crude samples by means of strong anion high performance liquid chromatography and other methods, and respectively purifying the crude samples to obtain four hexasaccharide segments and three octasaccharide fragments; analyzing the disaccharide constituent of each purified oligosaccharide by compound enzymolysis with heparinase I, heparinase II and heparinase III and strong anion chromatography, and primarily deducing sequence structures of the four hexasaccharide and the three octasaccharide in combination with heparinase I substrate specificity; and finally, identifying the structure by electrospray ionization-ion trap-time of flight mass spectrometry (ESI-IT-TOF-MS). The preparation method provided by the invention can be used for solving the problem of difficulty in preparation and structure determination of oligosaccharide containing N-acetylated heparin, which makes research on a relationship between a special structure and functions of heparin/heparan sulfate developed further.

Description

technical field [0001] The invention belongs to the field of separation and purification of natural products, and more specifically relates to a preparation method of heparin oligosaccharides containing N-acetylated structure. Background technique [0002] Heparin (HP) is a linear glycosaminoglycan with a large number of negative charges, which is widely expressed on the cell surface and in the cell matrix. HP interacts with numerous proteins and regulates various biological functions. In addition to anticoagulant and antithrombotic functions, heparin also has anti-smooth muscle cell proliferation, anti-inflammation, anti-tumor and anti-virus functions, and these biological activities are closely related to the specific structure of heparin. The heparin glycan chain is composed of 50-200 repeating disaccharide units. Heparin hexasaccharides and octasaccharides interact with various proteins and enzymes through special sequences to regulate the physiological functions of hep...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
Inventor 魏峥林江慧
Owner FUZHOU UNIV
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