Method for preparing low molecular weight heparin with high activity

A low-molecular-weight heparin, active technology, applied in the field of biomedicine, to achieve the effects of increased activity, reduced production costs, and huge industrial application value

Inactive Publication Date: 2009-12-02
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, low molecular weight heparins are also problematic when used

Method used

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  • Method for preparing low molecular weight heparin with high activity
  • Method for preparing low molecular weight heparin with high activity
  • Method for preparing low molecular weight heparin with high activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, PAPS regeneration system

[0023] The concentration of components in the PAPS regeneration system is: the concentration of PNPS is 5mM, the concentration of PAP is 20μM; the dosage of AST-IV in the PAPS regeneration system is 10mg / mM PAPS. The solution prepared according to the above composition is added to the solution for preparing high-activity low-molecular-weight heparin as a sulfate group donor for the enzyme modification reaction of heparin biosynthetic enzyme 3-O-sulfatyltransferase. Such as figure 1 shown.

Embodiment 2

[0024] Example 2. Preparation of high-activity low-molecular-weight heparin using low-molecular-weight heparin Fragamine as raw material

[0025] The commercial product Fragmin (Pfizer, USA, potency) was used as the raw material, and the reaction conditions were 1mg of low molecular weight heparin Fragmin in 20ml of reaction liquid and shaking (300rpm) at room temperature for 6h. The reaction solution contained 50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 1% BSA, 1 mM MgCl2, 1 mM MnCl2, 1 mM PNPS, 40 μM PAP, 8 mg 3-OST-1 and 4 mg AST-IV. Heat the reaction solution at 100°C for one minute to terminate the reaction, centrifuge to obtain the supernatant, mix the supernatant with twice the volume of 2.0% NaCl aqueous solution, and load the sample on the DEAE column; sequentially use 2.0% NaCl aqueous solution and 3.0 % NaCl aqueous solution for gradient elution, and collect the eluate obtained from the second elution to obtain high-activity low-molecular-weight heparin. The resultin...

Embodiment 3

[0026] Example 3. Preparation of high-activity low-molecular-weight heparin using low-molecular-weight heparin tinzaparin as raw material

[0027]The commercial tinzaparin (DuPont, titer) was used as the raw material, and the reaction conditions were 1mg of low molecular weight heparin tinzaparin in 20ml of the reaction solution at room temperature for 6h with shaking (300rpm). The reaction solution contained 50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 1% BSA, 1 mM MgCl2, 1 mM MnCl2, 1 mM PNPS, 40 μM PAP, 8 mg 3-OST-1 and 4 mg AST-IV. Heat the reaction solution at 100°C for one minute to terminate the reaction, centrifuge to obtain the supernatant, mix the supernatant with twice the volume of 2.0% NaCl aqueous solution, and load the sample on the DEAE column; sequentially use 2.0% NaCl aqueous solution and 3.0 % NaCl aqueous solution for gradient elution, and collect the eluate obtained from the second elution to obtain high-activity low-molecular-weight heparin. The resulting ...

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Abstract

The invention discloses a method for preparing low molecular weight heparin with high activity, which is characterized in that the prior low molecular weight heparin with low anticoagulant activity is used as a substrate, a PAPS (3'-phosphoadenosine-5'-phosphoric acid sulfuric acid) regeneration system is adopted, and a heparin biosynthetic enzyme 3-O-sulfate-transferase is used for modifying the substrate to obtain the low molecular weight heparin with high anticoagulant activity; and the PAPS regeneration system can use extremely cheap PNPS (p-nitryl potassium phenolsulfonate) as an enzyme-modified reactive sulfate donor to further save the cost and make the industrialization possible. The method adopts the 3-O-sulfate-transferase with high enzyme activity to combine with the PAPS regeneration system, performs selective modification on the low molecular weight heparin, and increases the number of anticoagulant activity centers so as to greatly improve the anti-thrombosis activity of the heparin. The method provides a path for the enzymatic industrial production of the low molecular weight heparin with high activity.

Description

technical field [0001] The invention relates to a method for preparing high-activity low-volume heparin. It belongs to the field of biomedicine. Background technique [0002] Heparin is a polysaccharide formed by hexuronic acid (L-iduronic acid, D-glucuronic acid) and D-glucosamine sulfate alternately with 1-4 glycosidic bonds, its molecular weight is between 3000-40000Da, and the average molecular weight is 15000Da; Heparin has been used as an anticoagulant and antithrombotic agent for more than 60 years. The anticoagulant activity of heparin is mainly through the binding of antithrombin (AT-III) at the pentose sugar active center containing 3-glucosamine at position 3-position, inducing its conformational change, thus showing anticoagulant activity. The anticoagulant activity of heparin includes antithrombotic activity (anti-factorXa) and anticoagulant (anti-factor IIa) activity. Anticoagulant activity is also related to the molecular weight of heparin. However, becaus...

Claims

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Application Information

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IPC IPC(8): C08B37/10A61P7/02
Inventor 陈敬华金坚许正宏
Owner JIANGNAN UNIV
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