| [Objective]Heparin is linear polysaccharides consisting of repeating disaccharide unit backbones of N-acetyl-D-glucosamine that is linked to D-glucuronic acid. The disaccharide repeat unit can be modified to include N-and O-sulfation (6-O and 3-O sulfation of the glucosamine and 2-O sulfation of the uronic acid). These specific sulfate patterns are involved in the interaction of heparin with other molecules. Heparin is known to bind and modulate the activity of various proteins, including cell growth factors, cytokines, angiogenic factors, complement components, adhesion molecules, heparin exerts its activities via interaction with some proteins. Potent anticoagulant activity of heparin by the interaction with antithrombin III is extensively used in medicine. In addition to its traditional anticoagulant activity, heparin has been shown to inhibit various aspects of the inflammatory response such as lymphocyte trafficking and neutrophil chemotaxis, modulate various aspects of immune function via multiple mechanisms and inhibit the metastasis of tumour cells via inhibiting the activities of endoglycosidase heparanase and angiogenesis. In addition, heparin has been shown to potently inhibit HIV -1 replication and affect cell growth, gene expression, modulate clinically relevant event such as diabetes and atherosclerosis. However, the clinical application of heparin for the treatment of both cancer and various inflammatory is limited by its anticoagulant activity. So we want to know whether LMW heparin and some polysaccharides from algae substitute heparin at some functions avoiding its side effects.A Sepharose-heparin column (a affinity chromatoraphic column) was used to study the binding of heparin to protein previously. In the 1990's, a sensitive technique developed for the study of the potential interaction of cytokine with heparin and related polysaccharides is affinity coelectrophoresis (ACE). Butthese techniques can not provide physiological pH and ionic strength, and radio-iodination of heparin required for ACE endangers health of human and environment. For avoiding the radiological hazards and reflecting directly the binding characteristic of heparin to protein. Synthesis of heparin-BSA complex used a chemistry method, established an ELISA technique to detect the interaction of heparin with protein in vitro. ELISA is a simple, effective method to study the interaction of polysaccharides and proteins.[Methods]Consideration of free heparin, unlike polypeptides, adsorb poorly onto plastic surfaces. In order to study the interaction of heparin with IFN- Y using ELISA assay, heparin-bovine complex (HBC) was synthesized by conjugating heparin to BSA in the presence of sodium cyanoborohydride. For removing the free heparin and free BSA, The reaction products were resolved by a 1 X 90 cm gel filtration column on Sepharose 4B, the elution of high Mr HBC was identified by SDS-PAGE, stained for heparin with Azure A and for protein with Coomassie blue. HBC was quantified for protein using the bicinchoninic acid assay (BCA reagent Kit).ELISA plate wells were coated either with HBC (0.5 g BSA equivalent )or with the same amount of mock-treated BSA, detected the binding of HBC to IFN- . For further observation the influence of some polysaccharides including free heparin, LMW heparin, CS-A, CS-C, HA, DxS, fucoidan and carrageenan family on the interaction of heparin with IFN-, 1.5 ng IFN- y waspreincubated at 37 for 30 mins in the presence and absence of different concentration of these polysaccharides before addition to coated well.[Results]We got HBC with high purity by Sepharose-4B gel filtration chromatography, and confirmed the important role of heparin in the interaction of HBC withIFN- γ with ELISA assay. The binding of IFN-γ to HBC is readily detectable at as little as 0.25 ng IFN- γ /well, increases in a cytokine dose-dependent manner, and saturates at around 2ng/well. Free heparin, LMW heparin and other polysaccharides inhibit the binding of HBC to IFN-γ with significant... |
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