Heparin characterization

Neuropilin-1 (NRP1), a molecule that had been previously shown to be implicated in axon guidance as a receptor for members of collapsin/semaphorin family, has been characterized as a which interacts with the heparin-binding VEGF isoforms. 

Grainger DW, Kim SW, and Feijen J. Poly(dimethyl siloxane)-poly(ethylene oxide)-heparin block copolymers. I Synthesis and characterization. J Biomed Mater Res, 1988, 22, 231-242. 

Sobel M, Soler DF, Kermode JC, Harris RB. Localization and characterization of a heparin binding domain peptide of human von Willebrand factor. J Biol Chem 1992 267 8857-8862. 

For detailed description and discussion of methods of separation and characterization of GAG, the reader is referred to specific mono-graphs38-42-46-47 dealing with the advantages and drawbacks of different colorimetric, titrimetric, electrophoretic, chromatographic, spectroscopic, and enzymic methods for the qualitative and quantitative characterization of heparin and its most common contaminants. The present article is concerned only with analytical aspects of relevance to the structural characterization of heparin. 

Chromatographic methods (essentially using ion-exchange resins) are especially useful for separating heparin from other glycosaminoglycans, for further analytical and structural characterization. However, it should be realized that recovery of heparin from resins is seldom quantitative,42 probably because of irreversible absorption of heparin fractions in the... 

Among the spectroscopic methods applicable to polysaccharides, u.v. spectrophotometry is of little value for characterizing heparin, whose main, electronic chromophore (the C02 group) displays a band at 220 nm, that is, in a region where all glycosaminoglycans absorb (also through their N-acetyl chromophores), and where minor proportions of unsaturated or aromatic contaminants cause serious interference.77 With pure heparin preparations, the carboxylate chromophore is most useful for chiroptical measurements, and a semi-quantitative evaluation of the extent of N-acetylation of 2-amino-2-deoxy-D-glucose residues is also possible.78... 

Infrared spectra are commonly used for characterizing glycosaminoglycans, especially solid samples thereof. However, being dominated by strong and broad bands due to polar and ionized groups (OH, C02 , and S03 ), the i.r. spectrum of heparin often contains concealed bands from contaminants.77 More-reproducible spectra, obtainable for solutions in H20 (Ref. 79) and D20 (Ref. 80) provide a better characterization, as well as quantitative information about such functional groups as carboxyl, acetamido, and sulfate groups. 

A classical, and still most useful, physicochemical parameter for characterizing glycosaminoglycans is their specific optical rotation. For pure heparins from common sources, [a]80 = + 45 to + 53 ° (see Table II). The actual value depends on the relative proportions of monosaccharide constituents (that is, the L-idosyluronic acid residues make a less positive contribution than the n-glucosyluronic residues), and on the degree of sulfation of the preparation (that is, although sulfate groups may have little direct influence on the molecular rotation, they lower the value by acting as diluents ). 

In contrast to L-iduronic acid residues, most of which are sulfated at C-2, D-glucuronic acid residues in heparin and heparan sulfate are largely or exclusively nonsulfated. This was especially proved by their susceptibility to periodate oxidation,123 and through characterization of D-glucuronic acid-containing di- and tetra-saccharides from deamina-tive104 109 110 138 or heparinase - heparanase cleavage137,145 of heparin. 

The scope of the deamination reaction was extended to the characterization of heparin segments containing 2-acetamido-2-deoxy-D-glucosyl residues by removing (by hydrazinolysis) the N-acetyl groups and cleaving with nitrous acid (at pH 4) the otherwise resistant segments.113,232... 

Plasma-derived antithrombin concentrates have been used medically since the 1980s for the treatment of hereditary and acquired antithrombin deficiency. Hereditary (genetic) deficiency is characterized by the presence of little/no native antithrombin activity in plasma and results in an increased risk of inappropriate blood clot/emboli formation. Acquired antithrombin deficiency can be induced by drugs (e.g. heparin and oestrogens), liver disease (decreased antithrombin... 

Linhardt RJ, Ampofo SA, Fareed J, Hoppensteadt D, Mulliken JB, Folkman J (1992) Isolation and characterization of human heparin. Biochemistry 31 12441-12445. 

Lohse DL, Linhardt RJ (1992) Purification and characterization of heparin lyases from Flavobacterium heparinum. J Biol Chem 267 24347-55. 

NHH Heegaard. A heparin-binding peptide from human serum amiloid P component characterized by affinity capillary electrophoresis. Electrophoresis 19 442-447, 1998. 

CC Griffin, RJ Linhardt, CL Van Gorp, T Toida, RE Hileman, RL Schubert, SE Brown. Isolation and characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin. Carbohydr Res 276 183-197, 1995. 

Degradation of heparin followed by affinity chromatography on immobilized AT III led to the identification of a pentasaccharide unit with high antithrombin affinity. This pentasaccharide 3 is characterized by a imique highly sulfated central glucosamine unit with a 3-0-sulfate group (Structures 2). 

Santos, J. C, Mesquita, J. M. F., Belmiro, C. L. R., Carolina da Silveira, . M., Viskov, C., Mourier, P. A., and Pavao, M. S. G. (2007). Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects on venous and arterial models of thrombosis. Thromb. Res. 121,213-222. 

Cesaretti, M., Luppi, E., Maccari, M., and Volpi, N. (2004). Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes philippinarum. Evidence for the presence of a high content of antithrombin Ill-binding site. Glycobiology 14,1275-1284. 

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