A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities

On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca 2+

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca2+-heparin, in which alpha-L-iduronate-2-O-sulfate residues adopt C-1(4) conformation preferentially, is a substrate, while Na+-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates beta-elimination by abstracting the C5 proton of the alpha-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)National High Magnetic Field LaboratoryUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilUniv Liverpool, Dept Struct & Chem Biol, Liverpool L69 7ZB, Merseyside, EnglandUniv Fed Rio Grande do Sul, Ctr Biotecnol, BR-91500970 Porto Alegre, RS, BrazilDiamond Light Source Ltd, Didcot OX11 ODE, Oxon, EnglandUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, Ctr Ciencias Tecnol, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilCAPES: 172/2012Web of Scienc