Affinity chromatography of β-N-acetylhexosaminidases on columns with mixed charge and affinity functions |
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Authors: | Luana C. B. B. Coelho Winston Hutchinson Achamma Koshy Donald Robinson John L. Stirling |
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Affiliation: | 1. Departmento de Bioquímica, Centro de Ciěncias Biológicas, Universidade Federal de Pernambuco, 50730, Recife, PE, Brazil 2. Division of Biomolecular Sciences, King’s College London, W8 7AH, Campden Hill, London
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Abstract: | Analysis was made of the nature of interactions between β-N-acetylhexosaminidase and affinity chromatography gels made by coupling 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-β-D-glucopyranosylamine (ANAG) to CNBr-Sepharose columns. This showed that although specific binding of the enzyme to the immobilized ligand was too weak to cause retention, compound affinity in which charge interactions were involved could be exploited for purification of the enzyme. Evidence is given for the specificity of interaction of the enzyme with immobilized ligand and for biospecific desorption of β-N-acetylhexosaminidases from ANAG-Sepharose columns. A method was developed for the purification of placental β-N-acetylhexosaminidase A in mg amounts starting from crude extracts. |
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