Biospecificity chromatography the investigation of the active site ofN-acetyl-β-d-hexosaminidase with affinity chromatography
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Affinity chromatography of different natural biopolymers, including enzymes, is perhaps the most widely used modern technique for isolation and purification of these molecules. We have synthesized three biospecific sorbents with weak ion exchange properties by coupling ligands to carriers through hydrazide groups. These sorbents have no ion exchange or hydrophobic groups, thereby minimizing the influence of nonspecific binding on the process of affinity chromatography. These biospecific sorbents have been used for purification ofN-acetyl-β-d-hexosaminidase (EC 18.104.22.168), and for study of its active site and of its sorption and elution mechanisms in affinity chromatography. Biospecificity of sorbents was suggested by adsorption ofN-acetyl-β-d-hexosaminidase at optimum catalytic pH and by elution of the enzyme with minimal variation of pH.
By comparing the known data for mapping the active site ofN-acetyl-β-d-hexosaminidase and energetic contributions of functional groups of inhibitor molecules with the obtained results, one can infer that sorption of the enzyme on biospecific sorbents is realized only by hydrogen bonds between the ligand used and ionizable groups ofN-acetyl-β-d-hexosaminidase (pK 5.5).
KeywordsAffinity Chromatography Thioglycolic Acid Energetic Contribution Selective Sorbent Hydrazide Group
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