title: Adsorption of a therapeutic enzyme to self-assembled monolayers: effect of surface chemistry and solution pH on the amount and activity of adsorbed enzyme.
abstract: The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functionalities (X = CH(3)-, OH- and COOH-) was evaluated as a function of solution pH. Radiolabelling studies showed that the enzyme has higher affinity for hydrophobic surfaces than for hydrophilic surfaces, and that the highest adsorption was obtained at the more acidic pH values (4.5 and 5.5), despite the type of surface. IRAS and XPS measurements confirmed this tendency. Dye-binding studies and fluorescence quenching were used to investigate if a pH variation induces any conformational changes on the enzyme. Both methods suggest that lowering the pH from physiological to acidic values triggers an increased exposure of non-polar sites in the enzyme, which may modulate its adsorption behaviour to the more hydrophobic surfaces. At pH 4.5, the enzyme carries a substantial positive net charge and therefore relatively low native-state stability. As a consequence, surface binding may be favoured, irrespective of the type of surface, by providing increased conformational entropy to the enzyme. The specific activity (SA) of the adsorbed enzyme was strongly dependent on the conditions used. A decrease in SA (ca. 30% of control) was observed after adsorption on CH(3)-SAMs for all the pH tested. Adsorption on gold and on the more hydrophilic SAMs (OH- and COOH-) resulted in different degrees of inactivation at the more acidic pH (4.5), and in enzyme activation (up to ca. 230% of control) at higher pH (7-8), near the isoelectric point of the enzyme.
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Laboratório de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. ccbarrias@ineb.up.pt
authors.forename: Cristina C
authors.initials: CC
authors.lastname: Barrias
authors.forename: M A Cristina L
authors.initials: MA
authors.lastname: Martins
authors.forename: M A Clara
authors.initials: MA
authors.lastname: Sá Miranda
authors.forename: Mário A
authors.initials: MA
authors.lastname: Barbosa
mesh_terms: D000327:Adsorption; D020099:Coated Materials, Biocompatible / Q000032:analysis / Q000737:chemistry*; D016503:Drug Delivery Systems / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D005962:Glucosylceramidase / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008422:Materials Testing; D008567:Membranes, Artificial; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013499:Surface Properties
chemical_list: D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D012996:Solutions; D005962:Glucosylceramidase