Adsorption of Glucose Oxidase at Organic-Aqueous and Air-Aqueous Interfaces Dimitra G. Georganopoulou* and David E. Williams Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, U.K. Carlos M. Pereira and Fernando Silva Departamento de Quı ´mica, Faculdade de Cie ˆ ncias, Universidade do Porto, P-4169-007 Porto, Portugal Tsueu-Ju Su and Jian R. Lu Department of Physics, UMIST, P.O. Box 88, Manchester M60 1QD, U.K. Received June 5, 2002. In Final Form: January 6, 2003 The adsorption of glucose oxidase (GOx) was studied at the interface between two immiscible electrolyte solutions (ITIES) by interfacial capacitance and surface tension measurements and at the air/water (phosphate buffer) interface by surface tension and neutron reflection measurements. The adsorption at both interfaces was found to be time, enzyme concentration, and ionic strength dependent. There was a switch from one interfacial adsorption state to another, as the enzyme concentration was increased. At the ITIES, there was evidence of an interaction between the adsorbed enzyme and the hydrophobic cation in the organic phase (1,2-dichloroethane). The enzyme adsorbed at the air/water interface was found to dissociate into monomers at the lower buffer total concentration of 2 mM while, at the higher buffer concentration of 0.2 M, the adsorbed enzyme retained its dimer structure. The adsorption mostly formed monolayers and the layer thickness varied with bulk concentration, indicating deformation related to the packing of the enzyme at the interface. For enzyme concentrations above 1 μM, in high ionic strength medium, bilayers of enzyme started to form, and the interlayer interactions resulted in a less densely packed second layer forming on the aqueous side of the first one. The switch in properties of the adsorbed layer observed in interfacial tension and capacitance measurements at the ITIES occurred over the same enzyme concentration range as the formation of a more densely packed layer detected from neutron reflection at the air/water interface. Introduction Glucose oxidase (GOx) is an enzyme that has attracted immense interest, due to its applicability in biosensors for the determination of glucose in body fluids, as well as for removing glucose and oxygen from beverages and food products. Because it is easily obtainable and robust, it is convenient to use as an initial model for exploring the reactivity of redox enzymes at interfaces. 1 As part of such a study, we report here an exploration of the adsorption of glucose oxidase at both an organic solvent/water interface and the air/water interface, comparing the results of electrochemical adsorption measurements at the organic/ aqueous interface with those of structural studies using neutron reflection at the air/water interface. Interfacial tension measurements at both interfaces are used to provide a link. GOx is a dimeric globular glycoprotein 2 of dimensions 60 × 52 × 77 Å 3 , made up from two identical subunits, each of molecular weight ∼75 kDa, that are bound with disulfide bridges, salt linkages, and hydrogen bonds. 3 GOx has one redox coenzyme, flavin adenine dinucleotide (FAD), per monomer. The FAD is not covalently bound to the protein and can be released under denaturing condi- tions. The enzyme has a diffusion coefficient of 4.94 × 10 -7 cm 2 s -1 in 0.1 M NaCl and a considerable part of hydrophobic side chains located near the surface. 4 The mean diameter of the native enzyme in solution, according to photon correlation spectroscopy data, is 76 Å at pH 7.4. 5 The same group gives the Stokes radius for the molecule to be 43 Å with a frictional ratio of 1.21, from which they conclude that the enzyme in solution is an elongated protein with rigid structure. Hecht et al. 3 state that each monomer is a compact spheroid of dimensions 60 × 52 × 37 Å 3 . The native protein is acidic with an isoelectric point of 4.44. At pH 7 it is negatively charged with 11 charges. Duinhoven et al. 6 summarized four categories of in- teraction between proteins and surfaces: (i) Electrostatic and Van de Waals interactions, which are dependent on the net charge on both protein and * To whom correspondence should be addressed: Currently at University of North Carolina. Phone: 919-9620458; Fax: 919- 9621381; E-mail: degeorgan@email.unc.edu. (1) Georganopoulou, D. G.; Caruana, D. J.; Strutwolf, J.; Williams, D. E. Faraday Discuss. 2000, 116, 109. (2) Kriechbaum, M.; Heilmann, H. J.; Wientjes, F. J.; Hahn, M.; Jany, K.-D.; Gassen, H. G.; Sharif, F.; Alaeddinoglu, G. F. FEBS Lett. 1989, 255, 63. (3) Hecht, H. J.; Kalizs, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (4) Hecht, H. J.; Schomburg, D.; Kalizs, H. M.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197. (5) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1999, 209, 302. (6) Duinhoven, S.; Poort, R.; Vandervoet, G.; Agterof, W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170, 340. 4977 Langmuir 2003, 19, 4977-4984 10.1021/la0205248 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/10/2003