1096 DOI: 10.1021/la904114u Langmuir 2010, 26(2), 1096–1106 Published on Web 12/08/2009 pubs.acs.org/Langmuir © 2009 American Chemical Society Adsorption Induced Enzyme Denaturation: the Role of Polymer Hydrophobicity in Adsorption and Denaturation of r-Chymotrypsin on Allyl Glycidyl Ether (AGE)-Ethylene Glycol Dimethacrylate (EGDM) Copolymers Challa Lahari, † Lakshmi S. Jasti, † Nitin W. Fadnavis,* ,† Kalpana Sontakke, ‡ Ganesh Ingavle, ‡ Sarika Deokar, ‡ and Surendra Ponrathnam ‡ † Biotransformations Laboratory, Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India and ‡ Polymer Science & Engineering, Chemical Engineering Division, National Chemical Laboratory, Pashan Road, Pune 411008 India Received July 8, 2009 Effects of changes in hydrophobicity of polymeric support on structure and activity of R-chymotrypsin (E.C. 3.4.21.1) have been studied with copolymers of allyl glycidyl ether (AGE) and ethylene glycol dimethacrylate (EGDM) with increasing molar ratio of EGDM to AGE (cross-link density 0.05 to 1.5). The enzyme is readily adsorbed from aqueous buffer at room temperature following Langmuir adsorption isotherms in unexpectedly large amounts (25% w/w). Relative hydrophobicity of the copolymers has been assessed by studying adsorption of naphthalene and Fmoc-methionine by the series of copolymers from aqueous solutions. Polymer hydrophobicity appears to increase linearly on increasing cross-link density from 0.05 to 0.25. Further increase in cross-link density causes a decrease in naphthalene binding but has little effect on binding of Fmoc-Met. Binding of R-chymotrypsin to these copolymers follow the trend for Fmoc-methionine binding, rather than naphthalene binding, indicating involvement of polar interactions along with hydrophobic interactions during binding of protein to the polymer. The adsorbed enzyme undergoes extensive denaturation (ca. 80%) with loss of both tertiary and secondary structure on contact with the copolymers as revealed by fluorescence, CD and Raman spectra of the adsorbed protein. Comparison of enzyme adsorption behavior with Eupergit C, macroporous Amberlite XAD-2, and XAD-7 suggests that polar interactions of the EGDM ester functional groups with the protein play a significant role in enzyme denaturation. Introduction Protein molecules spontaneously adsorb on solid surfaces due to their intrinsic surface activity. 1 This phenomenon is widely used for protein separation and purification by hydrophobic interac- tion chromatography 2 and in medical diagnostics e.g., protein detection on biochips. 3 Enzyme immobilization on solid supports through covalent bonding is another major area of research for industrial applications. 4 In all these cases, studies in forces involved in the interaction of the protein with the solid support and the effect of these interactions on the structure and biological activity of the protein are crucial for a successful application. During the last 20 years, several polymeric supports have been developed to achieve immobilized enzyme preparations with desirable properties such as a fairly uniform particle size, good mechanical strength, high stability toward changes in pH and temperature etc. 4 Among these, polymers carrying epoxy groups such as Eupergit C are considered to be highly useful since the technique of immobilization is very simple, e.g., an enzyme solution in aqueous buffer of pH 7-8 is contacted with the poly- mer for a few hours to achieve covalent linkage between the polymer and the protein via nucleophilic attack by amino groups on the enzyme surface on the epoxide. 5 Recently, new macro- porous polymers containing epoxy groups such as copolymers of ethylene glycol dimethacrylate (EGDM) with allyl glycidyl ether or glycidyl methacrylate have been employed in enzyme immo- bilization. 6 Although a lot of data is available on the stability and recycle of the enzymes after covalent binding on these supports, very little information is available on the initial interactions which drive the enzyme toward the polymer surface from bulk aqueous phase. Since the epoxy-activated polymers are uncharged, initial binding of the protein to the polymer presumably occurs through hydrophobic interactions. However, most of the polymers also possess polar functional groups such as an amido group in Eupergit C and ester groups in methacrylates. Contributions from polar interactions (hydrogen bond and van der Waals) should also be playing some role in protein binding. Whether the two types of interactions work in tandem to destabilize the protein or in opposite direction to compensate the effects so that the *To whom correspondence may be addressed. Tel. 91-40-27191631. Fax: 91-40-27160512. E-mail: fadnavisnw@yahoo.com; fadnavis@iict.res.in. (1) Horbett, T. A.; Brash, J. L. Proteins at Interfaces II, ACS Symposium Series 602; American Chemical Society: Washington DC, 1995. (2) Lienqueo, M. E.; Mahn, A.; Salgado, J. C.; Asenjo, J. A. J. Chromatogr. B 2007, 849, 53–68. (3) Weinberger, S. R.; Morris, T. S.; Pawlak, M. 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