Interfacial Complexes Between a Protein and Lipophilic Ions at an Oil-Water Interface

Anal. Chem. 2010, 82, 7699–7705 Interfacial Complexes between a Protein and Lipophilic Ions at an Oil-Water Interface Rune A. Hartvig,† Manuel A. Me´ ndez,‡ Marco van de Weert,† Lene Jorgensen,† Jesper Østergaard,† Hubert H. Girault,*,‡ and Henrik Jensen*,† Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark, and Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Station 6, CH-1015 Lausanne, Switzerland The interaction between an intact protein and two lipo- adsorption and ion transfer processes.8-12 In relation to protein philic ions at an oil-water interface has been investigated research, electrochemistry at ITIES have been used to study using cyclic voltammetry, impedance based techniques protein adsorption,13 protein adsorption kinetics,7 the effects on and a newly developed method in which the biphasic transfer of aqueous ions,14 and the assisted transfer of proteins oil-water system is analyzed by biphasic electrospray to the oil phase.15-18 Recently, electrochemistry at ITIES has been ionization mass spectrometry (BESI-MS), using a dual- used for detecting proteins in solution by means of a protein channel electrospray emitter. It is found that the protein assisted transfer of organic anions into the water phase at positive - forms interfacial complexes with the lipophilic ions and potentials.19 22 A suggested mechanism for the organic anion that it specifically requires the presence of the oil-water transfer involves the formation of a protein-anion complex at the 22 interface to be formed under the experimental conditions. interface. Furthermore, impedance based techniques and BESI-MS Usually electrospray ionization mass spectrometry (ESI-MS) with a common ion to polarize the interface indicated that involves an aqueous solution which by application of a high voltage the Galvani potential difference across the oil-water is sprayed into a mass analyzer. Recently, the development of a interface significantly influences the interfacial complex- new biphasic electrospray interface has allowed interfacial com- ation degree. The ability to investigate protein-ligand plexes in two phase systems to be analyzed using a mass complexes formed at polarized liquid-liquid interfaces is spectrometer; the new technique is termed biphasic electrospray 23-26 thus a new analytical method for assessing potential mass spectrometry (BESI-MS). The combined use of BESI- dependent interfacial complexation using a structure MS and electrochemistry at ITIES is attractive as it allows studying elucidating detection principle. quantitative mechanistic aspects of adsorption and ion transfer processes as well as structural characteristics of complexes formed - The behavior of biological macromolecules at liquid liquid at the interface. However, the usual experimental conditions in interfaces has important implications in the pharmaceutical sci- (7) Thomsen, A. E.; Jensen, H.; Jorgensen, L.; van de Weert, M.; Østergaard, ences. It is for example well-known that proteins adsorb at a wide J. Colloid. Surface. B 2008, 63, 243–48. variety of interfaces,1-4 a process that can have unwanted (8) Arrigan, D. W. M. Anal. Lett. 2008, 41, 3233–52. consequences such as a loss of therapeutic activity due to (9) Vanysek, P.; Ramirez, L. B. J. Chil. Chem. Soc. 2008, 53, 1455–63. (10) Reymond, F.; Fermin, D.; Lee, H. J.; Girault, H. H. Electrochim. Acta 2000, 4,5 denaturation and aggregation. Two-phase systems are at equi- 45, 2647–62. librium generally associated with an interfacial potential differ- (11) Samec, Z. Pure Appl. Chem. 2004, 76, 2147–80. (12) Vanysek, P. Electrochim. Acta 1995, 40, 2841–47. ence,6 in the following referred to as the Galvani potential (13) Vanysek, P.; Sun, Z. J. Electroanal. Chem. 1990, 298, 177–94. difference between a water (w) and oil (o) phase at equilibrium; (14) Vanysek, P.; Reid, J. D.; Buck, R. P. J. Electrochem. Soc. 1984, 131, C107. w w (15) Vagin, M. Y.; Trashin, S. A.; Ozkan, S. Z.; Karpachova, G. P.; Karyakin, ∆o φ. The value of ∆o φ can influence adsorption kinetics and/ 7 A. A. J. Electroanal. Chem. 2005, 584, 110–16. or adsorption isotherms. Electrochemistry at Interfaces be- (16) Vagin, M. Y.; Malyh, E. V.; Larionova, N. I.; Karyakin, A. A. Electrochem. tween two immiscible electrolyte solutions (ITIES) provides Commun. 2003, 5, 329–33. an effective methodology for studying potential dependent (17) Osakai, T.; Shinohara, A. Anal. Sci. 2008, 24, 901–06. (18) Shinshi, M.; Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 5937– 44. * To whom correspondence should be addressed. E-mail: [email protected] (19) Herzog, G.; Moujahid, W.; Strutwolf, J.; Arrigan, D. W. M. Analyst 2009, (H.J.); hubert.girault@epfl.ch (H.H.G.). 134, 1608–13. † University of Copenhagen. (20) Herzog, G.; Kam, V.; Arrigan, D. W. M. Electrochim. Acta 2008, 53, 7204– ‡ Ecole Polytechnique Fe´de´rale de Lausanne (EPFL). 09. (1) Dickinson, E. J. Chem. Soc., Faraday Trans. 1998, 94, 1657–69. (21) Kivlehan, F.; Lanyon, Y. H.; Arrigan, D. W. M. Langmuir 2008, 24, 9876– (2) Walstra, P.; Deroos, A. L. Food Rev. Int. 1993, 9, 503–25. 82. (3) Kleijn, M.; Norde, W. Heterog. Chem. Rev. 1995, 2, 157–72. (22) Scanlon, M. D.; Jennings, E.; Arrigan, D. W. M. Phys. Chem. Chem. Phys. (4) Burke, C. J.; Steadman, B. L.; Volkin, D. B.; Tsai, P. K.; Bruner, M. W.; 2009, 11, 2272–80. Middaugh, C. R. Int. J. Pharm. 1992, 86, 89–93. (23) Prudent, M.; Mendez, M. A.; Girault, H. H. Anal. Sci. 2008, 24, 1399–404. (5) Sluzky, V.; Tamada, J. A.; Klibanov, A. M.; Langer, R. Proc. Natl. Acad. Sci. (24) Mendez, M. A.; Prudent, M.; Su, B.; Girault, H. H. Anal. Chem. 2008, 80, U.S.A. 1991, 88, 9377–81. 9499–507. (6) Kakiuchi, T. Liquid-Liquid Interfaces: Theory and Methods, 1st ed.; Volkov, (25) Oyama, H.; Ohashi, A.; Watarai, H. Anal. Sci. 2004, 20, 1543–47. A. G.; Deamer, D. W., Eds.; CRC Press, Inc.: Boca Raton, 1996; Chapter 1. (26) Watarai, H.; Matsumoto, A.; Fukumoto, T. Anal. Sci. 2002, 18, 367–68. 10.1021/ac101528r 2010 American Chemical Society Analytical Chemistry, Vol. 82, No. 18, September 15, 2010 7699 Published on Web 08/24/2010 Chemie) or lithium tetrakis(pentafluorophenyl)borate (LiTPFB) (Boulder Scientific Company, Mead, CO), respectively. Electrochemical Setup. Voltammetric and potential scan methods were carried out as previously described,7 using a four electrode potentiostat PGSTAT30 (Autolab, Eco Chemie B.V., Utrecht, The Netherlands) and a custom-made glass cell from EuroGlas (Karlslunde, Denmark). The surface area of the organic- aqueous interface was 1.4 cm2 and the temperature was main- tained at 25 ± 0.5 °C by placing the cell in a water jacket. The w Galvani potential difference (∆o φ) was controlled using two Ag/ AgCl reference electrodes, one in the aqueous phase and one in a reference phase in contact with the 1,2-DCE phase (Scheme 1). The two counter electrodes were made of pure platinum and situated at each side of the interface. All potentials are taken with point of reference in the aqueous phase, so that a positive potential corresponds to a more positive potential for the aqueous phase relative to the potential of the oil phase. Figure 1. The red circle represents the organic anion (TPBCl- or The potential is adjusted to the Galvani potential scale by fixing - TPFB ), the colored groups on lysozyme represents positively (blue) the potential of zero charge (pzc) of the cell without lysozyme and negatively (red) charged amino acids at the investigated pH value at ∆wφ ) 0.28 of 2.81, as calculated from reference.27 The horizontal line corre- o sponds to the interface separating water (top) and 1,2-DCE (bottom) The transfer of ions was measured as an increase or decrease and also represent increasing interfacial potential difference in the in direct current, where a positive current corresponds to transfer w direction to the right. In the order of increasing ∆oφ the events depicted of a positive charge from the aqueous phase to the organic phase, thus correspond to, (i) adsorption of lysozyme which happens within or a negative charge transferred in the opposite direction. w > the entire potential window, but increasingly so at ∆o φ pzc, as Capacitance-potential curves were obtained by measuring the real evident from capacitance scans (ii) formation of an interfacial complex between lysozyme and one or more organic anion molecules, as and imaginary current response from a sinusoidal voltage ampli- investigated by BESI-MS (iii) assisted transfer of the organic anion tude of 5 mV (rms) and a frequency of 6 Hz at a range of potential to the water phase where it is bound to adsorped lysozyme, gives values. In the absence of ion transfer across the interface, the rise to the forward prepeak in the cyclic voltammogram (iv) the forward total impedance can be considered as a resistor and capacitor in main peak is the result of an assisted ion transfer which is less series. The interfacial capacitance (C) is then obtained as follows: favorable, and which ultimately leads to precipitation of lysozyme (v) the complex then may diffuse to the bulk phase. ) 1 C × (1) electrochemistry (high salt concentration) and BESI-MS (volatile ω ZIm buffer systems) are not compatible, and great care should thus be taken when combining data using the two techniques. where ZIm is the imaginary part of the impedance and ω is the In this work we have studied the electrochemical responses angular frequency calculated as the applied frequency times obtained at an ITIES under conditions which are directly compat- 2π. ible with those used in a BESI-MS experiment. Further, we Biphasic Electrospray MS.

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