Binding of the volatile general anesthetics halothane and isoflurane to a mammalian b-barrel protein Jonas S. Johansson 1,2,4 , Gavin A. Manderson 1 , Roberto Ramoni 5 , Stefano Grolli 5 and Roderic G. Eckenhoff 1,3 1 Department of Anesthesia, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA 3 Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA 4 Johnson Research Foundation, University of Pennsylvania, Philadelphia, PA, USA 5 Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita ` e Sicurezza degli Alimenti, Universita ` di Parma, Parma, Italy A molecular understanding of volatile anesthetic mechanisms of action will require structural descrip- tions of anesthetic–protein complexes. Because the in vivo sites of action remain to be determined, the structural features of anesthetic binding sites on proteins are being explored using well-defined model systems, such as the serum albumins and four-a-helix bundle proteins [2,3]. Studies with these model systems have suggested that volatile general anesthetics prefer- entially bind to pre-existing appropriately sized Keywords anesthetic–protein interaction; halothane; isoflurane; isothermal titration calorimetry; porcine odorant binding protein Correspondence J. S. Johansson, 319C, John Morgan Building, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104, USA Fax: +1 215 349 5078 Tel: +1 215 349 5472 E-mail: JohanssJ@uphs.upenn.edu (Received 18 October 2004, revised 19 November 2004, accepted 24 November 2004) doi:10.1111/j.1742-4658.2004.04500.x A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic–protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large b-barrel internal cavity (515 ± 30 A ˚ 3 ) lined predomin- antly by aromatic and aliphatic residues. Halothane binding to the b-barrel cavity was determined using fluorescence quenching of Trp16, and a com- petitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using iso- thermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the b-barrel of porcine odorant binding protein with dissociation constants of 0.46 ± 0.10 mm and 0.43 ± 0.12 mm determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calori- metry revealed that halothane and isoflurane bound with K d values of 80 ± 10 lm and 100 ± 10 lm, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by )0.9 ± 0.1 kcalÆmol )1 . In addition to indicating specific binding to the native protein conformation, such stabilization may repre- sent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 A ˚ resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules. Abbreviation AMA, 1-aminoanthracene. FEBS Journal 272 (2005) 573–581 ª 2004 FEBS 573 packing defects, or cavities, within the protein matrix [4,5]. In addition, favorable polar interactions with hydrophobic core side chains can further enhance anesthetic binding affinity [6,7]. Previous work has demonstrated that volatile anes- thetics bind to a-helical proteins such as bovine serum albumin [8–10] and the synthetic four-a-helix bundles [4,6,7,11,12]. Helical proteins are known to be able to bind a variety of ligands due to their relative conform- ational flexibility [13]. In contrast, b-sheet secondary structure forms a rigid fold, which may result in a better-defined binding site, and is represented in the lipid-spanning b-barrel domains of mitochondrial outer membrane proteins [14–16]. The roles played by these b-barrel membrane proteins include active ion trans- port, passive nutrient intake, and enzymatic activity. One member of this group of proteins, the voltage- dependent anion channel-1 from rat brain, has recently been identified as a target for both neuroactive steroids [17] and halothane [18]. Porcine odorant binding protein (Fig. 1) is a 157 residue member of the lipocalin family that features a large b-barrel internal cavity [1]. The b-barrel cavity has a volume of 515 ± 30 A ˚ 3 , and is lined predomin- antly by aromatic and aliphatic residues. The ability of this cavity to bind anesthetic molecules was explored. Halothane binding to the b-barrel cavity was determined using fluorescence quenching [10] of the single tryptophan residue (Trp16). Halothane and isoflurane binding were also characterized thermody- namically using isothermal titration calorimetry [12]. The ability of halothane to displace the fluorescent probe 1-aminoanthracene (AMA) bound in the por- cine odorant binding protein cavity was also exam- ined. Finally, hydrogen exchange [19] was used to evaluate the effect of bound halothane and isoflurane on global protein dynamics, with the goal of further defining a potential mechanism of volatile general anesthetic action. Results Binding of the volatile anesthetic halothane to the hydrophobic core of porcine odorant binding protein The binding of halothane to the porcine odorant bind- ing protein hydrophobic core was followed by trypto- phan fluorescence quenching [10] as shown in Fig. 2. Halothane causes a concentration-dependent quench- ing of the intrinsic Trp16 fluorescence, without chan- ging the emission maximum, indicating that halothane binding in the cavity does not alter the local dielectric environment of the indole ring. Furthermore, the lack of a red-shift in the tryptophan fluorescence emission maximum upon halothane binding suggests that the anesthetic does not promote unfolding of the protein, which would lead to increased water exposure of the indole ring. Figure 3 (curve a) shows a plot of the Trp16 fluorescence as a function of the halothane concentration. Fitting the data using Eqn (1) yields a K d ¼ 0.99 ± 0.06 mm with a Q max ¼ 0.27 ± 0.01, indicating that the fluorescence of the single trypto- phan residue in the porcine odorant binding protein is only partially quenched by bound anesthetic. Fig. 1. The X-ray crystal structure of the porcine odorant binding protein dimer at 2.25 A ˚ resolution (PDB entry 1A3Y). The side chains Trp16 and Tyr82 are indicated as stick structures. The figure was generated using RASMOL v2.7.2.1 (http://www.bernstein-plus- sons.com/software/rasmol). Fig. 2. Quenching of the porcine odorant binding protein (1 lM) Trp16 fluorescence by halothane. Excitation was at 295 nm, with the emission maximum at 339 nm. The concentrations of halothane were (a) 0, (b) 0.6, (c) 1.5, and (d) 5.0 m M. General anesthetic binding to a b-barrel protein J. S. Johansson et al. 574 FEBS Journal 272 (2005) 573–581 ª 2004 FEBS The effect of halothane on Trp16 fluorescence follow- ing excitation at 305 nm was examined in order to understand why only partial quenching was observed. With excitation at 305 nm, no contribution to Trp16 fluorescence secondary to energy transfer from any of the five tyrosine residues present in porcine odorant binding protein should be observed. With excitation at 305 nm, halothane causes a small linear decrease in the fluorescence intensity of Trp16 (Fig. 3, curve b), which is attributed to collisional quenching (the Stern– Volmer collisional quenching constant, K sv ,is 22 ± 1 m )1 ) because it is comparable to the effect of ha- lothane on free N-acetyl-tryptophanamide fluorescence (K sv ¼ 25±1m )1 ) [7]. This indicates that halothane does not bind in close proximity to Trp16, but rather in the vicinity of one of the five tyrosine residues. Halo- thane is able to quench tyrosine fluorescence with the same efficiency as tryptophan fluorescence [7]. Of the five tyrosine residues, Tyr82 is located within the por- cine odorant binding protein cavity (Fig. 1), and the fluorescence quenching results in Fig. 3 suggest that this may be one of the residues that halothane binds to adjacently. Subtraction of the collisional quenching contribution to the decrease in Trp16 fluorescence inten- sity results in curve c in Fig. 3, which yields a K d of 0.46 ± 0.10 mm and a Q max of 0.17 ± 0.01. Binding of the volatile anesthetics halothane and isoflurane to the porcine odorant binding protein as determined by isothermal titration calorimetry Representative calorimetric titrations at pH 7.0 of por- cine odorant binding protein with halothane and iso- flurane are shown in Figs 4 and 5. Each peak in the binding isotherm (upper panels, Figs 4 and 5) repre- sents a single injection of halothane and isoflurane. The negative deflections from the baseline on addition of halothane and isoflurane indicate that heat was evolved (an exothermic process). The enthalpy change associated with each injection of anesthetic was plotted vs. the anesthetic ⁄ porcine odorant binding protein molar ratio (lower panels, Figs 4 and 5), and the DH°, K d , the free energy change associated with binding (DG°), and the change in entropy associated with bind- ing (DS°) were determined from the plots. The K d value for halothane of 80 ± 10 lm is quite comparable to the value of 0.46 ± 0.10 mm obtained using trypto- Fig. 4. Titration of porcine odorant binding protein (pOBP) with halothane, showing the calorimetric response as successive injec- tions of ligand are added to the reaction cell. The lower panel depicts the binding isotherm of the calorimetric titration shown in the upper panel. The continuous line represents the least-squares fit of the data to a single-site binding model. Fig. 3. (a) Quenching of Trp16 fluorescence by added halothane with excitation at 295 nm. (b) Collisional quenching of Trp16 fluor- escence by halothane with excitation at 305 nm. (c) Replot of data in (a) after subtracting the collisional quenching contribution to Trp16 fluorescence. The porcine odorant binding protein concentra- tion was 1 l M. Data points are the means of three experiments on separate samples with error bars representing the SD. For curves (a) and (c) the lines through the data points has the form of Eqn (1). Error bars are omitted from curve (c) for clarity. J. S. Johansson et al. General anesthetic binding to a b-barrel protein FEBS Journal 272 (2005) 573–581 ª 2004 FEBS 575 phan fluorescence quenching, supporting the validity of the results. Isoflurane binds to porcine odorant binding protein with a K d ¼ 100 ± 10 lm. The other thermo- dynamic parameters underlying halothane and isoflura- ne binding to the porcine odorant binding protein are given in Table 1. Halothane displaces 1-aminoanthracene (AMA) bound to the internal cavity in the hydrophobic core of porcine odorant binding protein Figure 6 shows that halothane can displace AMA from the porcine odorant binding protein cavity. The competition curve was treated as a two parameter hyperbolic decay (R ¼ 0.97) and gave an EC 50 of 0.86 ± 0.24 mm. The true dissociation constant ( K d, true ), calculated using Eqn (2) resulted in a value of 0.43 ± 0.12 mm, in agreement with the results obtained using Trp16 fluorescence quenching and iso- thermal titration calorimetry. Effect of bound halothane and isoflurane on the dynamics of the porcine odorant binding protein Figure 7 shows the terminal hydrogen exchange rates for the porcine odorant binding protein. Because these terminal hydrogens exchange in about 100 min (6000 s), and freely exposed amide hydrogens exchange in % 0.1 ms, protection factors can be estimated to have values of 6 · 10 5 . Assuming that these slow hydrogens exchange only through global unfolding events, the stability of the porcine odorant binding protein is estimated to be %8 kcalÆmol )1 . The folded conformation of the porcine odorant binding protein was stabilized further by the addition of halothane or isoflurane. Both anesthetics stabilized the porcine odorant binding protein by )0.9 ± 0.1 kcalÆmol )1 , consistent with the premise of preferential binding to the native folded conformation of the porcine odorant binding protein. Discussion Halothane binds to the hydrophobic cavity in the b-barrel of porcine odorant binding protein with a K d of 0.46 ± 0.10 mm as determined by the quenching of the fluorescence of Trp16. Isothermal titration Fig. 5. Titration of porcine odorant binding protein (pOBP) with iso- flurane, showing the calorimetric response as successive injections of ligand are added to the reaction cell. The lower panel depicts the binding isotherm of the calorimetric titration shown in the upper panel. The continuous line represents the least-squares fit of the data to a single-site binding model. Table 1. Dissociation constants and thermodynamic data for the binding of halothane and isoflurane to the porcine odorant binding protein. The entropy unit (eu) is calÆmol )1 Æ K )1 . Anesthetic K d (lM) DG° (kcalÆmol )1 ) DH° (kcalÆmol )1 ) DS° (eu) Halothane 80 ± 10 )5.5 ± 0.1 )1.4 ± 0.1 14.0 Isoflurane 100 ± 10 )5.4 ± 0.1 )2.4 ± 0.1 10.3 Fig. 6. Competition between halothane and 1-aminoanthracene (AMA) for binding to porcine odorant binding protein. The fluores- cence intensity at 480 nm (a measure of bound AMA) is plotted as a function of the halothane concentration. See text for details. General anesthetic binding to a b-barrel protein J. S. Johansson et al. 576 FEBS Journal 272 (2005) 573–581 ª 2004 FEBS calorimetry indicates that halothane binds to porcine odorant binding protein with a K d of 80 ± 10 lm. The energetics underlying binding are therefore about 10 times more favorable than the interaction with human serum albumin [10]. The affinity with which porcine odorant binding protein binds halothane is quite comparable to the affinity with which the four-a- helix bundles bind this anesthetic [6,12]. Previous stud- ies have shown that volatile general anesthetics can bind to a-helical proteins [5–12,27,28], but this is the first study to demonstrate binding to a b-barrel protein using direct binding assays. Isoflurane has been shown to bind to bovine serum albumin with K d values of 1.4 ± 0.2 and 1.3 ± 0.2 mm using 19 F-NMR spectroscopy [29,30]. Using a competitive photoaffinity labeling approach, Eckenhoff & Shuman [9] reported a K d value of 1.5 ± 0.2 mm for isoflurane binding to bovine serum albumin. Similarly, a tryptophan fluorescence aniso- tropy study determined that isoflurane bound to bovine serum albumin with a K d of 1.6 ± 0.4 mm [31]. In addition, isoflurane has been shown to bind to nico- tinic acetylcholine receptors from Torpedo nobiliana with an average K d value of 0.36 ± 0.03 mm using 19 F-NMR spectroscopy and gas chromatography [32]. Finally, isoflurane was shown to bind to the four- a-helix bundle (Aa 2 -L38M) 2 with a K d ¼ 140 ± 10 lm using isothermal titration calorimetry [12]. The affinity of the interaction of isoflurane with porcine odorant binding protein (K d ¼ 100 ± 10 lm) is therefore com- parable to the findings in the latter two studies. For both anesthetics, the free energy of binding (D G °) exceeded the heat of binding (DH°) by more than a factor of two (Table 1), indicating that binding to por- cine odorant binding protein is entropy driven, in con- trast to the results obtained with the four-a-helix bundle (Aa 2 -L38M) 2 [12]. AMA has been shown to bind to porcine odorant binding protein [21,22] and to be competitively dis- placed by other ligands [23] shown by X-ray crystallo- graphy to localize in the hydrophobic cavity [24]. The results presented in Fig. 6 indicate that halothane is able to displace the bound AMA with a K d of 0.43 ± 0.12 mm. This value is quite comparable to the K d of 0.46 ± 0.10 mm determined for the binding of halothane to the protein, using fluorescence spectrosco- py. This result suggests that volatile general anesthetics may exert some of their physiological effects by displa- cing endogenous ligands from their receptors as sug- gested earlier based upon studies with firefly luciferase [33] and bovine rhodopsin [34]. The majority of proteins adopt a unique three- dimensional structure (the native state) under physiolo- gical conditions. The native structure is maintained by the hydrophobic effect and electrostatic contributions, with entropic terms tending to favor unfolding of the polypeptide [35]. The balance between these opposing energetic components is responsible for the overall sta- bility of the native folded protein conformation. The effect of halothane binding to (Aa 2 ) 2 on the four- a-helix bundle scaffold stability was examined using chemical denaturation with guanidinium chloride as shown by circular dichroism spectroscopy [27]. The bound anesthetic stabilized the native bundle confor- mation by )1.8 kcalÆmol )1 at 25 °C, and increased the m-value (the slope of the unfolding transition) from 1.6 ± 0.2 to 2.0 ± 0.1 kcalÆmol )1 Æ m )1 . The latter effect is compatible with improved hydrophobic core packing [36], and supports anesthetic binding to the cavity in the core of (Aa 2 ) 2 . Using hydrogen exchange [6], halothane was also shown to stabilize the folded conformation of the four-a-helix bundle (Aa 2 -L38M) 2 by approximately )0.9 kcal Æmol )1 . Thus, binding of anesthetic to the four-a-helix bundle scaffolds is associ- ated with a stabilization of the folded conformation of the protein. Halothane has been shown to increase the stability of the native folded conformation of bovine serum albumin using differential scanning calorimetry and hydrogen exchange [37]. Furthermore, both halothane and isoflurane stabilize the native folded state of albumin to thermal denaturation as determined by circular dichroism spectroscopy [31]. Fig. 7. Effect of halothane (b, 4 mM, d) and isoflurane (c, 7 mM, h) on terminal hydrogen exchange rates in porcine odorant binding protein. Curve a (s) is the control rate of hydrogen exchange in the absence of anesthetic. The y-axis is the molar ratio of unexchanged hydrogen to protein. The most rapid time point acheivable in these studies is between 5 and 7 min after mixing the protein with anes- thetic-containing solution. J. S. Johansson et al. General anesthetic binding to a b-barrel protein FEBS Journal 272 (2005) 573–581 ª 2004 FEBS 577 Binding of halothane and isoflurane is associated with a stabilization of the native folded conformation of the porcine odorant binding protein by )0.9 ± 0.1 kcalÆ- mol )1 . In addition to indicating specific binding to the native protein conformer, such stabilization may con- stitute a fundamental mechanism whereby anesthetics reversibly alter protein function. There are relatively few X-ray crystal structures to date that involve a protein with a bound anesthetic. All involve model proteins such as myoglobin, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, human serum albumin, and the enzyme firefly luciferase [2]. No high-resolution structure that involves any of the modern halogenated ether anesthetics has yet been published. However, a 2.4 A ˚ resolution X-ray crystal structure of human serum albumin with several bound halothane molecules has recently been reported [38]. Six of the binding sites involve a combination of ali- phatic and charged residues, such as arginine or lysine, with the remaining two composed of aliphatic and somewhat polar residues such as serine, phenylalanine, and asparagine. The crystallographic results are in accord with earlier solution studies using fluorescence spectroscopy and photoaffinity labeling that indicated that halothane bound in close proximity to Trp214 and Tyr411 in human serum albumin [10,28]. Because porcine odorant binding protein has been successfully crystallized and analyzed by X-ray diffrac- tion to 2.25 A ˚ resolution [1], the current results suggest that it represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macro- molecules, and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins. Experimental procedures Protein purification Porcine odorant binding protein was purified from an aque- ous extract of fresh pig nasal mucosa as described [1]. The protein was shown to be pure by SDS ⁄ PAGE, yielding a single band at 28 kDa. Steady-state fluorescence measurements Binding of halothane to the porcine odorant binding pro- tein was determined using steady-state intrinsic tryptophan fluorescence measurements [10] on a K2 multifrequency cross-correlation phase and modulation spectrofluorometer (ISS Inc., Champaign, IL, USA). Tryptophan was excited at either 295 nm or 305 nm (bandwidth 2 nm) and emission spectra (bandwidth 4 nm) recorded with peaks at 339 nm. The quartz cell had a pathlength of 10 mm and a Teflon stop- per. The temperature of the cell holder was controlled at 25.0 ± 0.1 °C. The buffer was 130 mm NaCl, 20 mm sodium phosphate, pH 7.0. Protein concentrations were determined with a UV ⁄ Vis Spectrometer Lambda 25 (PerkinElmer, Nor- walk, CT, USA), using a e 278 of 12 200 m )1 Æcm )1 [20]. Halothane-equilibrated porcine odorant binding protein, in gas-tight Hamilton (Reno, NV, USA) syringes, was diluted with predetermined volumes of plain protein (not exposed to anesthetic, but otherwise treated in the same manner) to achieve the final anesthetic concentrations indicated in the Figures. As described previously [10], the quenched fluorescence (Q) is a function of the maximum possible quenching (Q max ) at an infinite halothane concentration ([Halothane]) and the affinity of the anesthetic for its binding site (K d )in the vicinity of the tryptophan residue. From mass law con- siderations, it then follows that Q ¼ ðQ max [Halothane]Þ ðK d þ [Halothane]Þ ð1Þ Halothane displacement of bound AMA The dissociation constant of the complex between halot- hane and porcine odorant binding protein was determined using a competitive binding assay with the fluorescent lig- and AMA [21,22]. The approach has previously been employed for the determination of the dissociation con- stants for other ligands [23] shown crystallographically to occupy the internal cavity of the protein [24]. Briefly, por- cine odorant binding protein samples (1 lm), containing a fixed amount of AMA (1 lm), were incubated overnight at 4 °C in the presence of increasing concentrations of halot- hane in 20 mm Tris ⁄ HCl buffer, pH 7.8. The displacement of AMA from the porcine odorant binding protein was monitored as a progressive decrease in the fluorescence intensity at 480 nm (upon excitation at 380 nm) using an LS 50 Luminescence Spectrofluorometer (PerkinElmer, Milan, Italy). The resulting competition curve was analyzed as a two parameter hyperbolic decay using sigmaplot 5.0 (Cambridge Soft Corporation, Cambridge, MA, USA) and the EC 50 for halothane was determined. The true value of the dissociation constant of the halothane–porcine odorant binding protein complex was finally calculated using the following equation [23,25]: K d;true ¼ EC 50 Á 1 À 1 þ À 1 K d;AMA Á [AMA] ÁÁ ð2Þ which takes into account the concentration of AMA and the K d,AMA of the AMA–porcine odorant binding protein complex (1 lm). General anesthetic binding to a b-barrel protein J. S. Johansson et al. 578 FEBS Journal 272 (2005) 573–581 ª 2004 FEBS The stock solution of halothane contains the stabilizing agent thymol, which can also bind to porcine odorant bind- ing protein. However, control experiments showed that thy- mol alone, at the concentrations present in the experiments (< 0.0001% or < 5 pm), was unable to displace AMA from the porcine odorant binding protein. In addition, halothane (at concentrations less than 200 mm) does not directly quench AMA fluorescence. Isothermal titration calorimetry Isothermal titration calorimetry was performed using a MicroCal VP-ITC titration microcalorimeter (Northamp- ton, MA, USA) at 20 °C. Porcine odorant binding protein at a concentration of 87 lm in 130 mm NaCl, 20 mm sodium phosphate, pH 7.0, was placed in the 1.4 mL calori- meter cell, and anesthetic (5 mm in 130 mm NaCl, 20 mm sodium phosphate, pH 7.0) was added sequentially in 10 lL aliquots (for a total of 29 injections) at 5 min inter- vals. The heat of reaction per injection (microcalories per second) was determined by integration of the peak areas using the origin v5.0 software (http://www.microcal.com/). This software provides the best-fit values for the heat of binding (DH°), the stoichiometry of binding (n), and the association constant (K a ) from plots of the heat evolved per mol of anesthetic injected vs. the anesthetic ⁄ porcine odor- ant binding protein molar ratio [26]. The heats of dilution were determined in parallel experiments by injecting either 130 mm NaCl, 20 mm sodium phosphate, pH 7.0 into an 87 lm porcine odorant binding protein solution or 5 mm anesthetic (in 130 mm NaCl, 20 mm sodium phosphate, pH 7.0) into the 130 mm NaCl, 20 mm sodium phosphate, pH 7.0 buffer. These heats of dilution are subtracted from the corresponding porcine odorant binding protein-anes- thetic binding experiments prior to curve-fitting. The overall shape of the titration curve depends upon the c-value ([porcine odorant binding protein] ⁄ K d ) [26] and is rectangular for high c-values (> 500) and flat for low c-values (< 0.1). The results using Trp16 fluorescence quenching (Fig. 3) indicate that halothane binds to the por- cine odorant binding protein with a K d of 0.46 ± 0.10 mm. To achieve a c-value in the ideal range for isothermal titra- tion calorimetry (5–50) would therefore require prohibi- tively high concentrations of protein (on the order of 2.3– 23 mm). The porcine odorant binding protein concentration used was 87 lm (c ¼ 0.2), resulting in shallow hyperbolic titration curves for halothane and isoflurane. During curve- fitting, n was initially set as 1.0 and then increased in whole increments if the resulting chi square analysis indicated an improved description of the data. With this approach, deconvolution of the resulting isotherms only required the K a and DH° values to be minimized. Allowing all three var- iables to float simultaneously during the curve-fitting proce- dure may be associated with more variable results because of the potential for multiple minima [26]. Hydrogen exchange Porcine odorant binding protein (3–5 mg) was dissolved in 1mLof1m guanidinium chloride and 50 mm sodium phos- phate, pH 8.5, with 40 lL 3 HOH added (100 mCiÆmL )1 , ICN, Costa Mesa, CA, USA), and allowed to equilibrate overnight at 20 °C to permit complete exchange-in of tritium. The porcine odorant binding protein solutions were then passed through a PD-10 gel filtration column (Sigma Chem- ical Co, St Louis, MO, USA) to remove free 3 HOH, and to switch to the exchange-out buffer (50 mm sodium phosphate, pH 7.0). The protein fraction was collected and immediately placed in gas-tight Hamilton syringes prefilled with exchange-out buffer, with or without 4.0 mm halothane or 7.0 mm isoflurane. The syringe contents were mixed with microstir bars, and 100 lL aliquots were precipitated with 2 mL 20% trichloroacetic acid at regular intervals, immedi- ately filtered through Whatman (Hillsboro, OR, USA) GF ⁄ F filters, and washed with 8 mL 2% trichloroacetic acid. Filters were equilibrated with 10 mL fluor overnight and counted using liquid scintillation. Parallel aliquots allowed determin- ation of protein concentration using UV ⁄ Vis absorption spectroscopy at 280 nm. Protection factors for given hydrogens were determined from the exchange-out curves (Fig. 7). Assuming the hori- zontal equivalence of hydrogen exchange (the n -th hydro- gen to exchange is the same with and without anesthetic), protection factor ratios were estimated by dividing the time required for a given hydrogen to exchange under differing conditions (i.e. with and without anesthetic), and were determined for the last hydrogens in common for the two conditions. Protection factor ratios (Pfr) were averaged, and DDG values (the change in the free energy favoring the folded conformation) determined, using the relationship DDG ¼ –RTln(Pfr), where R is the gas constant, and T is the absolute tem- perature. Negative values reflect stabilization of the native folded porcine odorant binding protein conformation (slower exchange), and positive values indicate destabiliza- tion (faster exchange). Acknowledgements Work supported by NIH GM55876 (JSJ and RGE), and by MIUR, Progetto Giovani Ricercatori, Ricerca- tori Singoli (RR and SG). 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