Báo cáo khoa học: Binding of the volatile general anesthetics halothane and isoflurane to a mammalian b-barrel protein doc

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:
(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 (nstein-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 ( />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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