Studies with these model systems have suggested that volatile general anesthetics prefer-entially bind to pre-existing appropriately sized Keywords anesthetic–protein interaction; haloth
Trang 1and isoflurane to a mammalian b-barrel protein
Jonas S Johansson1,2,4, Gavin A Manderson1, Roberto Ramoni5, Stefano Grolli5
and Roderic G Eckenhoff1,3
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
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 Kd 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.
Trang 2packing 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
Kd¼ 0.99 ± 0.06 mm with a Qmax 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 l M ) 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
Trang 3The 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, Ksv, is
22 ± 1 m)1) because it is comparable to the effect of
ha-lothane on free N-acetyl-tryptophanamide fluorescence
(Ksv¼ 25 ± 1 m)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 Kd of 0.46 ± 0.10 mm and a Qmaxof 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 theDH,
Kd, the free energy change associated with binding (DG), and the change in entropy associated with bind-ing (DS) were determined from the plots The Kdvalue 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.
Trang 4phan fluorescence quenching, supporting the validity of
the results Isoflurane binds to porcine odorant binding
protein with a Kd¼ 100 ± 10 lm The other
thermo-dynamic parameters underlying halothane and
isoflura-ne binding to the porciisoflura-ne 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 (R¼ 0.97) and gave an EC50 of 0.86 ± 0.24 mm The true dissociation constant (Kd, 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 · 105 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 Kd
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)1Æ K)1.
Anesthetic Kd(l M ) 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.
Trang 5calorimetry indicates that halothane binds to porcine
odorant binding protein with a Kd 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
1.3 ± 0.2 mm using 19F-NMR spectroscopy [29,30]
Using a competitive photoaffinity labeling approach,
Eckenhoff & Shuman [9] reported a Kd 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 Kdof 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 Kd value of 0.36 ± 0.03 mm using
19F-NMR spectroscopy and gas chromatography [32]
Finally, isoflurane was shown to bind to the
four-a-helix bundle (Aa2-L38M)2with a Kd¼ 140 ± 10 lm
using isothermal titration calorimetry [12] The affinity
of the interaction of isoflurane with porcine odorant
binding protein (Kd¼ 100 ± 10 lm) is therefore com-parable to the findings in the latter two studies For both anesthetics, the free energy of binding (DG) 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 (Aa2-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 Kd of 0.43 ± 0.12 mm This value is quite comparable to the
Kd 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 (Aa2)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)1at 25C, and increased the m-value (the slope of the unfolding transition) from 1.6 ± 0.2 to 2.0 ± 0.1 kcalÆmol)1Æ m)1 The latter effect is compatible with improved hydrophobic core packing [36], and supports anesthetic binding to the cavity in the core of (Aa2)2 Using hydrogen exchange [6], halothane was also shown to stabilize the folded conformation of the four-a-helix bundle (Aa2-L38M)2
by approximately )0.9 kcalÆmol)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 m M , d) and isoflurane (c, 7 m M , 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.
Trang 6Binding 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˚ resoludiffrac-tion [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 ± 0.1C 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 e278 of 12 200 m)1Æcm)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 (Qmax) at an infinite halothane concentration ([Halothane]) and the affinity of the anesthetic for its binding site (Kd) in the vicinity of the tryptophan residue From mass law con-siderations, it then follows that
Q¼ðQmax[Halothane]Þ
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
4C 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 EC50for 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]:
Kd;true¼ EC50 1
K d;AMA [AMA] ð2Þ which takes into account the concentration of AMA and the Kd,AMAof the AMA–porcine odorant binding protein complex (1 lm)
Trang 7The 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 20C 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 (Ka) 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]⁄ Kd) [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 Kdof 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 (c¼ 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
KaandDH 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
1 mL of 1 m guanidinium chloride and 50 mm sodium phos-phate, pH 8.5, with 40 lL 3HOH added (100 mCiÆmL)1, ICN, Costa Mesa, CA, USA), and allowed to equilibrate overnight at 20C 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 free3HOH, 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 concentrdetermin-ation 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
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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