Binding and hydrolysis of soman by human serum albumin

Article abstract of DOI:10.1021/tx700339m

Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25°C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, ³¹P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 ± 3 M^-1 min^-1. MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and ³¹P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25°C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h ^-1, indicating that the adduct is quite stable (t_1/2 = 6.5 days). At pH 7.4 and 22°C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable (t_1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.

Full text of DOI:10.1021/tx700339m

Chem. Res. Toxicol. 2008, 21, 421–431
421
Binding and Hydrolysis of Soman by Human Serum Albumin
Bin Li,† Florian Nachon,‡,§ Marie-Thérèse Froment,‡,§ Laurent Verdier,#
Jean-Claude Debouzy,‡,4 Bernardo Brasme,‡, Emilie Gillon,‡,§ Lawrence M. Schopfer,†
Oksana Lockridge,† and Patrick Masson*,‡,§
UniVersity of Nebraska Medical Center, Eppley Institute, Omaha, Nebraska 68198-6805, Centre de Recherches
du SerVice de Santé des Armées, Département de Toxicologie, Unité d’Enzymologie, Unité de Biophysique,
SerVice de Biospectroscopie, BP 87, 38702 La Tronche cédex, France, 3, and Département Analyse Chimique,
Centre d’Etudes du Bouchet, BP 3, 91710 Vert-le-Petit, France
ReceiVed September 15, 2007
Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 °C.
Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the
aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass
spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 ( 3 M-1
min-1. MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin
was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent
docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for
the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 °C, measured as regaining
of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin)
by NMR, occurred at a rate of 0.0044 h-1, indicating that the adduct is quite stable (t1/2 ) 6.5 days). At
pH 7.4 and 22 °C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry,
was more stable (t1/2 ) 20 days). Though the concentration of albumin in plasma is very high (about 0.6
mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a
major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the
bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could
lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts
detected by mass spectrometry could be useful for the diagnosis of soman exposure.
sequence Arg-Tyr-Thr-Lys (9). Arg and Tyr were subsequently
identified as Arg 410 and Tyr 411, the key residues of the
esteratic site of HSA (4). Recently, DFP-inhibition of the aryl
acylamidase (AAA) activity of FAF-HSA confirmed that Tyr
411 is also the nucleophilic pole of the albumin AAA activity
(2). Finally, MALDI-TOF mass spectrometry provided direct
evidence that the OPs chlorpyrifos-oxon, dichlorvos, DFP, and
sarin bind covalently to human albumin at Tyr 411 (19).
1. Introduction
Albumin is an abundant protein that represents 50–60% of
the total protein in human plasma and body fluids. Its concentra-
tion in plasma is about 0.6 mM. Albumin displays both an
esterase activity (1) and an aryl acylamidase activity (2, 3). The
topology of the esterase/amidase active site of albumin has been
probed by site-directed mutagenesis (4) and X-ray structure
determination of several drug-albumin complexes (5). Tyr411
was determined to be the catalytic nucleophile in these reactions.
Albumin is also known to bind organophosphates (OPs1) and
carbamates (6–8) and to react with them (9–11). OPs and certain
carbamates are actually hydrolyzed by albumin through transient
phosphylation/alkylation of its active site (12–17). The residue
that reacts with OPs was proven to be a tyrosine (9-11, 14, 18).
DF32P-labeling of human albumin followed by peptide sequenc-
ing showed that the labeled tyrosine is in the tetrapeptide
In the present work, we investigated the reaction of human
albumin with soman. MALDI-TOF and quadrupole tandem MS/
MS mass spectrometry of the soman-albumin adduct showed
that Tyr411 was phosphonylated. Unlike soman adducts of
butyrylcholinesterase, the albumin-soman adduct did not age,
that is, lose its pinacolyl chain. Covalent docking simulations
and 31P NMR experiments indicated that there was no enan-
tiomeric preference of albumin for the stereoisomers of soman.
Kinetic parameters of albumin phosphonylation by soman and
subsequent dephosphonylation were determined.
* To whom correspondence should be addressed. Patrick Masson,
CRSSA, Département de Toxicologie, Unité d’Enzymologie, BP 87, 38702
La Tronche cédex, France. Tel: +33 (0)4 76 63 69 59. Fax: +33 (0)4 76
63 69 62. E-mail: pmasson@unmc.edu.
Our results could have application for the detection of soman
exposure in humans. Mass spectrometry could be used to detect
soman-albumin adducts. In addition, antibodies to the soman-
albumin adduct could be generated for use in a rapid antibody-
based assay of soman exposure. Lastly, mutants of albumin
could lead to a new class of scavengers against OP poisoning.
† Eppley Institute.
‡ Centre de Recherches du Service de Santé des Armées.
§ Département de Toxicologie, Unité d’Enzymologie.
# Centre d’Etudes du Bouchet.
4 Unité de Biophysique.
Service de Biospectroscopie.
1 Abbreviations: AAA, aryl acylamidase; AChE, acetylcholinesterase;
BuChE, butyrylcholinesterase; DFP, diisopropylfluorophosphate; FAF-HSA,
fatty acid-free human serum albumin; HSA, human serum albumin; MP,
methylphosphonate; PMP, pinacolyl methylphosphonate; o-NTFNAC, N-(o-
nitrophenyl)trifluoroacetamide; OP, organophosphorus ester.
2. Experimental Procedures
2.1. Chemicals. Fatty acid-free human albumin (FAF-HSA) and
porcine pepsin were from Sigma Chemical Co. (Saint Quentin
10.1021/tx700339m CCC: $40.75
2008 American Chemical Society
Published on Web 12/29/2007

422 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Li et al.
Fallavier, France). o-NTFNAC was a gift from Dr. Sultan Darvesh
(Dalhousie University, Halifax, Canada). Racemic soman (pinacolyl
methylfluorophosphate) dissolved in isopropanol was from CEB
(Vert-le-Petit, France). All other chemicals were of biochemical
grade.
The final enhanced product ion scan was the average of 105 scans.
Ions were identified by manual sequencing.
2.4. 31P NMR Spectroscopy. 31P NMR spectra for the reaction
between albumin (0.78 mM or 1.3 mM) and soman (1.3 mM) in
60 mM Tris/HCl buffer at pH 8.0 were recorded at 27 °C on a
Bruker AM400NB spectrometer operating at 162 MHz. The spectra
were acquired using successive 8 acquisition blocks of 5,000
accumulated scans (acquisition time: 3 h/spectrum) using 30 kHz
spectral width, 32,000 acquisition points, and a composite pulse
proton decoupling (CPD mode). The external reference for chemical
shifts was 85% (w/v) H3PO4. Spectra were compared to the
spectrum of soman in buffer and the spectrum of methyl pinacolyl
phosphonate (MPP) in the presence and absence of albumin. MPP
was made by the complete hydrolysis of soman in 5 N sodium
hydroxide.
The concentration of racemic soman in the stock solution was
determined by programmed temperature gas chromatography after
hydrolysis into methylpinacolyl phosphonic acid and derivatization
with pentafluorobenzyl bromide (20). It was 4.73 mg/mL (26 mM).
31P NMR spectra of 2 mM soman in deuterated dimethylsulfoxide
were recorded at 27 °C on a Bruker spectrometer (AM400, 9.4 T).
The spectra showed that the four stereoisomers are present in nearly
equimolar amounts. Soman has two chiral centers. The absolute
configuration of the four diastereoisomers is known. PS, PR, CS,
and CR correspond to the old notation P- P+, C+, and C-,
respectively. Thus, isomers PSCS ) P-C+, PSCR ) P-C-, PRCR
) P+C-, and PRCS ) P+C+. Stereoisomers PSCS and PSCR are
the most active toward the biological target acetylcholinesterase
(21).
2.5. Kinetic Studies of the Reaction of Albumin with
Soman. 2.5.1. Residual AAA Activity. FAF-HSA (780 µM) was
incubated with 120 to 1300 µM concentrations of racemic soman
in 60 mM Tris/HCl buffer at pH 8.0 at 25 °C. The time dependence
of the inhibition of albumin by soman was monitored by following
the residual AAA activity of albumin. Measurements of the AAA
residual activity were performed on 100 µL aliquots of reaction
mixture, using the sampling method (23).
2.5.2. Monitoring the Release of Fluoride from Soman. The
release of fluoride, that is, the leaving group of soman, upon the
reaction of soman with albumin and spontaneous hydrolysis was
monitored by ionometry using a thermostatted ionometer (Radi-
ometer IONcheck 45) equipped with an ion-selective electrode for
fluoride (ISE 301F). FAF-HSA (780 µM) was incubated with 520,
780, and 1300 µM concentrations of racemic soman in 60 mM
Tris/HCl buffer at pH 8.0 at 25 °C, and the concentration of released
fluoride was assayed. The spontaneous hydrolysis of soman was
determined under the same conditions.
2.2. Aryl Acylamidase Enzymatic Assay of Albumin. The
AAA activity of albumin was assayed with o-NTFNAC (2 mM)
as the substrate in 60 mM Tris/HCl buffer at pH 8.0 at 25 °C. A
60 mM stock solution of o-NTFNAC was prepared in 50% water/
acetonitrile (v/v). Because isopropanol is the solvent for soman,
isopropanol was also added to the buffer in the control assay for
the AAA activity of albumin in the absence of soman. The final
concentration of acetonitrile in the assay was 3.3% and that of
isopropanol was 2%. The release of the phenolic product (o-
nitroaniline) was monitored for 5 min at 430 nm (ε ) 3954 M-1
cm-1) according to Darvesh et al. (22). Because of the low
enzymatic activity of albumin with o-NTFNAC (3), high concentra-
tions of FAF-HSA were used in assays (0.075 mM final). Measured
rates were corrected for spontaneous hydrolysis of o-NTFNAC.
2.3. Mass Spectrometry (MALDI-TOF and Quadrupole
MS/MS) of the Soman-Albumin Adduct from Human Plasma.
One hundred microliters of human plasma was mixed with 2.3 µL
of stock soman (26 mM in isopropanol) to give a reaction mixture
containing 600 µM soman (in 2.3% isopropanol). The mixture was
incubated at room temperature for 3–7 days before processing. No
buffer was added to the plasma during this step. The pH of a 10
µL aliquot was reduced to pH 2.3 by adding 10 µL of 1%
trifluoroacetic acid. Proteins were digested with 0.5 µg of pepsin
for 2 h at 37 °C. At pH 2.3, selective proteolysis at the C-terminal
side of the leucine and phenylalanine residues is expected. There
was no need to denature the proteins or to reduce and alkylate the
disulfide bonds because the peptides of interest were released
without these added steps. Peptides were separated on a C18 reverse
phase column on a Waters 625 LC system with a 40 min gradient
starting with 85% buffer A (0.1% trifluoroacetic acid in water),
15% buffer B (acetonitrile containing 0.07% trifluoroacetic acid),
and ending with 65% buffer A and 35% buffer B. One milliliter
fractions were reduced in volume to 200 µL in a vacuum centrifuge,
and 1 µL was analyzed by MALDI-TOF with a 2,5-dihydroxy-
benzoic acid matrix. Mass spectra were acquired with the Applied
Biosystems Voyager DE-PRO MALDI-TOF mass spectrometer in
linear positive ion mode. The spectrometer was calibrated using
standard peptides (Applied Biosystems Mixture 1). A control plasma
sample was identically treated, except that it was incubated with
3.1% isopropanol rather than with soman.
2.5.3. Kinetic 31P NMR Spectroscopy. To follow the phos-
phonylation reaction, 1.33 mM FAF-HSA was incubated with 1.35
mM soman in 60 mM Tris/HCl buffer at pH 8.0 at 25 °C in the
presence of deuterated DMSO (10% v/v final). The successive
spectra were recorded every 20 min (800 scans) over 4 h.
Concentrations of the different species were plotted as a function
of time. To follow the reactivation of phosphonylated albumin, 1.3
mM FAF-HSA was reacted with one equivalent of racemic soman
in 60 mM Tris/HCl buffer at pH 8.0. Then, the phosphonylated
albumin solution was maintained at 25 °C, and spectra were
recorded every 2 h over 60 h. Kinetic constants for the spontaneous
hydrolysis of soman, phosphonylation of albumin, and reactivation
of albumin (dephosphonylation) were determined by fitting data
against the numeric solution of differential equations that describe
Scheme 1, using Mathematica 5 (Wolfram Research).
2.5.4. Kinetic Mass Spectrometry. The stability of the covalent
soman-albumin adduct was measured on a MALDI-TOF-TOF
4800 mass spectrometer (Applied Biosystems). Human albumin was
labeled by incubating a 1 mg/mL solution (15 µM) in 10 mM Tris/
HCl at pH 8.0 with 200 µM soman for 24 h at room temperature.
After this time, the concentration of active soman was negligible
as determined by measuring the inhibition of human butyrylcho-
linesterase. The albumin solution was diluted to 0.1 mg/mL with
0.01% sodium azide in water and the pH adjusted to 7.4. The 200
µL solution was stored at 22 °C. Every 2 or 3 days, a 10 µL aliquot
was acidified with 10 µL of 1% trifluoroacetic acid and digested
with 2 µL of 1 mg/mL porcine pepsin (dissolved in 10 mM HCl).
The digest was incubated at 37 °C for 1–4 h, and then 0.5 µL was
spotted on a MALDI target plate. The dry spot was overlaid with
0.5 µL of 10 mg/mL R-cyano-4-hydroxycinnamic acid in 50%
acetonitrile and 0.1% trifluoroacetic acid. MS scans in reflector
mode were acquired at 3000 V by summing 500 laser shots per
scan. The percent label on Tyr 411 was calculated from cluster
areas. Peptide masses acquired in reflector mode are monoisotopic,
which makes their mass about 1 amu lower than the average mass
acquired in linear mode.
MS/MS spectra were acquired on a Q-Trap 2000 triple quadru-
pole linear ion trap mass spectrometer (Applied Biosystems, MDS
Sciex, Foster City, CA) with a nano electrospray ionization source.
Samples were infused into the mass spectrometer at 0.35 µL/min
via a fused silica emitter (360 µm o.d., 20 µm i.d., 15 µm taper,
New Objective, Woburn, MA) using a Harvard syringe pump to
drive a 25 µL Hamilton syringe equipped with an inline 0.25 µm
filter. Samples were sprayed with 50% acetonitrile and 0.1% formic
acid. Mass spectra were calibrated using fragment ions generated
from collision-induced dissociation of Glu fibrinopeptide B (Sigma).
Enhanced product ion scans were obtained with a collision energy
of 50 ( 5 V and a pure nitrogen gas pressure of 4 × 10-5 Torr.

Reaction of Albumin with Soman
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 423
2.6. Molecular Modeling. 2.6.1. Noncovalent Docking. Dock-
ing calculations were carried out using two programs: (1) Autodock,
version 3.0.5, with the Lamarckian genetic algorithm (LGA (24))
and (2) Gold, version 3.1 (CCDC Software, Ltd). Docking with
Autodock employed the following procedure. The molecular models
of the four diastereoisomers of soman were built and minimized
with the MM2 force field of Chem3D (Cambridge soft.). The
structure of HSA was prepared from the crystal structure of its
complex with indoxyl sulfate (pdb code 2bxh) or warfarin (pdb
code 2bxd) to take into account the heterogeneity of conformation
of Val433. Molecules of water and ligands were removed from
the model. Soman and HSA were further prepared using Autodock
Tools 1.4 (25). The 3D affinity grid box was designed to include
the full pocket near Tyr411. The number of grid points in the x-,
y-, and z-axes was 60, 60, and 60 with grid points separated by
0.375 Å. Docking calculations were set to 100 runs. At the end of
the calculation, Autodock performed cluster analysis. Docking
solutions with ligand all-atom root-mean-square deviation (rmsd)
within 1.0 Å of each other were clustered together and ranked by
the lowest energy representative. The lowest-energy solution was
accepted as the one most representative of the soman-HSA
complex. Docking with Gold employed the following procedure.
Protein structures were protonated and minimized by conjugate
gradients using Gromacs 3.3 (26, 27) and the parameter set 53A6
(28). Ligands were built into the Sybyl 7.2 package (Tripos Inc.).
The Mopac-PM3 (29) semiempirical method was chosen for
geometry optimization and calculation of atomic charges. The cavity
was defined as residues having atoms up to 15 Å from the Tyr411
hydroxyl group. We used long search settings for the Gold GA: 40
docking with parameters corresponding to the default 200% search
efficiency settings. The final selection of conformers was based on
the two scoring functions, Goldscore (30) and Chemscore (31).
2.6.2. Covalent Docking. This simulation was carried out using
Gold, version 3.1 with GA settings similar to those for noncovalent
docking. We used the same proteins as before, and the results were
scored using both Goldscore and Chemscore functions. The fluorine
atom from each of the four soman stereoisomers was replaced by
a hydroxyl group, and the phosphorus stereochemistry was inverted
to get the right configuration when the phosphorus atom binds to
the Tyr411 Oγ. The highest score isomers were chosen as the ones
giving the lowest ∆Hf when estimating the energy of the system
with Mopac-PM3. To reduce the number of atoms in our system,
we removed residues having all atoms further than 5 Å from any
soman atoms.
Figure 1. MALDI-TOF mass spectra of peptic peptides from the
soman-albumin adduct. Panels A and C, control human plasma
digested with pepsin and fractionated by HPLC, but not treated with
soman. Panels B and D, human plasma treated with soman before
digestion with pepsin and fractionation by HPLC. Soman-labeled
albumin peptides of 1880 and 1993 amu include 162 amu from soman.
sensitivity. Tyr411 is present in both peptides (indicated by the
star), supporting the proposal that soman binds covalently to
Tyr411 of human albumin in plasma. Peptides with an added
mass of 162 from soman, but not with an added mass of 78
were found. This indicates that the soman adduct does not lose
the pinacolyl group when soman is bound to albumin. Thus,
the soman-albumin adduct does not age.
An additional experiment was performed to demonstrate by
a second mass spectrometry method that soman was covalently
bound to Tyr 411 of human albumin. HPLC fraction 21
containing the soman-labeled albumin peptide at 1880 m/z was
infused into the Q-Trap, quadrupole mass spectrometer. The
enhanced mass spectrum showed a peak at 940.7 m/z, which is
consistent with the doubly charged form of the 1880 peptide.
This peptide was subjected to collision-induced dissociation.
The resulting enhanced product ion spectrum yielded amino acid
sequence information consistent with the sequence VRYT-
KKVPQVSTPTL, with soman covalently bound to tyrosine (see
Figure 2).
3. Results
3.1. Mass Spectrometry to Identify the Covalent Bind-
ing of Soman to Tyrosine 411. Peptic digests of soman-labeled
and control human plasma were separated by HPLC. Matched
fractions from the two preparations were examined by MALDI-
TOFmassspectrometry.MALDI-TOFsignalsforsoman-albumin
and control albumin peptides are shown in Figure 1. A peptide
with mass 1880 amu is present in fraction 21 from the soman-
labeled preparation (Figure 1, panel B). This mass is consistent
with the peptide (L)VRY*TKKVPQVSTPTL(V) (1718 amu,
from residues 409–423 of the mature albumin sequence) with
an added mass of 162 amu from soman. A peptide with mass
1993 amu is present in fraction 23 from the soman-labeled
preparation (Figure 1, panel D). This mass is consistent with
the peptide (L)LVRY*TKKVPQVSTPTL(V) (1831 amu, from
residues 408–423 of the mature albumin sequence, representing
a missed peptic cleavage) with an added mass of 162 amu from
soman. The parallel fractions from control human plasma do
not have peptides of these masses (Figure 1, panels A and C).
However, other prominent peaks are present at identical masses
in both the control and soman-labeled spectra (compare panels
A to B, and C to D), confirming that the fractions are matched
with regard to HPLC elution and relative mass spectral
The prominent peak at 898.6 m/z is a doubly charged ion, 42
mass units less than the parent ion at 940.7 m/z. This is
consistent with the loss of the pinacolyl group (84 mass units)
from the doubly charged parent to form a doubly charged
product retaining methylphosphonic acid. A comparable 42 amu
loss from the parent ion was seen in the enhanced product ion
spectrum of the LVRYTKKVPQVSTPTL peptide (data not
shown). Preferential loss of the pinacolyl group seems to be a
characteristic feature of soman-labeled peptides when they are
fragmented in the mass spectrometer, as the soman-labeled
butyrylcholinesterase peptide also lost the pinacolyl group during
the collision process (32, 33).
The 940.7 m/z parent ion in Figure 2 included the pinacolyl
group of soman. This is a significant point because it demon-
strates that soman had not lost the pinacolyl group while bound
to intact albumin protein or during digestion and HPLC
separation. The pinacolyl group only dissociated when the
peptide was subjected to collision-induced dissociation in the

424 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Li et al.
Figure 2. Product ion spectrum of the soman-labeled human albumin peptic peptide. The doubly charged parent ion at 940.7 m/z yielded fragments
consistent with the sequence VRY*TKKVPQVSTPTL, where * indicates soman covalently bound to tyrosine 411. The accession number for
human albumin is GI:28592. In this resource, Tyr 411 is given as Tyr 435 because the numbering begins with the 24-amino acid signal peptide.
mass spectrometer. The presence of the pinacolyl group in the
parent ion demonstrates that soman did not age when bound to
albumin.
At least four separate ion series from the VRYT-
KKVPQVSTPTL peptide could be extracted from the spectrum
in Figure 2. Only the y and b ion series are indicated in the
Figure for the sake of clarity.
The peptide [VRY*T]KKV[PQ] was seen as part of the b-ion
series. The masses of all five observed fragments were consistent
with the indicated amino acid sequence plus 78 amu for the
added mass of a methyl phosphonic acid. The only residue in
this sequence capable of forming a methyl phosphonic acid
adduct is the tyrosine, establishing this as the modified amino
acid in the peptide. The presence of a 78 amu added mass on
the observed fragments, rather than 162 amu, indicates that the
pinacolyl group is dissociated from the peptide, in the collision
chamber, prior to the onset of backbone fragmentation.
The peptide [PQ][VS]TP[TL] was seen as part of the y-ion
series. The masses of the observed fragments were consistent
with only the amino acids, unencumbered with any adduct mass.
Thus, the potentially phosphonylated serine was not labeled,
supporting the assignment of the tyrosine as the modified amino
acid.
and DFP (10), a low soman phosphonylation rate was expected.
Therefore, the phosphonylation of albumin by soman was carried
out under second-order conditions (3, 34), that is, the initial
concentrations of soman, [S]0, and albumin, [A]0, were not very
different. As a result, the concentrations of the uncomplexed
forms of both reactants varied with time during the reaction.
Albumin (0.78 mM) was phosphonylated by sub and super-
stoichiometric amounts of racemic soman (0.32 to 1.3 mM) that
inhibited albumin’s aryl acylamidase activity. Reaction was
monitored by following albumin’s aryl acylamidase (AAA)
activity. Inspection of the inhibition time courses (Figure 3)
indicated that less albumin was inhibited by soman than would
have been expected from a stoichiometric reaction with the total
amount of soman in the reaction. For example, in the presence
of a 2-fold excess of soman, 100% inactivation of the AAA
activity of albumin was never reached. The titration plot (not
shown) for 0.78 mM albumin incubated with different concen-
trations of soman (0.38, 0.65, 0.81, and 1.30 mM) confirmed
this observation. This suggests either that a large part of soman
spontaneously hydrolyzed in the nucleophilic Tris/HCl buffer
at pH 8.0 or that albumin reacted stereoselectively with specific
stereoisomers of soman.
3.2.2. Determination of Fluoride Released during the
Reaction of Soman with Albumin. In an effort to decide
whether the substoichiometric reaction of soman with albumin
was due to a competing spontaneous hydrolysis of soman or to
a stereoselective reaction of albumin with soman, ionometric
measurements of fluoride released from soman in the presence
and absence of albumin were performed (Figure 4). It was found
that spontaneous hydrolysis of soman in 60 mM Tris/HCl buffer
at pH 8.0 at 25 °C was high. The rate constant for hydrolysis
The complete amino acid sequence of peptide VRY*TKKV
PQVSTPTL was represented by these two ions. These results
confirm the conclusion that soman covalently binds to Tyr 411
of human albumin.
3.2. Kinetics for the Phosphonylation of Albumin by
Soman and for the Reactivation of Phosphonylated Albumin.
3.2.1. Phosphonylation of Albumin by Soman. Because of
the low reactivity of albumin with esters, aryl amides (1, 3),

Reaction of Albumin with Soman
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 425
8.0 is faster in the presence of albumin than in buffer alone.
However, dephosphonylation of the Tyr411 adduct is far too slow
(0.0044 ( 0.0008 h-1; see section 3.2.5) to account for this
albumin-dependent hydrolysis of soman. Thus, this suggests that
this catalysis takes place at a different site. However, only one
soman adduct on albumin was detected by MALDI-TOF. This
finding is consistent with reports on the phosphonylation of bovine
serum albumin by FP-biotin (18) and the phosphorylation of human
serum albumin by diisopropylfluorophosphate (9). Therefore, it may
be hypothesized that albumin-mediated hydrolysis of soman occurs
via a mechanism that does not involve a covalent intermediate.
Nevertheless, this catalytic effect is extremely weak, about 2 times
higher than spontaneous hydrolysis, and may be tentatively
regarded as a nonenzymatic, physicochemical interfacial reaction.
However, we cannot rule out the existence of a second active center.
3.2.3. Is There Enantioselectivity in the Reaction of
Albumin with Soman? Racemic soman is an equimolar mixture
of four stereoisomers. Though experiments on the release of
fluoride argue against the enantioselectivity of albumin for certain
soman isomers, the possibility that albumin’s reaction with soman
is stereoselective was tested directly using 31P NMR. Each of the
Figure 3. Progressive inhibition of the AAA activity of FAF-HSA ([E]
) 0.78 mM) by different concentrations of racemic soman (b, 0.324
mM; O, 0.520 mM; π, 0.780 mM) in 60 mM Tris/HCl buffer at pH
8.0 at 25 °C. A plot of % residual AAA activity of albumin vs time.
The progress curves reach a plateau after consumption of the reactive
soman.
intact isomers of soman (Ps- and Pr-) generates a doublet in 31
P
NMR (Figure 5, panel A). Soman completely hydrolyzed in 5 N
sodium hydroxide gives methylphosphonate (MP) (Figure 5, panel
B); the hydrolysis of soman at pH 8.0 gives pinacolyl methylphos-
phonate PMP (Figure 5, panel D) (35).
The reaction between 1.3 mM racemic soman and 0.78 mM
albumin was monitored over 24 h by recording 31P NMR
spectra. Initial spectra showed the progressive disappearance
of the intact soman peaks with the concomitant appearance of
three broader resonance peaks, of 80 Hz line width (Figure 5,
panel C). After 2 h of incubation, signals for intact soman had
vanished. At this point in the reaction, the spectrum of the
products was compared to a spectrum of methylpinacolyl
phosphonate (MPP). MPP, created under mild conditions of
hydrolysis, showed a peak at 23.74 ppm. That peak was
unaffected by the presence of albumin (Figure 5, panel D). The
most prominent peak in the soman/albumin reaction mixture
(the 23.74 ppm peak, f, in Figure 5, panel C) can therefore
be assigned to MPP. For longer reaction times (up to 24 h),
the MPP peak increased at the expense of the other two peaks
in Figure 5, panel C. This strongly suggests that peaks e and d
(at 25.59 and 27.56 ppm) correspond to the Ps- and Pr-
covalent adducts of soman bound to albumin and that there is
a slow, spontaneous release of MPP from the of phosphonlyated
albumin. Such a release can be described as reactivation.
Figure 4. Time course of fluoride ions released from soman, in the
presence and absence of FAF-HSA. Soman ([S]0 ) bO, 0.52, 24,
0.78, 90, 1.3 mM) was incubated with albumin ([E] ) 0.78 mM) (filled
symbols), or soman was incubated by itself through spontaneous
hydrolysis (open symbols on continuous curves). Both reactions were
in 60 mM Tris/HCl at pH 8.0.
The fact that after 2 h of reaction between albumin and soman,
adduct peaks d and e have the same intensity indicates that
albumin reacted with the same probability with both the Ps-
and Pr- soman enantiomers. Thus, there is no enantiomeric
preference of albumin for the soman isomers.
3.2.4. Effect of MPP on Phosphonylation of Albumin
by Soman. Because a large part of soman was hydrolyzed into
MPP in Tris buffer at pH 8.0 during the time course of the
phosphonylation reaction (Figure 5, panel C), we tested the
hypothesis that MPP could compete with soman for noncovalent
binding to the Tyr411 site and thereby slow down the phos-
phonylation reaction. We found that phosphonylation of (0.75
mM) albumin by soman (1.3 mM) in the presence of 1.3 mM
MPP caused no change in the progressive inhibition of the AAA
activity of albumin. Thus, MPP does not compete with soman.
of soman (kh0) was 0.00342 ( 0.00007 min-1. In the presence
of albumin, the rate of fluoride release was significantly higher.
Semiquantitative analysis of progress curves shows not only
that spontaneous hydrolysis of soman occurred at rates com-
parable to the phosphonylation rates of albumin but also that
albumin itself provides a physicochemical environment that
enhances the spontaneous hydrolysis of soman. The following
is an example of this semiquantitative analysis.
The filled squares in Figure 4 describe the release of fluoride
after mixing 1.3 mM soman with 0.78 mM FAF-HSA. After 100
min of incubation, 0.3 mM albumin was inhibited (see Figure 3)
so that the phosphonylation of albumin accounts for 0.3 mM of
released fluoride. Over this time period, about 0.9 mM fluoride
was released. Spontaneous hydrolysis of 1.3 mM soman (0 in
Figure 4) accounts for about 0.35 mM of released fluoride. This
leaves about 0.25 mM fluoride unaccounted for. Thus, the release
of this extra fluoride suggests that the hydrolysis of soman at pH
3.2.5. Spontaneous Reactivation of Soman-Inhibited
Albumin Monitored by 31P NMR and MALDI-TOF. Fluoride
has been shown to promote dephosphonylation of albumin (36).

426 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Li et al.
Figure 6. Reactivation kinetics by 31P NMR. Each point represents
the integrated area of the NMR peaks (d + e). The areas, in turn,
correspond to the relative amounts of adduct. The line is from a fit of
the data to eq 1.
Scheme 1
0.0044 ( 0.0008 h-1 ) 0.000073 ( 0.000013 min-1 ) 0.74
(0.13week-1.Thusthehalf-timefordecayofthesoman-albumin
adduct at pH 8.0 and 25 °C is about 1 week.
Figure 5. 31P NMR spectra of soman in the presence and absence of
albumin. Panel A shows 2 mM racemic soman in dimethylsulfoxide-
d6. The four stereoisomers are indistinguishable. The four resonance
peaks correspond to the two doublets of the two pairs of diastereo-
isomers (35). Panel B shows methyl phosphonate (MP) (a product of
soman hydrolysis in 5 N NaOH). Panel C shows 1.3 mM racemic soman
after reaction (2 h) with 0.78 mM albumin, in 60 mM Tris/HCl at pH
8; d and e correspond to the PR and PS adducts of albumin, and f is the
product of the spontaneous hydrolysis of soman and methyl pinacolyl
phosphonate (MPP). Panel D shows 1.3 mM methyl pinacolyl phos-
phonate (MPP) in the presence of 1.3 mM albumin. The observed peak
is that of uncomplexed MPP (35).
The stability of the soman-albumin adduct was also mea-
sured by mass spectrometry. Soman-labeled albumin was
digested with pepsin to release labeled and unlabeled Tyr411
peptides after various periods of incubation. The peptide masses
and the area of each peak were determined by MALDI-TOF in
reflector mode. Figure 7A shows a scan, after 24 h, where the
highest peaks at 1992.1 and 1879.0 have soman bound to Tyr411
in peptides LVRYTKKVPQVSTPTL and VRYTKKVPQ-
VSTPTL. At a later time point after the adduct had been
incubated at pH 7.4 and 22 °C for 768 h, the unlabeled peptides
at 1717.0 and 1830.1 have a higher intensity than the labeled
peptides (Figure 7B). The % label on Tyr411 was calculated
from cluster areas and plotted in Figure 7C as a function of
time. The half-life for decay of the soman-albumin adduct at
pH 7.4 and 22 °C was 20 days. The time course in Figure 7C
is first order for nearly two half-lives, supporting the proposal
that reactivation is a simple first order process. The difference
in rate constants obtained from the mass spectrometry and the
NMR experiments is consistent with expectation for the
difference in pH.
However, because of the low nucleophilicity of fluoride ion, it
seems unlikely that reactivation was mediated by the fluoride ions
released during the phosphonylation step ([F-] e 1.3 mM for the
highest soman concentration used). Following the reactivation rate
by measuring the recovery of the AAA activity was not accurate;
therefore, 31P NMR spectral kinetics was used instead.
FAF-HSA (1.3 mM) was inhibited by 1.3 mM soman (10%
isopropanol, final). We followed the decrease of the integrated
area of the NMR peaks corresponding to the soman-albumin
adduct (peaks d + e in Figure 5, panel C) from 10 to 60 h
(Figure 6). Because no soman was left after 10 h of incubation,
because of its spontaneous hydrolysis (S f S′′ + F-) and to
its reaction with albumin (see Scheme 1), the change in the
concentration of the soman-albumin conjugate (A-S′) was due
only to reactivation (kr). It was assumed that the process was
uncompromised and followed pseudo-first-order kinetics (eq 1):
3.2.6. Progressive Binding of Soman to Tyr411 of
Albumin Monitored by 31P NMR Kinetics. The complete
reaction path of albumin (A) with soman (S) can be depicted
by Scheme 1, which includes the competing spontaneous
hydrolysis of soman, second-order phosphonylation of albumin,
followed by hydrolytic dephosphonylation (reactivation of the
AAA activity of albumin).
[A - S′]t ) [A - S′]10h · e-k ·(t-10)
(1)
r
The relative intensity at t ) 10 h was 0.54 ( 0.01; this is
equivalent to 0.700 ( 0.015 mM of conjugate. Fitting the data
from Figure 6 to eq 1 provided the reactivation constant, kr )
In Scheme 1, kp is the rate constant of phosphonylation, kr is
the rate constant of dephosphonylation (spontaneous reactivation
of phosphonylated albumin A-S′), and kh is the rate of the

Reaction of Albumin with Soman
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 427
Figure 8. Time course of the reaction between 1.3 mM soman and 1.3
mM albumin monitored by 31P NMR: b, soman; O, adduct; 2, MPP.
The points were measured data. The lines are from numerical fitting
of the data to eq 2.
same order of magnitude, it is not possible to make assumptions
allowing a simple integration of the system. However, a
numerical solution of the system can be obtained using
mathematical software such as Mathematica.
Following the residual AAA activity of albumin allows only
the measurement of d[A]/dt ) -d[AS′]/dt, while measuring the
release of F- gives only d[F–]/dt ) d[S]/dt. However, following
the reaction kinetics by 31P NMR gives d[S]/dt ) -d[F-]/dt,
d[AS′]/dt ) -d[A]/dt, and d[S′′]/dt in one single experiment
(Figure 8). kr was measured separately under the same experi-
mental conditions (kr ) 0.0044 ( 0.0008 h-1; see section 3.2.4
and Figure 6).
Fitting the numerical solution of the differential equation
system against 31P NMR data gave kp ) 15 ( 3 M-1 min-1
and kh ) 0.0076 ( 0.005 min-1. Thus, the rate constant for
spontaneous hydrolysis of soman in the presence of albumin
(kh) is about 2 times the rate for the spontaneous hydrolysis of
soman in the absence of albumin (kh0 ) 0.00342 ( 0.00007
min-1, from section 3.2.2). This value for kh is in agreement
with the estimate for kh made from the measurement of [F-]
release, as described in section 3.2.2.
Figure 7. Stability of the soman-albumin adduct calculated from mass
spectrometer data. Panels A and B are MALDI-TOF spectra of pepsin-
digested, soman-labeled human albumin after 24 and 768 h incubation,
respectively, at pH 7.4 and 22 °C. Panel C is a plot of % Tyr411 labeled
by soman, as a function of time of incubation of the soman-albumin
adduct. The soman-albumin adduct decays with a half-life of 20 days.
3.3. Molecular Modeling. The crystal structure of HSA with
numerous ligands has been described (5). A comparison between
these structures shows that Val433 located in the pocket
containing Tyr411 might adopt two different conformations
depending on the occupant of the pocket. These alternate
conformations change the diameter of the pocket and may affect
the binding of soman in the pocket. Therefore, we docked soman
with both representative crystal structures: 2bxd displaying a
narrower pocket in absence of ligand and 2bxh displaying a
wider pocket that develops when indoxyl sulfate is bound. The
four diastereoisomers of soman were docked in both structures.
3.3.1. Noncovalent Docking by Autodock. Key values from
the noncovalent docking results are summarized in Table 1. All
four stereoisomers of soman bind better to HSA when the pocket
is narrowed by the conformational change of Val433 (2bxd).
A narrower pocket provides more stabilizing van der Waals
interactions between soman and the residues of the pocket.
Therefore, it is expected that the actual conformation of HSA
when in complex with soman is similar to that of 2bxd. For
2bxd, the estimated affinity constants for all four stereoisomers
spontaneous hydrolysis of soman. F- is the fluoride leaving
group and S′′ the hydrolysis product, methyl pinacolyl phos-
phonate (MPP). It follows then that the concentration of all
species as a function of time is described by a system of
differential equations (eq 2) as sollows:
d[A]
dt
d[S]
dt
d[AS′]
dt
d[S′′]
dt
d[F-]
dt
) -kp · [A][S] + kr · [AS′]
) -kp · [A][S] + kh · [S]
d[A]
dt
) -
(2)
) -kr · [AS′] + kh · [S]
d[S]
) -
dt
Because the concentration of soman and albumin are of the
same order of magnitude and because the rates of spontaneous
hydrolysis of soman and phosphonylation of albumin are of the

428 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Li et al.
Table 1. Non Covalent Docking of Soman in the Tyr411
Pocket of HSA Performed by Autodock
difference between the structures; therfore, we describe here
only the results obtained with the minimized 2bxh protein.
soman
isomer
HSA pdb
structure
docked energy
estimated Kd
(µM)
We docked the four enantiomers of soman with the minimized
2bxh protein using Gold/Goldscore. The best conformers
obtained were similar to the ones obtained by Autodock. The
pinacolyl moiety is located at essentially the same place obtained
by Autodock (Figure 9); the difference is only a small translation
of Tyr411 in the direction of the phosphorus atom (P-OHY411
distances vary from 6.37 to 6.67 Å). As described for Autodock,
the rotation of the P (O, F, Me) moiety is relatively free; this is
not surprising because of the lack of specific interactions of
these atoms with residues of the active site pocket. Again, the
phosphorus atom is at a position that does not permit easy
phosphonylation of Tyr411. The scores obtained were in the
same range for all enantiomers: PSCS ) 39.93, PSCR ) 40.09,
PRCR ) 41.28, and PRCS ) 38.16. We obtained very similar
results in terms of conformers and energies when we used
Autodock or Gold with either 2bxh or 2bxd, which clearly shows
the robustness of the models in Figure 9.
(kcal.mol-1
)
PSCR
PSCS
PRCR
PRCS
2bxd
2bxh
2bxd
2bxh
2bxd
2bxh
2bxd
2bxh
-5.69
-5.56
-5.76
-5.48
-5.69
-5.50
-5.73
-5.42
148
167
115
206
147
176
128
226
are virtually identical: Kd ) 134 ( 16 µM (Table 1). The pocket
seems to be large enough to accommodate all enantiomers of
soman (Figure 9). Soman binds with the pinacolyl group stacked
against the disulfide bridge formed by Cys392-Cys438. The
pinacolyl group makes many hydrophobic contacts with the side
chains of Val433, Ile388, Ala449, Phe395, Phe403 and the main
chain of Gly434. The binding conformation of soman is largely
dictated by its carbon stereocenter. Indeed, PSCS and PRCS
display the same binding conformation (rmsd ) 0.07 Å), except
that the positions of the fluoride and phosphonate oxygen are
inverted as a result of the difference in stereochemistry at the
phosphorus. The same applies to the PSCR and PRCR stereo-
isomers (rmsd ) 0.13 Å). This inversion between the fluoride
and phosphonate oxygen does not cost much energy of binding
because of the lack of specific interactions between these atoms
and residues of the pocket. The phosphorus atom is separated
from the hydroxyl of Tyr411 by a distance ranging from 6.80
Å (CR enantiomers) to 7.24 Å (CS enantiomers). This distance
is not suitable for a nucleophilic attack of Tyr411 on the
phosphorus and explains the slow reactivity of soman toward
HSA. It looks like soman may randomly react with Tyr411 on
its way in/out of the pocket.
3.3.2. Noncovalent Docking by Gold. In order to check the
dependency of the results on the software, to test the robustness
of the models obtained by Autodock, and also to generate as
many conformers as possible, we decided to repeat the docking
simulations using another software, Gold, version 3.1. Gold gave
us the possibility of using two scoring functions, Goldscore
(force-field-based like Autodock) and Chemscore (empirical,
i.e., optimized on a test set of protein–ligand complexes). We
performed the docking simulations using both the 2bxh and the
2bxd protein structures. Our results did not show any significant
The Chemscore function is empirical. It can potentially
generate different binding modes because contributions of
intermolecular interactions are not the same as those with
Goldscore or Autodock. The docking using Gold/Chemscore
gave different kinds of conformers than Goldscore, but with
very small changes in energy. Within the different clusters, some
models displayed a phosphorus atom close to Tyr411 (P-OHY411
distances: PRCR ) 4.33 Å, PRCS ) 3.51 Å, PSCS ) 3.98 Å,
and PSCR ) 3.41 Å). The enantiomers with PR configuration
have a P-F in a favorable position for a nucleophilic attack
(OHY411-P-F angle: PRCR ) 155°, PRCS ) 138°, PSCS ) 42°,
and PSCR ) 45°). Chemscore docking simulation generated a
prephosphonylation complex (fluorine opposite OHY411 and
hydrogen bonding between P ) O and OGS489) for the two PR
enantiomers. These results suggest that the prephosphonylation
step would be energetically more favorable for the PR than for
the PS enantiomers. To compare these models with the previous
ones, we rescored the prephosphonylation complexes with
conformers PRCR and PSCS by the Goldscore function and
obtained 10.78 and 27.07, respectively. Compared to the Gold/
Goldscore results (41.28 and 38.16), the rescoring clearly
indicates that the ligand positions in the prephosphonylation
complexes are not energetically favorable. Therefore, the
Chemscore results do not contradict the previous conclusions
obtained from the Autodock and Gold/Goldscore dockings.
These docking results may explain why the reactivity of
soman with HSA is so poor. First, the binding affinity is weak
Kd ) 134 µM. Second, the phosphorus does not bind in a
position that is favorable for reaction with Tyr411. The docking
results also suggest that a higher bimolecular rate constant is
probable in the case of PR enantiomers. But because of the poor
reactivity and the limited measurement accuracy, an increased
reactivity of the PR enantiomers was not experimentally
observed.
3.3.3. Covalent Docking by Gold. The noncovalent docking
of soman to the HSA protein showed a low enantioselectivity.
In order to confirm this finding, we covalently docked the four
enantiomers of soman to the 2bxh form of albumin and
compared energies (or scores). We used the software Gold,
version 3.1, which permits covalent docking. As done before,
dockings were scored by the functions Goldscore and Chem-
score. To discriminate the conformers obtained by Gold/
Goldscore or Gold/Chemscore dockings, we calculated the
energies of soman-protein complexes using mopac-pm3, a
Figure 9. Models of soman noncovalently docked into albumin. The
various stereoisomers of soman were docked in the estero-amidase
active center (Tyr411) of HSA using Autodock software.

Reaction of Albumin with Soman
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 429
Table 2. Covalent Docking of Soman in HSA Performed by
Golda
mice were injected with small amounts of the organophospho-
nate, FP-biotin (10-fluoroethoxyphosphinyl-N-biotinamido pen-
tyldecanamide), amounts that produced no toxic signs, albumin
was the most abundantly labeled protein in plasma (46), making
it the dominant stoichiometric scavenger. In vitro pretreatment
of plasma with a variety of organophosphates (malaoxon,
paraoxon, chlorpyrifos oxon, methyl paraoxon, dichlorvos,
diisopropylfluorophosphate, diazoxon, and echothiophate) re-
duced the subsequent reaction of FP-biotin with albumin,
indicating that all of these other OPs also react with albumin
(46). This strongly suggests that albumin will also be a scavenger
for these OPs in vivo.
soman isomer
Goldscore/∆Hf
Chemscore/∆Hf
PSCR
PSCS
PRCR
PRCS
11.68/489.8
16.88/483.2
15.67/529.0
12.88/480.4
15.95/519.3
17.43/514.7
16.46/497.8
14.55/539.8
a Scores (Goldscore and Chemscore) have no units. Enthalpies of
formation (∆Hf) are in kcal·mol-1
.
Although albumin displays a low reactivity toward OPs
compared to that of cholinesterases and other known secondary
biological targets of OPs (37), the large quantity of albumin in
blood circulation and lymph may provide a substantial reservoir
for sequestering OPs. We also found that phosphonylated
albumin slowly self-reactivates. Though the catalytic OPase
activity is associated with HSA, and Tyr411 is slow and thus
toxicologically insignificant, it may be hypothesized that mutants
of albumin capable of hydrolyzing OPs at high rate could be
used for the detoxification of soman in vivo.
4.1.2. Biomarker. Phosphonylated albumin may prove to be
a useful biological marker of soman exposure (11). The sensitive
mass spectrometry methods used by Black et al. (11) and further
developed in the present article could prove useful for the
diagnosis of soman exposure. In particular, it has been shown
that soman tyrosine adducts can be detected at least up to 7
days post exposure in guinea pigs (38). Advantages of a
soman-albumin biomarker include its stability and the absence
of aging. The half-life for spontaneous reactivation is estimated
to be about 1 week at pH 8.0 and 25 °C, and 20 days at pH 7.4
and 22 °C, making the soman-albumin adduct suitable for
retrospective studies. The absence of aging means that soman
exposure can be unambiguously identified because the soman-
albumin adduct does not lose the diagnostic pinacolyl group.
Our finding that soman-albumin adducts do not age confirms
the reports that there is no loss of alkoxy groups from soman-,
sarin-, or DFP-albumin adducts (11, 19, 36). In addition, it
has recently been shown that OP-albumin conjugates do not
appear to be reactivatable by oximes used as antidotes of OP
poisoning. Therefore, the detection of Tyr411 adducts in plasma
may be possible, even though individuals exposed to OPs have
received effective oxime therapy (38).
Figure 10. Models of soman covalently docked into albumin. Various
enantiomers of soman were docked into Tyr 411 of HSA (form 2bxh)
using Gold software, version 3.1. PSCS (blue), PSCR (red), PRCR
(magenta), and PRCS (green).
semiempirical method, and selected those with the lowest ∆Hf.
The results are summarized in Table 2.
The four models are superimposed in Figure 10. The position
of the tertiary butyl group is well conserved for all enantiomers
(it points to Leu387, Ile388, Asn391, and Phe403), while the P
) O points to Leu430 and Leu457 for PSCR or PSCS enantio-
mers, Ser489 (hydrogen bonding with OH) for PRCR, and R410
for PRCS. The conformers PSCS and PSCR are nearly perfect
mirror images, the only difference being the position of the CH3
bond to the asymmetric carbon that points to Leu430 for CR
and Arg410 for CS. In the case of PR, there are more differences
between the two enantiomers because the PRCR enantiomer is
the only one that allows hydrogen bonding with Ser489 without
having too many disfavored van der Waals interactions. (Arg410
constrains the position of the CH3 bond to C*.) The mopac-
pm3 ∆Hf values are in the same order of magnitude (∼490 (
10 kcal.mol-1), showing no thermodynamic enantioselectivity.
Another potential method of monitoring for soman exposure
is through the use of antibodies. The information presented in
this work raises interesting possibilities regarding the generation
of antibodies to the soman-albumin adduct. The noteworthy
aspect of the soman-albumin adduct in this regard is that
Tyr411 is located on the surface of albumin. A soman-albumin
adduct at Tyr411 therefore becomes a viable target for antibod-
ies, unlike the soman adducts of cholinesterases, which are
buried in a deep gorge well below the enzyme’s surface. In
addition, the surface location for the soman-albumin adduct
suggests that people exposed to soman might normally produce
antibodies against the soman-albumin adduct. This in turn
suggests the possibility of monitoring exposure to soman by
looking for those antibodies, long after the exposure incident
has passed and long after the antigen has disappeared.
4. Discussion
4.1. Roles for Albumin and Albumin/Soman Adducts in
the Monitoring of and Protection against Soman Exposure.
4.1.1. Scavenging OP. The reaction of albumin with soman
was found to be slow (kp ) 15 ( 3 M-1 min-1 at pH 8.0).
This is in agreement with the slow rate of reaction of albumin
with the nerve agent simulant, DFP (75 M-1 min-1 at pH 8.2)
(10). Combined with the high concentration of albumin present
in plasma (0.6 mM), the apparent pseudo-first-order rate constant
for the reaction of albumin with soman would be 0.009 min-1
(t1/2 ) 77 min). Thus, despite its high concentration in blood,
stoichiometric scavenging of soman by albumin is not expected
to play a major role in protection against acute poisoning.
Regardless of the low reactivity, significant amounts of OPs
are bound to albumin upon in vivo exposure. For example, when
4.2. Perspectives: Would Mutant Albumins Be Good
OP Scavengers? 4.2.1. Stoichiometric Scavenging. The bi-
molecular rate constant for the phosphonylation of albumin, kp,
15 M-1 min-1, is about 6 × 106 times slower than that for the
inhibition of acetylcholinesterase and butyrylcholinesterase by

430 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Li et al.
soman (∼9 × 107 M-1 min-1). Because the concentration of
soman in the most severe cases of poisoning would be far less
than that of albumin in plasma, for example, <11 nM (39), the
reaction between albumin and soman would be pseudo-first-
order. The pseudo-first-order rate expression can be rearranged
(eq 3) so that the time, t, needed for a stoichiometric scavenger
to reduce a concentration of a toxicant such as soman from [S]0
to [S]t can be easily determined.
constant, kp·[E], is 0.009 min-1. The sum of the first-order
reaction rate constants for the reaction of soman with erythrocyte
AChE and plasma BuChE is 4.5 min-1. This number was
arrived at by taking the concentration of AChE in blood to be
about 2.5 nM, the concentration of BuChE to be 50 nM, and
k2/Kd to be about 9 × 107 M-1 min-1 for both enzymes. Thus,
in blood, cholinesterases react with soman about 500 times faster
than albumin does.
For an injected, mutated albumin to be used as a stoichio-
metric scavenger, it should react at least 500 times faster than
the endogenous albumin to be competitive with the cholinest-
erases. There is a limit to the amount of plasma-derived albumin
that can be put into the human body, that is, 25 g/100 mL (Albu
Rx25) by slow infusion or 500 mg in 2 mL by intravenous
injection. Injected into a human of 70 kg (3 L of plasma), 500
mg would give a final plasma concentration of mutant albumin
of 166 mg/L (2.55 × 10-6 M). To react faster than cholinest-
erases, the bimolecular rate constant of this amount of mutant
albumin should be at least 1.8 × 106 M-1 min-1. Compared to
endogenous albumin (15 M-1 min-1), this is an efficiency
improvement of about 5 orders of magnitude. Though there are
few examples of evolved enzymes, showing enhancement of
such a magnitude, site-directed mutagenesis and/or directed
evolution can lead to dramatic improvement in the catalytic
activity of enzymes. Improvements of 4 orders of magnitude
for the glyphosate tolerance protein (44) and 5 orders of
magnitude for R-lytic protease (45) have been reported.
Therefore, improving the reactivity of albumin against OPs for
the purpose of making a stoichiometric scavenger would be a
challenge, but it is possible.
ln([S]0/[S]t)
t )
(3)
kp · [E]
Since the concentration of albumin in plasma is about 0.6
mM, it would take about 8.5 h to reduce the soman concentration
by 100-fold (Ln [S]0/[S]t ) 4.6). Because, red blood cell
acetylcholinesterase, plasma butyrylcholinesterase, and plasma
paraoxonase react much faster than albumin with soman, the
contribution of albumin to soman detoxification would take place
only after the saturation of these enzymes (36).
4.2.2. Catalyic Scavenging. If we assume that the turnover
of soman by albumin fits a Michael mechanism with a high
Km, then phosphonylation could appear to be second-order under
our experimental conditions. If in addition, kr<<kp, which is
the case for the soman/albumin turnover, then kcat/Km is equal
to kp. Under these conditions, kp defines the catalytic efficiency.
Thus, the catalytic efficiency for the hydrolysis of soman by
albumin, at Tyr411, is 15 M-1 min-1. This value is about 4 to
5 orders of magnitude lower than that for the hydrolysis of
soman by Pseudomonas diminuta phosphotriesterase (6 × 105
M-1 min-1) (40) or allozyme Q192 of human paraoxonase (2.6
× 106 M-1 min-1) (41). Eq 4 gives the time needed for a
catalytic scavenger to drop the concentration of a toxicant from
[S]0 to [S]t.
In addition, the observed catalysis of soman hydrolysis on
the albumin surface at a site different from Tyr 411 could also
be improved. Work is in progress to identify this area on the
albumin surface. Thus, stoichiometric and catalytic bioscav-
engers based on modified human albumins have to be considered
as possible candidates.
ln([S]0/[S]t)
t )
(4)
(kcat ⁄ Km) · [E]
Thus, taking the estimated kcat/Km value, the time needed for
the catalytic activity of albumin (0.6 mM) to decrease the soman
concentration in blood by 100-fold would be about 9 h. It is
therefore obvious that the reaction of soman at Tyr411 of HSA
does not contribute significantly to the catalytic turnover of
soman molecules in plasma. However, the in vitro observation
that pure FAF-HSA promotes the hydrolysis of soman at a
second site (kh ) 0.0076 min-1) with a rate 100 times higher
than the turnover at Tyr411 is important. The in vivo toxicologi-
cal relevance of this chemical activity has to be demonstrated,
but if proved, this process could contribute to the degradation
of soman and other nerve agents in human plasma.
Acknowledgment. We thank D. S. Darvesh (Department of
Medicine, Dalhousie University, Halifax, Canada) for the gift
of o-NTFNAC. Mass spectra were obtained with the support
of the Protein Structure core Facility at the University of
Nebraska Medical Center. This work was supported by U.S.
Army Medical Research and Materiel Command W81XWH-
07-2-0034, Eppley Cancer Center grant P30CA36727, and NIH
grant 1 U01 NS058056-02 (to O.L.), and DGA grant 03co010-
05/PEA 01 08 7 (to P.M.).
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catalytic scavengers for prophylaxis and treatment of OP
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