TOXICOLOGICAL SCIENCES 90(1), 142148 (2006)

doi:10.1093/toxsci/kfj054
Advance Access publication December 1, 2005

Interaction of Arsine with Hemoglobin in Arsine-Induced Hemolysis

Leonard T. Rael,*,,1 Felix Ayala-Fierro, Raphael Bar-Or,*, Dean E. Carter, and David S. Barber
*Swedish Medical Center, Trauma Research Laboratory, Englewood, Colorado 80113; DMI BioSciences, Inc., Englewood, Colorado 80113;
The Dial Corporation, Product Safety, Regulatory and MicrobiologyClinical Studies and Toxicology, Scottsdale, Arizona 85254; Department of
Pharmacology and Toxicology, The Center for Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721; and Department of
Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611
Received June 22, 2005; accepted November 22, 2005

INTRODUCTION

Arsine gas (AsH3), the hydride of arsenic, is the most

acutely toxic form of arsenic (threshold limit value 50 ppb;

1
To whom correspondence should be addressed at Swedish Medical Center,
Trauma Research Laboratory, 501 E. Hampden Ave., Rm. 4454, Englewood,
CO 80113. Fax: (303) 788-4064. E-mail: lrael@dmibio.com.

1
In 1999, a new AsH3 TLV-TWA of 2 ppb was proposed but was not adopted
in 2000 or 2001. In 2001, ACGIH proposed to lower AsH3 TLV-TWA from 50
to 3 ppb, and to designate AsH3 as an A-1 (Confirmed Human Carcinogen). In
2003, AsH3 was placed on the ACGIH under study list. In 2004, AsH3
appeared with an ACGIH proposed (trial value) TLV-TWA of 5 ppb and A4
designation (Not Classifiable as a Human Carcinogen). This proposal remained
in the 2005 ACGIH booklet.

The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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The mechanism of arsine (AsH3) toxicity is not completely

understood, but hemoglobin (Hb) has long been recognized as
a necessary component of the overall mechanism of AsH3-induced
hemolysis. In this study, the role of Hb in AsH3-induced hemolysis
was investigated. The purpose was to determine whether exposure
to AsH3 altered the structure of the heme or globin constituents of
Hb. Arsine was incubated with isolated, human oxyhemoglobin
(oxyHb) and carboxyhemoglobin (carboxyHb), and the release of
heme and formation of AsH3-induced hemoglobin modifications
were examined. Arsine increased the amount of heme released
from oxyHb by 18%. When carboxyHb was incubated with AsH3,
there was no change in heme release, suggesting that the sixth
ligand position on the heme iron may be critical in the interaction
with AsH3. ArsineHb interactions were studied by mass spectral
analysis of heme, a-chain globin, and b-chain globin. Arsine had
no significant effect on the a- or b-chain LCMS spectra in oxyHb
and carboxyHb, but in oxyHb, arsine consistently increased the
frequency of methyl acetate ion fragment ( CH2OOH, m/z = 59)
loss from heme in the matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS) spectra. The formation of Hb
protein crosslinks was investigated by Western blotting using an
anti-Hb antibody in isolated membranes from AsH3-treated
erythrocytes, but no Hb-membrane adducts were found. These
results suggest that the interaction between AsH3 and hemoglobin
result in an increase in heme release which may contribute to the
hemolytic mechanism of AsH3.
Key Words: arsine; oxyhemoglobin; carboxyhemoglobin; heme;
hemolysis.

ACGIH 1982).1 Exposure to AsH3 is possible from the

accidental release of the gas during some manufacturing
processes. AsH3 is extensively used for epitaxial growth of
gallium arsenide and as a dopant for silicon-based electronic
devices in the semiconductor industry. Accidental exposure to
AsH3 may also occur from any situation where arseniccontaminated metals are treated with a strong acid, e.g., metal
mining, paint, and herbicides (Buchanan, 1962). The erythrocyte is the main target of AsH3, with exposure causing
hemolysis. In human exposures to toxic levels of AsH3, clinical
experience is consistent with intravascular hemolysis, and
dark-red urine (hemoglobinuria) is usually the first symptom.
This is followed by abdominal pain, jaundice, and anemia
(Romeo et al., 1997). Splenomegaly has been observed in mice
exposed to low levels of AsH3 for 60 days (Hong et al., 1989).
Exposure to AsH3 was fatal in up to 25% of the reported human
cases (Fowler and Weissberg, 1974).
The mechanism of AsH3 toxicity is not clearly understood.
It has been postulated that AsH3 exerts its toxic effects through
oxidative stress depleting reduced glutathione (Pernis and
Magistretti, 1960; Blair et al., 1990), but other investigations
have contradicted the importance of GSH (Hatlelid et al.,
1995; Winski et al., 1997). It has also been suggested that
AsH3 interacts with important sulfhydryl groups located on
the membrane Na,K-ATPase pump inhibiting the pump
and causing cell swelling and lysis (Levinsky et al., 1970).
However, the finding that dog erythrocytes are also hemolysed
by AsH3 (Hatlelid et al., 1995) makes the Na,K-ATPase pump
an unlikely target of AsH3 because most dog erythrocytes
lack this pump. In addition, AsH3 did not significantly alter
ATP levels or inhibit the ATPase in human erythrocytes
(Winski et al., 1997).

INTERACTION OF ARSINE WITH HEMOGLOBIN

MATERIALS AND METHODS

Chemicals
Zinc arsenide (99%) and pyridine (ACS grade) were obtained from Aldrich
Chemical Co. (Milwaukee, WI). All other chemicals, including purified human
hemoglobin, rabbit anti-human hemoglobin antibody, and goat anti-rabbit IgG
(alkaline phosphatase conjugate), were all purchased from Sigma Chemical Co.
(St. Louis, MO).
Arsine Generation
Arsine was generated by the method of Hatlelid et al. (1995). Briefly, zinc
arsenide was reacted with 50% sulfuric acid to generate AsH3 gas, which was
bubbled into 0.02 M phosphate buffer or PBS to the desired concentration using
argon as the carrier gas. Arsine concentration was determined by reaction with
0.55% diethyldithiocarbamate in pyridine, followed by spectrophotometric
determination of this product at 510 nm. Caution: AsH3 is a toxic gas and
appropriate precautions should be taken. All procedures should be performed in
an approved fume hood. A saturated potassium permanganate solution trap, inline after the aqueous trap, should be used to prevent the release of AsH3 during
its generation.
Generation of Hemoglobin Species
A dialyzed, 0.8 mM solution of human hemoglobin (MW 64 kDa) in
a 0.02 M phosphate buffer (pH 7.4) was used in all the heme-release studies, as
well as the studies involving interactions between AsH3 and hemoglobin.

Generation of metHb. Methemoglobin was formed by dissolving human

Hb in 0.02 M phosphate buffer and treating the Hb solution with sodium nitrite.
After addition of sodium nitrite, the solution was dialyzed overnight against at
least two changes of buffer at 4C in 8000 MW cutoff tubing (Spectrum
Medical Industries, Inc., Houston, TX). The purity and concentration of the
metHb solutions were verified by spectrophotometry with wavelength maxima
of 500 and 631 nm (Zijlstra and Buursma, 1987).
Generation of oxyHb. A total of 1 mg of sodium hydrosulfite was added to
6 ml of metHb solution. The solution was placed in a dialysis bag and dialyzed
overnight in phosphate buffer under constant stirring, changing the buffer at
least once. The solution turned red, and oxyHb formation and concentration
were verified by spectrophotometry with wavelength maxima of 542 nm and
577 nm (Zijlstra and Buursma, 1987).
Generation of carboxyHb. CarboxyHb was produced using the oxyHb
solution described above. The oxyHb solution was bubbled with carbon
monoxide gas for approximately 1520 min. The solution turned a bright red,
indicating the replacement of oxygen with carbon monoxide. CarboxyHb
formation and concentration were verified by spectrophotometry at 539 nm and
569 nm (Zijlstra and Buursma, 1987).
Determination of Heme Release
For these studies, all three Hb species (metHb, oxyHb, and carboxyHb) were
assessed for heme release in the presence of AsH3. For each Hb species, four
separate 0.5 ml incubations were performed in triplicate at room temperature.
Each incubation contained 0.25 ml of a 0.8 mM Hb solution (final 0.4 mM).
In each specific Hb species, the negative control was prepared by adding
0.25 ml of 0.02 M phosphate buffer to the hemoglobin solution. The positive
control consisted of 2.5 M urea in PBS and was prepared by adding 0.125 ml
of 10 M urea and 0.125 ml phosphate buffer to the 0.8 mM Hb solution. Two
different AsH3-treated samples were prepared. The first AsH3-treated incubation contained Hb and AsH3 only and was prepared by adding 0.125 ml of
1.6 mM AsH3 in phosphate buffer (final AsH3 concentration 0.4 mM) and
0.125 ml of phosphate buffer to each Hb solution. The second AsH3-treated
incubation contained Hb, AsH3, and urea, and was prepared by adding 0.125 ml
of 10 M urea and 0.125 ml of 1.6 mM AsH3 to each 0.8 mM Hb solution. An
aliquot of the incubation was diluted and scanned on the spectrophotometer to
determine if any changes in the Hb spectra occurred after incubation with AsH3.
The assay for the quantification of free hemin was based on the extraction
procedure given by Letarte et al. (1993). Briefly, after a 30-min incubation,
1 ml of 0.1 M ammonium acetate (pH 4.0) was added to each incubation
and stirred gently for 10 seconds. This process converted the free hemin to
hematin. The samples were then extracted with two consecutive 5-ml washes
with chloroform:methanol (2:1). After each wash, the samples were spun at
2000 rpm for 10 min. After the extraction process, the organic layer (bottom
layer) was collected. The organic extract was then evaporated to dryness in
a water bath with nitrogen gas at 3540C. The dry extract was re-dissolved
in 0.5 ml of DMSO:chloroform (1:4) and stirred vigorously for 30 s. Hematin
was then back-extracted into the aqueous phase using 1 ml of 0.1 N NaOH.
The samples were spun for 10 min at 2000 rpm. The aqueous phase (0.8 ml)
was transferred to a disposable polystyrene cuvette along with 1.6 ml 0.1 N
NaOH. The samples were then read on a Beckman DU-7 spectrophotometer
at 385 nm.
ArsineHemoglobin Interactions
The negative control and AsH3-treated incubation mixtures from the heme
release study for all three Hb types were analyzed by mass spectrometry. The
samples were diluted 1:50 in dH2O and analyzed by high performance liquid
chromqtography (HPLC; Waters Corporation, Milford, MA) coupled to
positive electrospray ionization time-of-flight mass spectrometry (ESI-TOF
MS, Micromass, UK). An aliquot of 25 ll of each sample was injected
onto a YMC-Pack Protein-RP, 150 mm 3 4.6 mm, 5l, HPLC column
heated to 50C (Waters Corporation) using a 20-min linear gradient method
using water/0.1% trifluoroacetic acid (A) and acetonitrile/0.1% TFA (B).

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Oxyhemoglobin (oxyHb) has long been recognized as

a necessary component to the overall mechanism of AsH3induced hemolysis, as conversion of oxyHb to carboxyHb
prevents hemolysis in erythrocytes exposed to AsH3 (Rael
et al., 2000). Considerable evidence exists indicating that
exposure to AsH3 causes Hb oxidation and denaturation
that may lead to toxicity (Hatlelid et al., 1996; Blair et al.,
1990). It has been proposed that heme-containing proteins
activate AsH3 to a toxic intermediate, leading to AsH3 toxicity
(Ayala-Fierro et al., 1999). The release of free heme (ferriprotoporphyrin IX, hemin) from Hb can produce hemolysis by
a colloid-osmotic mechanism (Chou and Fitch, 1981). Similar
effects are observed in erythrocytes exposed to AsH3, with
massive potassium loss occurring within 5 minutes of exposure, followed by hemolysis (Rael et al., 2000). Based on the
similarity of toxic events, we investigated whether exposure of
Hb to AsH3 led to increased release of free heme.
It has also been shown that erythrocytes exposed to AsH3
retain arsenic (As) in a non-dialyzable form (Graham et al.,
1946). This had led to the hypothesis that the formation of
an AsHb complex may occur and be involved in hemolysis
(Fowler and Weissberg, 1974). Finally, it is known that oxidation of Hb resulting in Hb denaturation and precipitation
can lead to abnormal association of Hb with erythrocyte
membrane proteins that increase the fragility of the erythrocyte
membrane (Murakami and Mawatari, 2003). In the present
research, human Hb and human erythrocytes treated with AsH3
were used as a model system to investigate these three potential
interactions of AsH3 with Hb.

143

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RAEL ET AL.

The chromatographic and mass spectrometry conditions were as previously

described (Bar-Or et al., 2005). The mass spectra for the a- and b-chains of
globin were deconvolved to the uncharged parent mass using MaxEnt 1
(Micromass, UK). The same samples were analyzed by matrix-assisted laser
desorption/ionization (MALDI)-qTOF (Micromass, UK) by spotting 1 ll of a
1:50 dilution of the negative control or AsH3-treated incubations mixed 1:1
with 5 lg/ml alpha-Cyano-4-hydroxycinnamic acid. Samples were analyzed in
positive V-optics reflectron mode with a scan range of 3001000 Da using
a collision energy of 10 eV.
Arsine-Induced Hemoglobin Associations with Membrane Constituents

Data Analysis
All data are expressed as mean standard deviation. Values are denoted
with an asterisk (*) if statistically different from controls ( p < 0.05), using
Student t-test (Microsoft Excel).

FIG. 1. Arsine-induced heme release from human hemoglobin. 400 lM

oxyHb (A) or carboxyHb (B) were incubated with phosphate buffer (control),
2.5 M urea, 0.4 mM AsH3, or 0.4 mM AsH3 2.5 M urea for 30 min.
Extractable heme was measured as explained in Materials and Methods. Values
are mean SD (n 3). An asterisk denotes significant difference from control
( p < 0.05).

RESULTS

Interaction of Arsine with Isolated Human Hemoglobin

Heme release. Treatment of isolated human Hb with AsH3
increased the amount of heme released from oxyHb by 0.88
lM versus controls (Fig. 1A). This increase corresponded to an
18.3% increase in the AsH3-treated oxyHb compared to
controls, whereas an 18.5% (0.9 lM) and 43.6% (2.1 lM)
increase were observed in urea-treated and AsH3urea-treated
oxyHb, respectively. For carboxyHb, there was no significant
difference between AsH3-treated carboxyHb and controls,
although the combination of AsH3 and urea caused an increase
in heme release, albeit an insignificant one (Fig. 1B). This trend
for carboxyHb was also observed for metHb in that AsH3 had
no significant effect on heme release (data not shown). Spectral
analysis of hemoglobin samples after incubation with AsH3
demonstrated that absorbance spectra were identical to those
obtained prior to the addition of AsH3 (data not shown).
Direct reaction between AsH3 and hemoglobin. To investigate any possible alterations of the Hb constituents, the
incubations from the heme release study were analyzed by
matrix-assisted laser desorption/ionization mass spectrometry

(MALDI-MS) and liquid chromatographymass spectrometry

(LCMS). The MALDI-MS analyses of the heme groups (m/z
616) derived from AsH3-treated human hemoglobin showed no
peaks that corresponded to the addition of the arsenic species to
heme. However, there was an increase in the abundance of
heme fragments in the AsH3-treated samples of oxyHb (Fig.
2A) and metHb (Fig. 2B), but not for carboxyHb (Fig. 2C). The
peaks at m/z 557.2 and m/z 498.2 have been previously
identified as the loss of one or two CH2COOH fragments (59
per fragment) from the native heme molecule (Demirev et al.,
2002). The LCMS analyses of the a- and b-chains of the AsH3treated hemoglobin were virtual identical to control (data not
shown). Both treated and control samples showed an a-chain
peak at 15,127 Da, which corresponded to the theoretical value
(15,126 Da). Likewise, both samples showed a b-chain peak at
15,868 Da, corresponding to the theoretical value of 15,867 Da.
No evidence of covalent modification was seen throughout the
spectra of the a- or b-chain of globin.

Arsine-induced hemoglobin associations with erythrocyte

membrane constituents. No differences in protein abundance
were observed between control and AsH3-treated samples

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Blood was collected by venipuncture from healthy male and female

volunteers (ages 2240 years). The blood was sedimented by centrifugation
(2000 rpm, 10 min) and rinsed twice with glucose-containing phosphatebuffered saline (glucose-PBS, pH 7.4) in order to remove plasma and buffy
coat. Packed erythrocytes were treated with PBS (control) or 0.5 mM AsH3
(final) for 5 or 30 min at 37C. At each time point, a 0.5-ml aliquot was
removed, and the erythrocytes were lysed and then centrifuged at 14,000 rpm,
after which the supernatants were removed. Membranes were washed three
more times and the membrane pellet was white. Total protein content was
determined using the BCA assay (Pierce Biotechnology, Rockford, IL). A total
of 35 lg of membrane protein was separated on a 6% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with Coomasie
blue. Proteins from a duplicate gel were transferred to a polyvinylidene
difluoride (PVDF) membrane and probed with a rabbit anti-human hemoglobin
antibody (Sigma, St. Louis, MO). An alkaline phosphatase conjugated goat
anti-rabbit IgG secondary antibody (Sigma) was used at a 1:10,000 dilution,
and the blot was developed with BCIP/NBT (Sigma Fast tablets, Sigma).

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INTERACTION OF ARSINE WITH HEMOGLOBIN

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FIG. 2. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of arsine-treated human hemoglobin. Using the incubations
from the heme-release experiment, oxyHb (A), metHb (B), and carboxyHb (C) were analyzed by MALDI-TOF MS. Controls are the top spectra, and AsH3-treated
samples are the bottom spectra. The theoretical molecular weight of native heme is 616.5 Da.

stained with Coomasie blue, indicating that no significant

protein degradation or cross-linking had occurred (data not
shown). Probing a Western blot of erythrocyte membrane
proteins with anti-human Hb produced no signal, indicating
that no Hb adducts had been formed with membrane proteins
(data not shown).

DISCUSSION

Arsine (AsH3) has been recognized as a potent hemolytic

agent for well over 100 years (reviewed by Fowler and
Weissberg, 1974); however, the mechanism responsible for
toxicity remains unknown. Although the mechanism is not

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RAEL ET AL.

1992; Hargrove et al., 1994). If arsenic forms a bond with Fe2,

it may weaken the bond with the histidine on globin and
increase heme release. This idea is supported by the observation that treatment of carboxyHb with AsH3 had no effect on
heme release, indicating that the nature of the sixth Fe2 ligand
is important in the effect on heme release. Matrix-assisted laser
desorption/ionization-MS analysis of heme from AsH3-treated
Hb, however, did not provide evidence for arsenic binding to
heme iron. However, the increased frequency of methyl acetate
ion fragment ( CH2OOH, m/z 59) loss from oxyHb treated
with AsH3 indicates that the reaction with AsH3 may have
altered electron distribution in the heme group such that the
bond strengths at these sites are lower.
A second possible explanation is that AsH3 modifies the
globin chain or heme moiety in a way that decreases the
strength of heme binding. Because AsH3 is a highly reduced
form of arsenic that appears to be oxidized during the reaction
with hemoglobin (Carter et al., 2003), it could cause a conformational change in the Hb molecule by reducing the globin
chains or the porphyrin rings, which results in decreased
affinity of the globin for heme. Another possibility is that
AsH3, or a breakdown product of AsH3, binds directly to the
globin chains, resulting in modified protein conformation and
eventual precipitation. Multiple targets exist on the Hb
molecule that are susceptible to arsenic adduction; however,
cysteine residues are the most likely targets. In human Hb, the
a-chain contains one cysteine residue, whereas while the bchain has two cysteine residues within its amino acid sequence
(Lehninger et al., 1993). Therefore, there are six sulfhydryl
groups per Hb molecule, of which two are readily reactive with
metallic sulfhydryl inhibitors (Ingram, 1955). Previous studies
have shown that lewisite (dichloro(2-chlorovinyl) arsine) and
phenyldichloroarsine form adducts with cysteine on the bglobin chains of human Hb (Fidder et al., 2000; Chong et al.,
1989). These sulfur groups are clearly important in heme
retention, as evidenced by increased heme release from
globins treated with NEM (Scheler et al., 1963). Modification
of the two b-globin thiols also increased the rate of autooxidation and precipitation in isolated Hb solutions (Allen
and Jandl, 1961). In the present study, no modifications of the
a- or b-globin chains were found by mass spectrometry,
suggesting that exposure to AsH3 does not cause significant
changes in the Hb molecule. However, AsH3-induced adducts
might be labile and susceptible to neutral loss within the mass
spectrometer.
This finding has implications for the proposed formation of
As-Hb adducts following exposure to AsH3. It is clear that
some arsenic is tightly associated with erythrocytic proteins
after AsH3 exposure, being present in a complex that is strong
enough to resist decomposition during dialysis (Graham et al.,
1946). The lack of arsenic binding to purified Hb in the present
study suggests that other cellular components are required to
convert AsH3 to the form that ultimately binds to Hb, or that Hb
itself is not the site of arsenic binding in these experiments.

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known, it is clear that oxyHb is required for induction of

hemolysis (Rael et al., 2000). The purpose of this study was to
investigate the interaction of AsH3 with Hb in order to
determine if exposure to AsH3 caused release of free heme,
produced AsHb complexes, or induced cross-links of Hb with
erythrocyte membrane proteins.
Hatlelid et al. (1996) reported formation of metHb, hemichromes, and Heinz bodies (protein precipitation, verified
spectrophotometrically) when AsH3 was incubated with isolated oxyHb. Blair et al. (1990) also showed the production of
metHb and Heinz bodies in circulating erythrocytes of rats
treated with AsH3. Hemoglobin oxidation has long been
recognized as a disrupter of erythrocyte function. Chiu and
Lubin (1989) described the process of Hb oxidation in a series
of steps beginning with the formation of metHb followed by
hemichrome formation and breakdown to precipitated globin
(Heinz bodies). During degradation of the Hb complex, the
toxic heme moiety is released, resulting in widespread damage
to the erythrocyte membrane. The current study investigated
this hemolytic scenario in AsH3-induced hemolysis. Because
heme is the proposed toxic species released during hemoglobin
degradation, free heme was measured directly using a protocol
based on the assay described by Letarte et al. (1993).
Treatment of human oxyHb with AsH3 increased the release
of free heme by nearly 1 lM from a 0.4 mM oxyHb solution.
Because the normal concentration of hemoglobin in intact
erythrocytes is about 15 times higher than that used in our
study, it would be reasonable to assume that the concentration
of free heme released in erythrocytes treated with AsH3 would
be significantly greater than 1 lM. Incubation of normal mouse
erythrocytes with as little as 1 lM ferriprotoporphyrin IX
(hemin) has been shown to cause potassium loss and swelling
within minutes, and concentrations above 2 lM cause hemolysis (Chou and Fitch, 1981). Previous studies have demonstrated that formation of carboxyHb in erythrocytes prevents
AsH3-induced hemolysis (Rael et al., 2000). Therefore, if heme
release is involved in the hemolytic mechanism of AsH3, treatment of carboxyHb with AsH3 should not affect heme release.
Indeed, treatment of human carboxyHb with AsH3 had no effect
on heme release. Taken together, these results suggest that
release of free heme may be responsible for the colloid-osmotic
type hemolysis produced by AsH3. However, colloid-osmotic
type hemolysis is also produced by sulfhydryl reagents, such as
N-ethylmaleimide (NEM) (Jacob and Jandl, 1962).
The present data suggest that AsH3 somehow weakens the
interaction between the globin chains and the heme molecule,
leading to heme release. There are several possible explanations for the AsH3-induced increase in heme release and its
effect on the overall hemolytic mechanism of AsH3 on erythrocytes. One possibility is that arsenic, binding to the sixth
ligand position of the Fe2 of heme, decreases the strength of
the bond between heme and histidine on the globin. Binding of
ligands, such as CO and CN, increase the stability of the heme
by increasing the strength of this bond (Traylor and Sharma,

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INTERACTION OF ARSINE WITH HEMOGLOBIN

erythrocyte membrane could potentially determine the mechanism of hemolysis caused by AsH3.
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Finally, the hypothesis concerning the generation of hemoglobinskeletal protein complexes as part of the mechanism
of AsH3-induced hemolysis was also investigated. According
to Fortier et al. (1988), this complex is formed after the
oxidation of Hb, which increases the affinity of Hb for
sulfhydryl-containing, cytoskeletal components. It was concluded that a quantitative relationship exists between the
membrane-associated protein complex and increased membrane rigidity. It has been demonstrated that increasing intracellular calcium concentration increases the amount of
membrane-attached Hb (Friederichs et al., 1992). Based on
the observations that AsH3 interacts with sulfhydryl groups and
increases intracellular calcium concentrations in erythrocytes
(Rael et al., 2000), the possibility of the formation of such
a complex in the case of AsH3 was explored. One of the
obvious cytoskeletal targets is spectrin, which has been shown
to have globin-binding domains (Bhown et al., 1989). Also, the
sulfhydryl inhibitor NEM has been shown to selectively alter
erythrocyte deformability via cross-linking of sulfhydryl
groups located on spectrin (Fischer et al., 1978). A compound
known to produce hemoglobincytoskeleton complexes is
hydrogen peroxide. Snyder and co-workers (1988) showed
the formation of such a complex verified by Western blot in
erythrocytes treated with hydrogen peroxide. Pretreatment of
erythrocytes with NEM resulted in decreased lipid peroxidation and spectrinhemoglobin cross-linking, as well as less
marked alterations in cell shape and membrane deformability.
Equally important was the finding that hydrogen peroxide
caused no significant decrease in intracellular glutathione
(GSH). In these experiments, no such complex was observed,
even when the cells were treated with hydrogen peroxide. This
result may be a false negative because hydrogen peroxide, used
as a positive control, also failed to induce hemoglobinprotein
adducts. The reason may be due to the antibody, which
recognizes intact Hb tetramers and possibly globin chains.
Hemoglobin in its native form does not bind to spectrin, but
when denatured, it exhibits a strong binding (Bhown et al.,
1989). Based on the Western blot findings presented herein, Hb
and its globin components do not appear to form complexes
with the erythrocyte membrane in the presence of AsH3.
Because of the increase in heme release in isolated Hb treated
with AsH3 demonstrated in this study, the possibility of heme
membrane complexes in erythrocytes should be probed.
In conclusion, AsH3 caused a significant increase in heme
release from isolated oxyHb, but it had no effect on carboxyHb
and metHb. There was no in vitro formation of arsenic-Hb
adducts or globin chain modifications as evidenced by electrospray mass spectrometry although the heme MALDI-MS
fragmentation pattern changed when AsH3 was added. Using
an antibody that detects Hb only, no Hbcytoskeletal protein
adducts were observed in erythrocytes treated with AsH3. This
does not rule out the possibility of heme interacting with the
erythrocyte cytoskeleton. Based on the data presented in this
study, further evaluations of the interactions of heme with the

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