Fast-reacting Thiols in Rat Hemoglobins Can Intercept Damaging
Species in Erythrocytes More Efficiently Than Glutathione*
Ranieri
Rossi
,
Donatella
Barra§,
Andrea
Bellelli§,
Giovanna
Boumis§,
Silvia
Canofeni§,
Paolo
Di Simplicio
,
Lorenzo
Lusini
,
Stefano
Pascarella§, and
Gino
Amiconi§¶
From the
Istituto di Clinica delle Malattie Nervose e
Mentali, Sezione di Farmacologia, Università di Siena,
53100 Siena, the § Dipartimento di Scienze Biochimiche"
A. Rossi Fanelli," Università "La Sapienza," 00185 Roma,
and Centro di Biologia Molecolare, CNR, 00185 Roma, Italy
 |
ABSTRACT |
The S-conjugation rates of the
free-reacting thiols present on each component of rat hemoglobin with
5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a
variety of conditions. On the basis of their reactivity with DTNB (0.5 mM), three classes of thiols have been defined as follows:
fast reacting (fHbSH), with t1/2 <100 ms; slow
reacting (sHbSH), with t1/2 30-50 s; and very slow
reacting (vsHbSH), with t1/2 180-270 s. Under
paraphysiological conditions, fHbSH (identified with Cys-125
(H3))
conjugates with DTNB 100 times faster than glutathione and ~4000
times more rapidly than (v)sHbSH (Cys-13
(A11) and Cys-93
(F9)).
Such characteristics of fHbSH reactivity that are independent of the
quaternary state of hemoglobin are mainly due to the following: (i) its
low pK (~6.9, the cysteinyl anion being stabilized by a
hydrogen bond with Ser-123
(H1)) and (ii) the large exposure to the
solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the
high cellular concentration (8 mM, i.e. four
times that of glutathione), fHbSHs are expected to intercept damaging
species in erythrocytes more efficiently than glutathione, thus adding
a new physiopathological role (direct involvement in cellular
strategies of antioxidant defense) to cysteinyl residues in
proteins.
 |
INTRODUCTION |
Human but not rat erythrocytes are reported (1) to be able to
restore the cellular pool of GSH, which is strongly decreased after
treatment with diazenedicarboxylic acid
bis(N,N-dimethylamide), a thiol-oxidizing agent known by the
trivial name diamide (2). Such a difference in redox behavior was
attributed to a lower enzymatic capacity in reducing disulfides of rat
red cells relative to the human ones (3). However, recent work in this
laboratory demonstrated that the reversibility of such process can be
observed also in rat erythrocytes, depending on diamide dose (4).
Additional evidence (3) may suggest that these differences in behavior between rat and human erythrocytes could be related to diversities in
reactivity of sulfhydryl groups of the corresponding hemoglobins.
Studies of the reactivities of the sulfhydryl groups of oxygen carriers
(5-8) are mainly restricted to hemoglobins containing only two
accessible thiols per tetrameric molecule (such as human hemoglobin),
located at the (F9)93 position of each
subunit. Many sulfhydryl
reagents can be bound to Cys-93
(F9), even in intact red blood cells
(8). Nevertheless, despite its high intracellular level (about half the
concentration of the hemoglobin tetramer (9)), glutathione is not
significantly combined with this protein-bound thiol under normal
conditions. GSSG in fact is reported (5-8) (i) to react very slowly
with human hemoglobin in vitro and (ii) to spontaneously
form with this protein adduct in vivo only under very
special conditions and in small amounts (2-7% of the total
hemoglobin), having been observed to date solely in hemolysates of
patients on long term anti-epileptic therapy (10). On the contrary,
evidence exists that rat erythrocytes produce large quantities of
glutathione-protein mixed disulfides under any oxidative stress (4,
11-13), most of them being adducts with hemoglobin thiols
(HbSSG).1 In particular,
treatment of rat red cells with t-BOOH leads to GSH
oxidation with formation of both GSSG and HbSSG, followed by a recovery
of the initial values within 60 min; on the other hand, addition of
diamide to the same system only causes an increase in HbSSG with no
formation of GSSG (4).
All these facts, as a whole, prompted us to investigate further some
structural and functional properties of rat hemoglobin thiols. Many
hemoglobin components (6-10) have been separated from hemolysates of
adult rats (14-16), and two types of
subunits and four types of
subunits have been identified (17). The primary structures reveal
the presence of 3 cysteinyl residues on the major
chain (All(13),
G11(104), and G18(111)) and up to 2 on the
chain (F9(93) common to
all 4
subunits, and H3(125) in 3 out of 4
subunits) (15-19).
In the present study, the reactivities of cysteinyl residues in rat
hemoglobin components have been characterized, and the differences have
been interpreted in terms of molecular structure. The reported results
are consistent with a major role played, in the transient adaptation of
rat erythrocyte to oxidative stress, by thiols that do not show
conformational-associated changes in their microenvironment.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Glutathione (reduced and oxidized forms),
nicotinamide adenine dinucleotide phosphate (reduced form), and
glutathione reductase (EC 1.6.4.2) were purchased from Boehringer
Mannheim Italia (Milano, Italy). Glutathione transferase (EC 2.5.1.18),
tert-butylhydroperoxide, dithiothreitol, diamide,
iodoacetamide, 1-chloro-2,4-dinitrobenzene, 5,5-dithio-bis(2-nitrobenzoic acid), and guanidine hydrochloride (recrystallized from methanol) were Sigma products (Sigma Chimica, Milano, Italy). Iodo[2-14C]acetamide was obtained from
Amersham Pharmacia Biotech Italia (Milano, Italy); cyanogen bromide was
from Fluka Chimica (Milano, Italy), and trypsin (code TRTPCK) was from
Worthington. The liquid chromatography solvents, HPLC-grade, were
purchased from Carlo Erba Reagenti (Milano, Italy), and the
sequence-grade chemicals were from Perkin Elmer Italia (Monza, Milano,
Italy). All other reagents were of analytical grade purity.
Animals--
Blood was collected from the abdominal aorta of
male rats (Sprague-Dawley strain) fed ad libitum,
anesthesized with chloral hydrate; Na2EDTA was used to
prevent blood clotting.
Erythrocyte Counts and Indices--
Total number of erythrocytes
per unit blood volume, average volume of red cells, and hemoglobin
content per average red cell were determined by an Abbot Cell-dyn 300 apparatus.
GSH, GSSG, and HbSSG Determinations--
Samples were
deproteinized by addition of 4 volumes of a solution containing 6.25%
(w/v) trichloroacetic acid. After centrifugation (3 min at 14,000 rpm),
GSH and GSSG were determined in the clear supernatant. GSH was measured
by either an enzymatic (at pH 7.0) or colorimetric (at pH 7.4) assay
(20, 21). In the first case, sample aliquots and
1-chloro-2,4-dinitrobenzene (0.2 mM final concentration)
were added to the cuvette containing 0.1 M phosphate buffer, pH 7.0, and the reaction was started with 0.25 units of glutathione transferase (final concentration, one enzymatic unit conjugates 1.0 µmol of 1-chloro-2,4-dinitrobenzene with reduced glutathione per min at pH 6.5 at 25 °C) (readings at 340 nm,
= 9.6 mM
1 cm
1; end of reaction at
4-6 min). Colorimetric determination of GSH was performed by Ellman's
method, slightly modified (22), adding DTNB as the last reagent (0.1 mM final concentration, end of reaction at 2 min). GSSG was
assayed enzymatically by the procedure of Di Simplicio et
al. (21). To determine HbSSG, acid-precipitated proteins were
thoroughly washed with the precipitating solution until no trace of
soluble GSH or GSSG was detected. Alkaline pH (7.5 for rat samples;
12.0 for human samples) was then restored by adding 0.1 M
phosphate buffer, pH 7.8, or an appropriate amount of 0.125 N NaOH. The pellets were resuspended by stirring with a
glass rod. Under these conditions, GSH is released via an SH/SS exchange reaction as described previously by Rossi et al.
(22). The amount of GSH released was then determined enzymatically in the supernatant (see above).
Hemoglobin Preparation--
Hemoglobin was prepared from
stroma-free hemolysate by crystallization as described by Condò
et al. (23). Crystals were then dissolved in 25 mM Tris acetate buffer, pH 8.8, and stored under nitrogen.
The iron oxidation state was checked spectrophotometrically on the
basis of the absorbance ratio at 576/541 nm, taking 1.14 as normal
value. In all experiments, the concentration of the ferric derivative
was monitored as reported by Giardina and Amiconi (24).
Purification of Rat Hemoglobin Components--
Isolation and
purification of the rat hemoglobin components were carried out by
chromatofocusing on a MonoP column (0.5 × 20 cm) (Amersham
Pharmacia Biotech, Sweden). Hemoglobin solution, dialyzed against 0.025 M Tris acetate buffer at pH 8.3, was applied to the column
equilibrated with the same buffer. Elution was achieved at 4 °C by
application of a Polybuffer 96 exchanger (1 ml/min) previously adjusted
at pH 6.5 with 1 M acetic acid. Nine main peaks (A to I)
were resolved. Homogeneity of the fractions was checked by
polyacrylamide gel electrophoresis analysis. In general, components C,
E, and F were the main ones. In particular, the relative abundance of
the hemoglobin components was as follows: isoform A, 0.03-0.05;
isoform B, 0.025-0.035; isoform C, 0.08-0.12; isoform D, 0.02-0.03;
isoform E, 0.22-0.28; isoform F, 0.43-0.51; isoform G, 0.012-0.022;
isoform H, 0.018-0.028; and isoform I, 0.02-0.05.
Hemoglobin Thiol Titration--
Exposed sulfhydryl groups (HbSH)
of hemoglobin were assayed by a modification of Ellman's method (22)
(conditions, 0.1 M phosphate buffer pH 7.4; DTNB, 0.2 mM final concentration). Monitoring traces were recorded by
a stopped-flow apparatus (Applied Photophysics, UK) at room temperature
and read at 450 nm; at this wavelength the HbSH concentration was
calculated taking
= 7.0 mM
1
cm
1.
Cysteine Labeling--
In a typical experiment, a purified rat
hemoglobin component, e.g. isoform F (1 mg, i.e.
15 nmol in tetramer or 90 nmol in free-reacting thiols) in 0.4 ml of
0.1 M Tris-HCl, pH 8.0, was incubated for 5 min with 25 nmol of iodo[2-14C]acetamide plus 5 nmol of cold
iodoacetamide. The sample was then divided into two 0.2-ml aliquots as
follows: to the first aliquot was added 100 mg of guanidine and 100 µg of dithiothreitol and after 30 min 5 µmol of iodoacetamide; the
sample was left for 0.5 h at room temperature and then dialyzed
against a large volume of deionized water. The second aliquot was
treated in the same way, but with the addition of 10 nmol of
iodo[2-14C]acetamide in order to label all free-reacting
cysteines. After lyophilization, the two samples were separately
digested each with 10 µg of trypsin, and the peptide mixtures were
fractionated by HPLC using a Beckman System Gold chromatographer, on a
macroporous reversed-phase column (Aquapore RP-300, 4.6 × 250 mm
(Perkin-Elmer, Rome, Italy)), eluted with a 50-min linear gradient from
0 to 50% acetonitrile/isopropyl alcohol (4:1) in 0.2% (by volume)
trifluoroacetic acid, at a flow rate of 1 ml/min. Elution of the
peptides was monitored by a diode array detector (Beckman model 168) at
200 and 280 nm. Peaks were collected manually, and 10% aliquots were counted for radioactivity. Radiolabeled peptides were subjected to
automated Edman degradation on an Applied Biosystems 476A protein sequencer. Samples (0.1-0.5 nmol) were loaded onto polyvinylidene difluoride membranes, coated with 2 µl of Polybrene (100 mg/ml, 50%
methanol), and run on the sequencer with a Blott cartridge using an
optimized gas phase fast program. In parallel, the very same hemoglobin
component (about 8 nmol in tetramer or 48 nmol in free-reacting thiols)
was also incubated in 0.1 M Tris-HCl, pH 8.0, with 5 nmol
of iodo[2-14C]acetamide for 30 s in a syringe. Then
the mixture was applied to a Sep-Pak cartridge (Millipore, Milford,
MA), washed with 4 ml of water, 4 ml of 0.2% (by volume)
trifluoroacetic acid, and eluted with 2 ml of acetonitrile/isopropyl
alcohol (4:1) in 0.2% trifluoroacetic acid. After lyophilization, the
hemoglobin chains were separated (see below), and a 10% aliquot was
counted for radioactivity. Cyanogen bromide cleavage was obtained by
adding few crystals of the reagent to 20 µg of
chain dissolved in
70% formic acid.
Separation and Sequence Analysis of the Hemoglobin
Chains--
Hemoglobin chains were separated by HPLC under the
conditions described above (see "Cysteine Labeling").
The reversed phase column was eluted with a 40-min linear gradient from
30 to 50% of the same solvent systems as above. Amino acid
composition, sequence analysis, and peptide maps of chains from the
various hemoglobin components were performed as described previously
(25).
Electrophoretic Search for Hemoglobin Polymerization through
Disulfides (HbSSHb)--
Rat erythrocytes were separated by
centrifugation of the whole blood at 2,800 rpm for 10 min. The plasma
and buffy coat were removed, and then the red cells were washed three
times with 10 times their volume of isotonic phosphate-buffered saline,
pH 7.4, supplemented with 10 mM D-glucose.
After removal of the supernatant from the final wash, erythrocytes (2 ml, 40% v/v suspension in the washing solution) were incubated with 2 mM (final concentration) diamide or t-BOOH. A
200-µl aliquot of cell suspension was hemolyzed by adding 50 volumes
of 10 mM phosphate, pH 7.4, at various times (5, 15, and 30 min). After centrifugation (12,000 × g for 15 min) the
supernatant was diluted 1:1 and subjected to analysis by SDS (0.1%
(w/v)) polyacrylamide (12%) gel electrophoresis, according to the
method of Laemmli (26), using a sample buffer without
-mercaptoethanol and in the presence of 2 mM
N-ethylmaleimide. Chemicals for gel electrophoresis were
from Bio-Rad (Segrate, MI, Italy). A Bio-Rad model 220 (Bio-Rad) slab
gel apparatus was used.
Kinetic Experiments--
Samples of hemoglobin (0.1-0.3 mg/ml)
in 0.2 mM Tris acetate buffer, pH 8.6, were rapidly mixed
with equal volumes of a solution containing 1 mM DTNB in
0.2 M buffers (sodium acetate, pH 5.0-6.0, sodium/potassium phosphate, pH 6.0-8.0, and Tris acetate, pH
8.0-9.0), by means of an Applied Photophysics MV 17 stopped-flow
apparatus: tracings were recorded at 450 nm. Data were fitted to two
(e.g. isoform B) or three (isoforms D, E, and F)
exponentials, imposing the same amplitude for each kinetic process. The
kinetics of carbon monoxide binding to hemoglobin isoforms with free
and glutathione-blocked sulfhydryl groups were measured with the same
stopped-flow apparatus. Photolysis experiments were carried out using
an instrument developed in our laboratory. Briefly, the 5-ns pulse of a
Q-switched Quanta Systems Nd-YAG HYL101 laser equipped with a frequency
doubler (maximal output, 200 mJ at 532 nm) was focused onto a 4-face
fluorescence cuvette containing the hemoglobin component equilibrated
with 1 atm of carbon monoxide in the gas phase; oxygen was removed by
addition of a few grains of sodium dithionite. The transmittance of the
sample was probed continuously by a 300-watt lamp as light source, a
Spex Minimate monochromator, and a Hamamatsu R1398 photomultiplier tube. The light beam for observation was oriented at 90° from the
laser beam. The current generated by the photomultiplier was displayed
with a Tektronix TDS 360 digital oscilloscope triggered by the laser
Q-switch and output as a MS-DOS ASCII file. In a typical experiment,
the optical changes caused by 32-128 laser shots were averaged, in
order to improve the signal to noise ratio. The energy of the laser
pulse was usually kept to 20 mJ or less, and the degree of photolysis
ranged between 50 and 10%. The standard deviation for replicate
determinations of rate constants is approximately 30 to 60% of the
determined value, independently of pH and hemoglobin species.
Oxygen Binding Equilibria--
Values of p1/2
(the oxygen partial pressure required to half-saturate the hemes) and
n (the empirical Hill coefficient, a measure of the
cooperativity among the hemes) for oxygen association to hemoglobin
were determined at 20 °C from light absorbance changes accompanying
the oxygen binding by a tonometric method (24).
Measurement of Glucose-6P-Dehydrogenase (EC 1.1.1.49) and
Glutathione Reductase Activities--
Enzymatic determinations were
carried out at room temperature on hemolysate, previously passed
through a Sephadex G25 column, according to the standard methods (27).
Catalytic parameters were obtained by fitting experimental data to the
Michaelis-Menten equation by Sigmaplot software (Jandel Scientific,
version 2.01).
Prediction of Three-dimensional Structure of Isoform F--
The
three-dimensional models of the rat isoform F subunits were based on
the structural template of human ligated and unligated hemoglobin,
whose atomic coordinates were recovered from the protein (crystallographic) data distribution tape (Protein Data Base, codes
1hho and 3hhb, respectively; see Ref. 28). The models were built with
the HOMOLOGY and DISCOVER modules in the package Insight II (MSI, Inc.)
running on a Silicon Graphics Indigo 2 Extreme Graphics. Conformation
of the substituted side chains was manually adjusted to remove steric
strains, and the model was finally refined with 100 cycles of energy
minimization. Stereochemical properties, potential H bonds, solvent
accessibility surface, and secondary structures were calculated with
the programs PROCHECK (29), HBPLUS (30), NACCESS (31), and DSSP (32),
respectively. Drawing were produced with MIDAS (33) and MOLSCRIPT
(34).
 |
RESULTS |
Effects of Diamide Treatment on Intraerythrocyte
Thiols--
Treatment of human and rat blood with diamide evidenced
remarkable differences in the network of intraerythrocyte metabolic correlations (see Table I). In
particular, HbSSG, but not GSSG, was produced after drug addition to
rat erythrocytes; on the other hand, only GSSG, but not HbSSG,
concentration increase occurred in human erythrocytes after the same
treatment. Moreover, the latter cells, but not rat erythrocytes, were
able to restore (within 2 h incubation at 37 °C) control values
of GSH and HbSSG (see Table I).
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Table I
Kinetics of formation of oxidized glutathione (GSSG) and mixed
disulfides (HbSSG) after addition of diamide (2 mM, final
concentration) to 2-ml blood sample (hematocrit = 0.4) from rat or
from man at room temperature
Values are expressed in nmol/ml.
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Even though HbSSHb might form in rat erythrocytes, negligible or no
association of hemoglobin tetramers through disulfide bridges occurred
under the experimental conditions chosen, at least as measured (4) by
gel electrophoresis experiments (see Fig.
1); on the contrary, abnormal human
hemoglobin Porto Alegre (Ser-9
(A6)
Cys) spontaneously forms
disulfide polymers that can already be detected after 3 days of shelf
storage (35). Therefore, it appeared interesting to investigate the
molecular bases that limit the polymerization of rat hemoglobin. The
formation of disulfides from thiol groups only requires the presence of an electron acceptor, such as oxygen; the chemical nature of bond formation (possibly depending on metal ions that transiently bind to
the thiols (36)), however, is not completely understood, even though
sulfenic acid as electrophilic intermediate seems to occur. Since (i)
it is impossible to measure the reduction potential
(E'0) value of the hypothetical disulfide bond
in HbSSHb; (ii) E'0 values differ greatly for
these bonds, even if they are in the same sequence motif (37-39), and
(iii) disulfide bonds exist in a cell only if the overall reduction
potential value in the cytosol allows (40); the redox buffer capacity
of rat erythrocytes was set to zero. Thus, rat blood was incubated with
diamide at a concentration so high to generate HbSSG irreversibly (see
e.g. Table I); this event was taken as an index of complete
consumption of GSH (the chemical species that usually restores the
control values of HbSH and GSSG). Because HbSSHb was not observed in
rat erythrocytes even under the strongest (5 mM diamide)
oxidative stress, the factors that prevent its formation were
considered not strictly chemical. Fig. 2
reports computer-generated models of complexes between two
/
dimers of hemoglobin Porto Alegre (panel A) and of rat
isoform F (panel B), oriented in such a way to present their
potentially reactive thiols (Cys-125
(H3) and Cys-9
(A6),
respectively) at the least distance compatible with steric hindrance.
These thiols can approach each other 2.5 Å in the abnormal human
protein and no less than 4 Å in rat hemoglobin. This model-based
evidence suggests that the absence of polymerization into rat
erythrocytes treated with oxidants could mainly be a result of steric
factors.

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Fig. 1.
Electrophoretic pattern of rat hemolysate
from treated red blood cells. Rat erythrocytes (40% (v/v)) in
isotonic phosphate-buffered saline, pH 7, supplemented with 10 mM D-glucose, were treated with 2 mM (final concentration) diamide or t-BOOH for
different times at room temperature. Samples were then hemolyzed and
treated with Laemmli's solution without mercaptoethanol and in the
presence of 2 mM N-ethylmaleimide.
Lanes (from left to right): lane
1, hemolysate in 2 mM diamide, 5-min incubation;
lane 2, hemolysate in 2 mM diamide, 15 min;
lane 3, hemolysate in 2 mM diamide, 30 min;
lane 4, hemolysate in 2 mM t-BOOH, 5 min; lane 5, hemolysate in 2 mM
t-BOOH, 15 min; lane 6, hemolysate in 2 mM t-BOOH, 30 min; lane 7, control
hemolysate; lane 8, molecular mass standards (from
top to bottom: 94, 67, 43, 30, 20.1, 14.4 kDa).
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Fig. 2.
Space-filling models of two / dimers of
human hemoglobin mutant Porto Alegre (Ser-9 (A6) Cys)
(panel A) and isoform F of rat hemoglobin (panel
B), which were manually docked to present one in front of the
other potentially reactive thiols (Cys-125 (H3) and Cys-9 (A6),
respectively). In both cases, the two dimers are colored with
light and dark gray, and cysteines with
black.
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S-Thiolation of Hemoglobin Components in Rat Erythrocytes Treated
with Oxidants--
Titratable sulfhydryl groups in proteins react with
DTNB with different rate constants, depending on local environmental
factors (41, 42). The time course of DTNB combination with sulfhydryl groups of rat hemoglobin components (see below) was described by either
two or three pseudo first-order processes of equal amplitude, whose
rate constants fell within one of the three classes of thiols defined
as follows in kinetic terms (at a DTNB concentration of 0.5 mM): fast reacting HbSH (fHbSH) with a
t1/2 value <100 ms; slow reacting HbSH (sHbSH) with
t1/2 = 30-50 s; and very slowly reacting HbSH
(vsHbSH) with t1/2 = 180-270 s; on the other hand,
thiols in human hemoglobin (Cys-93
(F9)) reacted homogeneously with a
t1/2 value of about 30 s, a value corresponding
closely to sHbSH of the rat protein. Since all kinetic processes
described above induced similar or identical optical changes and were
second-order with respect to DTNB concentration, they were assigned to
the reaction of a couple of cysteinyl residues per tetramer;
accordingly, the end point of the thiol reaction traces allowed the
estimation of 4 or 6 titratable Cys/tetramer on rat hemoglobin
(depending on the component, see below) whereas, as expected, only two
were calculated on the human protein.
Nine main peaks (A to I) were resolved by chromatofocusing between pH
8.3 and pH 6.5 (Fig. 3, panel
A) of rat hemoglobin from blood of control animals. All peaks (see
Table II) showed two slow and two very
slow reacting thiols per tetramer; however, isoforms D-G also
contained fast reacting thiols as follows: two per tetramer were
present in D-F isoforms, whereas in the components G one
fHbSH/tetramer and one HbSSG were determined. The latter result was in
line with the evidence that after treatment of rat hemolysate with
dithiothreitol the peak corresponding to the G component disappeared
and peak F increased (Fig. 3, panel B) (meaning that isoform
G corresponded to the component F with one thiol blocked by
glutathione, this reactant being identified by colorimetric and HPLC
procedure (22)).

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Fig. 3.
Elution profiles (between pH 8.3 and 6.5) of
rat hemolysate, i.e. total hemoglobin components.
Panel A, control hemolysate (4 mg/ml); panel B,
hemolysate treated with 1 mM dithiothreitol; panel
C, hemolysate (4 mg/ml) treated with 30 µM diamide
plus 40 µM GSH; panel D, hemolysate (4 mg/ml)
treated with 175 µM diamide plus 200 µM
GSH.
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Table II
Association rate constants of DTNB to the oxyderivative of various
isoforms of rat hemoglobin and human hemoglobin in 0.1 M phosphate buffer, pH 7
Experimental conditions in the reaction vessel are as follows: [Hb] = 6.8 µM in heme; [DTNB] = 0.34 mM; 20 °C.
kf, ks, and
kvs refer, respectively, to second-order rate
constants with fast reacting, slow reacting, and very slow reacting
thiols of the investigated isoform; FG2 symbolizes the
component F with the two fast reacting sulfydryls blocked by
glutathione; F* indicates the component G after treatment with
dithiothreitol.
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The ability of some HbSH to produce HbSSG after diamide or
t-BOOH treatment was analyzed in more detail using both
erythrocytes and purified hemoglobins (human and rat). In the presence
of GSH, diamide was reported (3, 4) to form mixed disulfides with hemoglobin via a two-step reaction, i.e. (HbSH + diamide
HbS-diamide) followed by (HbS-diamide + GSH
HbSSG + diamide
(reduced form)). As shown (see Figs. 3, panels C and
D, 4, panel A, and
5, panel A), under these
conditions only fHbSH of rat hemoglobin was able to produce HbSSG. On
the other hand, treatment with t-BOOH formed GSSG that in
turn produced mixed disulfides through a thiol/disulfide exchange (3,
4), according to the following scheme: HbSH + GSSG
HbSSG + GSH. As
shown in Figs. 4, panel B and 5, panel B, both
HbSSG production and HbSH depletion were evident only with rat
hemoglobin. All hemoglobin isoforms were separated by chromatofocusing
after t-BOOH (not shown) or diamide treatment of rat
hemolysate (Fig. 3). Similar results were obtained with erythrocytes.
With both oxidants a marked increase in peak G area, a decrease in
concentration of isoforms D-F, and no changes in peak areas of A-C,
H, and I components were found. Furthermore, in both cases HbSSG
formation determined the appearance of new peaks retarded with respect
to the original components (in particular, see Fig. 3, panel
C), since the glutathione-hemoglobin adduct is more acidic
relative to the unreacted protein. In summary, thiol titration with
DTNB demonstrated that (i) fast reacting HbSH (present only in rat
hemoglobin) was depleted after oxidant addition, and (ii) neither rat
slow reacting HbSH nor human HbSH were grossly affected by the
treatment with diamide. These observations, as a whole, indicate that
only isoforms of rat hemoglobin with fHbSH are able to produce
disulfides either via thiol/disulfide exchange (after t-BOOH
treatment) or via diamide intermediate (after diamide addition).

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Fig. 4.
Comparison of time courses relative to HbSH
level changes in rat ( , ) and human ( ) blood (hematocrit, 0.4)
incubated with 2 mM diamide (panel A) or 2 mM t-BOOH (panel B) at room
temperature. After treatment, erythrocytes were washed with
isotonic saline and hemolyzed; then the solution was passed
through a Sephadex G25 column and HbSH determined. , rat (v)sHbSH;
, human HbSH; , rat fHbSH.
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Fig. 5.
Comparison of time courses relative to HbSSG
concentration changes in rat ( ) and human ( ) hemoglobin (0.5 mM in heme, in 0.1 M phosphate, pH 7.0),
incubated at room temperature with 1 mM diamide plus 1 mM GSH (panel A) or with 1 mM GSSG
(panel B).
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Identification of Fast Reacting Cysteinyl Residue--
All
cysteine-containing tryptic peptides were identified in the samples
radiolabeled under denaturating conditions, by sequence analysis. After
treatment with iodo[2-14C]acetamide and addition of cold
iodoacetamide, the identification of the radiolabeled peptides was made
on the basis of their chromatographic behavior. The specific
radioactivity of each peptide indicated that Cys-93
(F9) and
Cys-125
(H3) reacted faster than Cys-13
(A11).
In order to discriminate the fastest reacting sulfhydryl group between
Cys-93
(F9) and Cys-125
(H3), a second labeling experiment was
performed using a lower reagent/thiol ratio and a shorter incubation
time. Radioactivity measurements of the anilinothiazolinone derivatives
collected at each cycle of automated Edman degradation of a sample of
labeled
chain cleaved at methionines with CNBr revealed that, under
these conditions, only in the test tube corresponding to the
derivatives of Cys-125
(H3) (cycle 16) was there a significant increase in radioactivity.
Next, the samples that did not contain a fast reacting cysteinyl
residue (such as isoforms A-C) were treated with 4-vinylpyridine under
denaturating conditions, then cleaved with CNBr, and lastly subjected
to sequence analysis. The phenylthiohydantoin derivatives of the
fragments expected from CNBr cleavage were clearly identified during
the automated Edman degradation of the peptide mixture. In cycle 16, corresponding to position
125 of the pertinent CNBr fragment, no
pyridylethylcysteinyl derivative was detected, but a serine residue was
present, as expected on the basis of sequences (17). Therefore, the
S-thiolation rate of rat HbSH increases as follows:
Cys-13
(A11) < Cys-93
(F9) < Cys-125
(H3).
Functional Properties of Rat Hemoglobin Isoforms--
It was shown
(41, 42) that the thiolate anion form of the sulfhydryl group
preferentially reacts with DTNB. Therefore, the reactivity of thiols at
each pH will depend on the fractional population of the thiolate
anion.
The fast reacting sulfhydryls in rat oxyhemoglobin showed an ample
effect of pH, such that the rate constant observed at pH 8 was more
than five times higher than that observed at pH 6. The pH dependence of
the observed thiolation rate constant (k) of Cys-125
(H3)
can be given by Equation 1.
|
(Eq. 1)
|
where K represents the acid dissociation constant for
the thiol group, and ks
and
kSH indicate the rate constants for the reaction
of DTNB with the thiolate anion and the protonated sulfhydryl,
respectively. Allowing for experimental error (±10%), values of the
rate constants for the dissociated (3.9 × 104
M
1 s
1) and protonated
(practically zero) fHbSH can be calculated from Equation 1. The
undissociated Cys-125
(H3) therefore makes no significant
contribution to the observed rate.
The deprotonation profile of fHbSH is representative of only one
ionization (see Fig. 6, panel
B); this finding precludes the possibility of important
contributions to the reactivity from other ionizable groups on the
protein, because this would have appeared as an additional inflection
point in the pH profile (as observed, for instance, in dog hemoglobin
(43)). This conclusion was reinforced by the three-dimensional model of
rat isoform F, which does not predict the formation of any salt bridge
that could restrict access to Cys-125
(H3).

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Fig. 6.
Dependence on pH of the second-order rate
constant (k) for the reaction between DTNB and thiols
present on glutathione (panel A), rat hemoglobin components
( , isoform F; , isoform C), and human hemoglobin ( ).
Panel B, reports data of fHbSH; panel C, reports
those of sHbSH, and panel D reports those of (v)HbSH (values
in panel D were multiplied for 10). Experimental conditions
in the reaction vessel are as follows: hemoglobin concentration, 6.5 µM in heme; DTNB concentration, 0.8 mM;
temperature, 20 °C; 0.2 M buffers (acetate, pH 4.0-6.0;
phosphate, pH 6.0-8.0; Tris acetate, pH 8.0-9.0).
|
|
A pH dependence similar to that of fHbSH was also monitored for reduced
glutathione (Fig. 6, panel A), whose reactivity is known not
to be influenced by electrical charges on the molecule (41, 42). Since
fHbSH had an apparent pK equal to 6.89 (see Fig. 6,
panel B), it is not surprising that it reacted faster than
glutathione whose higher pK (equal to 8.9, under the very same conditions; see Fig. 6, panel A) should generate a much
smaller fractional population of anionic form of thiols at every pH
value between 6 and 8. Moreover, since under intra-erythrocyte
conditions (pH ~7.2) fHbSH is in the anionic form, i.e.
more nucleophilic and hence more reactive, for ~60% (see Fig. 6,
panel B), it conjugates with electrophilic compounds much
faster than (v)sHbSH which is dissociated for only a few percent (see
Fig. 6, panels C and D). In particular, around
neutrality the rate constant of (v)sHbSH with DTNB was approximately
lower by a factor of 5-10 than that of sHbSH and 4000-fold lower than
that of fHbSH (Table II). These considerations apply to glutathione
too; in fact, at pH 7.2 the value of S-thiolation rate
constant, as calculated from Equation 1, for fHbSH (2.7 × 104 M
1 s
1) is 1 order of magnitude higher than that for glutathione (2.6 × 103 M
1 s
1).
Such large differences in rate constants among the three kinds of
hemoglobin thiols suggest that, in addition to the ionization state,
accessibility could make a contribution to the observed reaction rate
(see below). By analogy with older observations (36, 44, 45), these
reactive thiols were assumed to correspond to partially buried
cysteinyl residues. The ratio between the rate constants reported in
Table II strongly suggests that the slow and very slow reacting thiols
should have a similar reactive nature (i.e. they should be
similarly embedded into the protein molecule and similarly protonated),
whereas the fast reacting thiols are expected to stand out as a
peculiar feature of some rat hemoglobin isoforms and cannot easily and
simply be interpreted as less buried (see below).
As a further step of this analysis, the rate constants of DTNB
combination with the deoxyhemoglobin derivative were measured. It is
well known that the reactivity of the only titratable couple of thiols
in human tetrameric hemoglobin (Cys-93
(F9)) depends on the
quaternary conformation, being slower in the deoxy state (44, 45).
Since DTNB is reduced by dithionite, studying its reaction with
unligated hemoglobin demands that oxygen be removed by evacuation and
equilibration with nitrogen. However, under these conditions some
contamination with oxygen occurs frequently, and therefore a systematic
check for the presence of the oxygenated derivative was done by
recording the optical change induced by mixing with dithionite; the
amount of oxyhemoglobin in the samples never did exceed 10%. Under
these conditions small deviations from the expected two or three
exponentials were observed; nevertheless, the data unequivocally showed
that fast thiols were insensitive to the ligation state of the heme
iron and the allosteric conformation of the protein (e.g. at
pH 7, k for fHbSH thiolation of rat isoform F was 2.94 × 104 M
1 s
1 and
2.91 × 104 M
1
s
1 for oxy and deoxy derivatives, respectively; see also
Table II).
A necessary complement to the results described so far was the analysis
of the functional properties of rat hemoglobin isoforms in the absence
and in the presence of reagents (glutathione or DTNB) bound to fHbSH.
The oxygen binding isotherm of rat isoform F (2 fHbSH/tetramer) was
demonstrated to be superimposable to that of the same hemoglobin
component with the fHbSH blocked by glutathione (isoform
FG2 in Table III; see also
Fig. 7). Moreover, the overall rates of
carbon monoxide combination with T state (as measured by stopped flow)
and that with R state (by partial photolysis) of rat isoform F were
insensitive to the presence of glutathione bound to fHbSH (Table III).
These results unequivocally demonstrate that, in rat isoform F, fHbSH
is not functionally linked to the hemes nor does it alter the ligand
affinity at hemes.
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Table III
Oxygen equilibrium (p1/2 and n) and carbon monoxide
kinetic (kR and kT) parameters for rat and human
hemoglobins at 20 °C
Experimental conditions were as follows: (i) equilibria were carried
out in 0.1 M HEPES, pH 7, and 20 µM
concentration in heme; (ii) kinetic data were collected in 0.1 M Tris/HCl, pH 7.4, and 5 µM in heme (value
in parentheses was obtained by photolysis experiments). The symbol
FG2 represents isoform F with the two fHbSH (i.e.
Cys-125 (H3)) blocked with glutathione. p1/2 is
the oxygen partial pressure required to yield half-saturation of hemes,
and n the Hill empirical constant (24);
kR and kT represent the overall
rate constants for carbon monoxide combination with R and
T quaternary state of hemoglobin, respectively.
|
|

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Fig. 7.
Oxygen binding curve of isoform F (4 mg/ml)
with fHbSH free ( ) and blocked with glutathione ( ) in 0.1 M HEPES, pH 7.4, and 20 °C. Y is the
fraction of hemes occupied with oxygen, and log
pO2 is the logarithm of the oxygen partial
pressure (proportional to the oxygen chemical potential).
|
|
Structural Bases of the High Reactivity of fHbSH Relative to
(v)sHbSH--
It is generally accepted that the major factors
influencing pK values in protein ionizable groups are the
interactions with other net charges or dipoles present on the
macromolecule surface (46). Therefore, the physical nature of the
potential interactions stabilizing the thiolate anion of fHbSH was
investigated by molecular graphics. A positively charged group in the
proximity of Cys-125
(H3) would be the most logical candidate, but
none was apparent on the model of the oxy as well as deoxy derivative
built on the structural template of human hemoglobin. However, in the
model (see Fig. 8, panel A)
the anion of Cys-125
(H3) is predicted to form a hydrogen bond with
Ser-123
(H1) (2.9 Å distance between sulfur and oxygen atoms in both
oxy and deoxy derivative), which therefore appears to be the most
likely candidate to explain the low pK value of fHbSH. This
interaction in fact can substantially contribute to the charge
stabilization of the thiolate form in fHbSH. In its turn, Fig. 8,
panel B, helps to make it clear why Cys-13
(A11) is the
least reactive among the free cysteinyl residues in rat hemoglobins. In
fact, this thiol is presumably H-bonded with His-113
(GH1) of the
same chain (see Fig. 8, panel B), the distance between
sulfur and nitrogen atoms being 3.5 and 4.1 Å in the deoxy and oxy
form, respectively. Such an interaction is expected to stabilize the
protonated form of Cys-13
(A11) and accordingly to decrease its
reactivity at neutral pH in comparison to Cys-93
(F9), which is not
involved in any interaction with net charges or polar groups on the
protein surface. As a whole, these structural data are consistent with,
and help to explain in rational terms, the functional behavior of the
three classes of thiols.

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Fig. 8.
Stereo drawing of predicted interactions
between Cys-125 (H3) and Ser-123 (H1) (panel A) and
between Cys-13 (A11) and His-113 (GH1) (panel B) in the
model of rat hemoglobin (isoform F). Residues in subunit in
panel A are linked by open bonds. Amino acids are
labeled with one-letter code. Potential hydrogen bonds are
represented with a dashed line.
|
|
Additional factors are expected to modulate rat HbSH reactivity toward
DTNB. Even though local conformational fluctuations are more useful for
the interpretation of chemical data, it is interesting that
static-accessible surface to solvent (47) shows a parallel trend to the
thiolate fraction of each HbSH class. Thus, Cys-125
(H3) (70% static
accessibility) is 10 to 15 times more exposed to the solvent than
Cys-93
(F9) and of Cys-13
(A11), respectively, indicating that, in
addition to the ionization state, steric factors contribute to the low
reactivity of (v)sHbSH toward DTNB.
Probing Some Aspects of Reducing Equivalent Transfer to Disulfide
in Rat Erythrocytes--
It is well known that NADPH is the primary
nucleotide that channels reducing equivalents to GSSG and/or other
oxidants (48). Therefore, the availability of NADPH should be the
ultimate determinant of cellular thiol redox status following an
oxidative stress. In the intact erythrocyte the supply of NADPH depends
on the pentose phosphate pathway, i.e. on
glucose-6-phosphate dehydrogenase activity (49). The factors
controlling the interconversion of thiols and disulfides are not fully
understood (50). In any case, glutathione reductase reduces the
disulfide bond in GSSG and therefore contributes to the cellular
mechanism for protection and repair under oxidative stress; even though
this enzyme itself contains a thiol group, it is not very susceptible
to oxidants (51). Therefore, some catalytic parameters (see Table
IV) of these two enzymes from rat and
human erythrocytes were determined. Whereas intracellular glucose-6-P-dehydrogenase activity was comparable for both animals, glutathione reductase appeared to work less efficiently in rat erythrocytes, the fraction of enzyme bound in any form whatsoever to
NADPH and/or GSSG (as measured by Km) being smaller in rat red blood cells relative to the human ones (at similar intracellular concentration of these substrates).
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|
Table IV
Comparison (n 5) of some catalytic parameters of two enzymes in rat and
human erythrocytes at pH 7.2 and 25 °C
G6P symbolizes glucose 6-phosphate.
|
|
 |
DISCUSSION |
Since amino groups generally remain protonated below pH 10, the
major nucleophilic functionality available in biological systems are
thiols, which, accordingly, are the chemical groups that provide strong
defense against electrophilic species. One of the objectives of the
present research was to characterize in kinetic terms the reactivity of
rat hemoglobin thiols under conditions mimicking the physiological
ones. Therefore, this study was limited to free reactive thiols,
whereas the so-called masked sulfhydryls, i.e. Cys-104
(G11) and Cys-111
(G18), which are located at the
1
1 interface and whose reactivity is
controlled by the tetramer dissociation beyond the dimer stage (52),
were neglected.
The peculiar reactivity of fHbSH present on most of the rat hemoglobin
components is clearly responsible for the formation of HbSSG after
oxidative stress by diamide. Thiols in proteins are integral to a
number of cellular functions, including protein folding, enzyme
catalysis, and metabolic regulation (36, 53). The reported findings
clearly suggest an involvement of fHbSH as a direct moderator of
oxidative stress at least in rat erythrocytes. Red cells are regularly
subjected to high oxygen tension and are among the first body cells
exposed to exogenous oxidative substances that are ingested, inhaled,
or injected. The importance of an antioxidant function in erythrocytes
is well known and is shown, e.g. by the massive
oxidant-induced hemolysis seen in subjects with a marked deficiency of
glucose-6-phosphate dehydrogenase (54).
The possible contribution of fHbSH to the intracellular detoxifying
mechanism appears to be in line with the definition of the term
antioxidant (55, 56) for both the intrinsic chemical properties and the
intraerythrocyte concentration. However, this thiol system seems to be
suited to resist acute episodes of oxidant fluxes but not the prolonged
ones, because its efficiency is somewhat impaired by the long recovery
time (see Table I). Since the reactivity of Cys-125
(H3) is not
linked to the oxygenation nor to the quaternary state of hemoglobin
(see Table III), fHbSH are expected to quickly produce
S-conjugates in both the arterial and venous blood; in other
words, flow in the blood circulation is expected not to induce a cycle
(speeding up and slowing down) in the reactivity of fHbSH, as described
for the highly conserved Cys-93
(F9) (and possibly true also for
Cys-13
(A11)) that, being oxygen-linked, modulates arterial-venous
differences in intraerythrocyte S-nitrosothiols (57, 58).
Moreover, the S-conjugation capacity of fHbSH is very high.
In fact, from the mean red cell volume (61.6 fl), the mean erythrocyte
hemoglobin concentration (21.1 pg), the molecular mass of tetrameric
hemoglobin (65 kDa), the fraction of isoforms carrying fHbSH (0.76),
and the number of fast reacting cysteinyl residues per tetramer (two),
an intracellular level of fHbSH equal to 8 mM can be
calculated; this value, which is four times that of glutathione (2 mM), is great enough to force the conclusion that fHbSH
works as a buffer system able to trap a large amount of attacking
(electrophilic) species in a very short time. Finally, in the
particular case investigated it can be also speculated that the
efficient nonenzymatic antioxidant defense offered by fHbSH is able to
compensate for the poor catalytic activity of glutathione reductase in
rat erythrocytes (see Table III).
In conclusion and from a more general point of view, the reported
results emphasize an additional function of some kinds of protein
cysteinyl residues, i.e. a direct detoxification role by
conjugation more efficient than low molecular weight thiols, such as
glutathione.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Ministero
dell'Università e della Ricerca Scientifica e Tecnologica (40 and 60%, respectively) and by the Consiglio Nazionale delle Ricerche of Italy.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biochemical Sciences, University of Rome "La Sapienza," Piazzale
Aldo Moro 5, 00185 Rome, Italy. Tel.: 0039-6-4450291; Fax:
0039-6-4440062; E-mail: amigino{at}axrma.uniroma1.it.
1
The abbreviations used are: HbSSG,
glutathione-hemoglobin mixed disulfide; t-BOOH,
tert-butylhydroperoxide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); HbSSHb, hemoglobin-hemoglobin disulfides; HbSH, reactive sulfhydryl groups on hemoglobin; fHbSH, fast reacting sulfhydryl groups
on rat hemoglobin components; vsHbSH, slow reacting sulfhydryl groups
on rat hemoglobin components; vsHbSH, very slow reacting sulfhydryl
groups on rat hemoglobin components; (v)sHbSH, very slow reacting
and/or slow reacting sulfhydryl groups on rat hemoglobin components;
HPLC, high pressure liquid chromatography.
 |
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