Hemoglobin-superoxide dismutase-chemical linkages that create a dual-function protein

Article abstract of DOI:10.1021/ja050339r

Chemical reagents were designed to cross-link and connect hemoglobin and superoxide dismutase, combining the oxygen transport and superoxide-removal capabilities of the red cell in a dual-function protein. Reaction of 1 with thiol-protected hemoglobin followed by reduction produces cross-linked hemoglobin with a free thiol on the cross-link. Reaction of SOD with 5 produces a cross-linked protein with a maleimide on the cross-link. Addition of the hemoglobin-thiol to the SOD-maleimide produces a protein with the desired dual properties. Hemoglobin's oxygenation cooperativity is lowered as a result of being in the conjugate, while SOD's activity is equal to that of the native protein.

Full text of DOI:10.1021/ja050339r

Published on Web 05/13/2005
Hemoglobin-Superoxide DismutasesChemical Linkages That
Create a Dual-Function Protein
Amer Alagic, Agnieszka Koprianiuk, and Ronald Kluger*
Contribution from DaVenport Laboratories, Department of Chemistry, UniVersity of Toronto,
Toronto, Canada, M5S 3H6
Received January 18, 2005; E-mail: rkluger@chem.utoronto.ca
Abstract: Chemical reagents were designed to cross-link and connect hemoglobin and superoxide
dismutase, combining the oxygen transport and superoxide-removal capabilities of the red cell in a dual-
function protein. Reaction of 1 with thiol-protected hemoglobin followed by reduction produces cross-linked
hemoglobin with a free thiol on the cross-link. Reaction of SOD with 5 produces a cross-linked protein with
a maleimide on the cross-link. Addition of the hemoglobin-thiol to the SOD-maleimide produces a protein
with the desired dual properties. Hemoglobin’s oxygenation cooperativity is lowered as a result of being in
the conjugate, while SOD’s activity is equal to that of the native protein.
Introduction
Our interest in the structurally defined chemical stabilization
of hemoglobin8,9 led us to seek a method that would produce a
defined conjugate of hemoglobin and SOD in order to assess
the effects of a link on the properties of the constituent proteins.
We report the preparation of a cross-linked hemoglobin with a
free thiol on its cross-link from reagent 1, formation of a cross-
Oxygen coordinated to ferrous hemes of hemoglobin within
red blood cells is subject to reduction to superoxide, a reactive
species that is removed by reactions promoted by intracellular
superoxide dismutase (SOD), converting superoxide to hydrogen
peroxide.1-4 Hemoglobin-based oxygen carriers (HBOCs) are
being evaluated as substitutes for red cells in transfusion
medicine. These materials also produce superoxide (and meth-
emoglobin) from coordinated oxygen.5 This phenomenon is
associated with reperfusion injury, tissue damage resulting from
transfusion of an oxygen donor after an organism has been
subject to an oxygen deficiency.6
The potential utility of a combination of hemoglobin and SOD
in a circulating oxygen carrier has been demonstrated by Chang
and co-workers, who combined an 8-fold excess of glutaral-
dehyde with hemoglobin and small amounts of SOD (and
catalase).6 The resulting material, which is a heterogeneous
mixture from its method of formation, was shown to decrease
the concentration of reactive oxygen species in circulation
through its enzymic activities. The material has been used
successfully in further studies of its physiological effects.7 This
work shows that only a small amount of SOD activity is
necessary to protect the system. The accessibility of intracellular
catalase may be sufficient to remove peroxide or it may be
necessary to form a specific conjugate.
linked SOD with a thiol-accepting maleimide group on its cross-
link from reagent 5, and formation of the conjugate from
reaction of the thiol and maleimide groups of the two modified
proteins.
Results
Synthesis of Cross-Linkers. Reagents for reaction with each
protein component were produced by activation of carboxylic
acids, followed by reaction with amines to form amides. Scheme
1 summarizes the route we used to produce 1, which we
designed to stabilize hemoglobin and to introduce an accessible
thiol. This material incorporates a disulfide that is converted to
a thiol by reduction. This feature simplifies synthetic steps as
well as protein modification. Scheme 2 represents the route used
(1) Sutton, H. C.; Roberts, P. B.; Winterbourn, C. C. Biochem. J. 1976, 155,
503-10.
(2) Winterbourn, C. C.; McGrath, B. M.; Carrell, R. W. Biochem. J. 1976,
155, 493-502.
(3) Lynch, R. E.; Lee, R.; Cartwright, G. E. J. Biol. Chem. 1976, 251, 1015-
9.
(4) Kosaka, H.; Tyuma, I.; Imaizumi, K. Biomed. Biochim. Acta 1983, 42,
S144-8.
(5) D’Agnillo, F.; Chang, T. M. S. Biomater., Artif. Cells, Immobilization
Biotechnol. 1993, 21, 609-21.
(8) a. Kluger, R. Synlett 2000, 12, 1708-1720. b. Schumacher, M. A.; Dixon,
N. M.; Kluger, R.; Jones, R. T.; Brennan, R. G. Nature (London) 1995,
375, 84.
(9) Kluger, R.; De Stefano, V. J. Org. Chem. 2000, 65, 214-219.
(6) D’Agnillo, F.; Chang, T. M. S. Nat. Biotechnol. 1998, 16, 667-671.
(7) Powanda, D. D.; Chang, T. M. S. Artif. Cells, Blood Substitutes, Im-
mobilization Biotechnol. 2002, 30, 23-37.
9
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J. AM. CHEM. SOC. 2005, 127, 8036-8043
10.1021/ja050339r CCC: $30.25 © 2005 American Chemical Society

Hemoglobin−Superoxide Dismutase
A R T I C L E S
Scheme 1. Synthesis of the Cross-Linking Reagent (1) for
Hemoglobin
We produced a peptide map from HPLC peptide elution
patterns resulting from tryptic digestion of modified globin
chains to determine the sites that had reacted with 1. The
products were separated by analytical C18 reversed phase
HPLC. The modification decreases the relative amounts of
peptides âT-1, âT-9, and âT-10. On the basis of the known
sequence of these peptides, the result establishes that the â
globin chains are cross-linked as amides from the R-amino group
of â-Val1 and from the ꢀ-amino group of the â-Lys82.
Modification of Superoxide Dismutase. SOD was reacted
with 5 to incorporate a thiol-reactive maleimide functional group
while cross-linking its subunits. The product mixture was
resolved by analytical C4 reversed phase HPLC (Figure 1).
SDS-PAGE analysis of the products gave bands at 32 and
16 kDa. A control lane containing native SOD shows only a
16 kDa band, which is the mass of a single subunit of the
enzyme. Native and modified SOD were digested with trypsin
to identify the residues that had reacted with 5. Individual
peptides formed during the digest were separated by C18
reversed phase HPLC, whose output was analyzed by electro-
spray ionization mass spectroscopy (ESI-MS). Comparison of
the peptide pattern of digested native and modified superoxide
dismutase showed that peaks T1-T2, T4, T6-T7, T5-T7, T11,
and T12-T13 were reduced in the modified protein (Figure 2).
This suggests that the ꢀ-amino groups of Lys3 (in T1-T2) and/
or Lys151 (in T12-T13) are cross-linked to the ꢀ-amino group
of Lys134 (in T11) or Lys67 (in T4) of the other subunit
(Supporting Information).
to produce 5, a cross-linker with a maleimide for stabilizing
SOD and introducing a coupling site. We note that there are
two types of electrophilic sites on 5 with orthogonal reaction
selectivity: acyl phosphate esters to react rapidly with amines
and maleimide to react with thiols. Scheme 3 summarizes the
overall reaction strategy for forming the protein-protein
conjugate.
Conjugation of Cross-Linked Hb and Cross-Linked SOD.
Cross-linked hemoglobin that been reacted with 1 and reduced
was combined with the maleimide-containing cross-linked SOD
in the presence of TCEP (Scheme 3). The isolated reaction
mixture was resolved by SDS-PAGE into bands at 64, 32, and
16 kDa (Figure 3, lane 2). Thus, the mass of 64 kDa corresponds
to the combined masses of cross-linked SOD (32 kDa) and
hemoglobin that is cross-linked at the two â subunits of
hemoglobin (32 kDa), separated from the R subunits.
These results show that the reaction between the Cys group
of SOD with a maleimide group of compound 5 is not prevalent.
Formation of a thioether bond between the Cys group and the
maleimide on compound 5 would block SOD for conjugation
to thiol-containing hemoglobin. Formation of hemoglobin-SOD
conjugates demonstrates that the potential side-reaction between
a Cys group of SOD and compound 5 is not interfering with
the conjugation reaction.
In order that only the chemically introduced thiol reacts with
the introduced maleimide, the two (â-Cys93) surface cysteinyl
thiols of hemoglobin were blocked with N-ethyl maleimide
(NEM). MALDI-TOF mass measurements indicated that
each â globin chain was modified with a single molecule of
NEM. Preliminary experiments showed that cross-linked
hemoglobin without a free thiol group on its cross-link
and with â93 Cys residues in their native state (not blocked
by NEM), did not produce conjugates when it was mixed
with maleimide-containing SOD. The â-82 lysyl ꢀ-amino
groups in the 2,3-diphosphoglycerate binding site of hemo-
globin react with a pair of adjacent acyl phosphate esters
of 1. The reagent introduces cross-links into each of two
hemoglobin tetramers, connecting them by a chain contain-
ing a disulfide. The reaction was readily monitored by
C4 reversed phase HPLC (disappearance of the NEM â
globin chains). We tracked the formation of reaction products
by size-exclusion chromatography, isolating the cross-linked
species. This was reduced with TCEP to liberate the thiol
(Scheme 4).
Native hemoglobin dissociates into four 16 kDa monomers
(2R and 2â) under the denaturing conditions of SDS-PAGE.
Analysis of the purified cross-linked species in the presence of
2-mercaptoethanol produced bands at 16 and 32 kDa, corre-
sponding to the masses of the expected unmodified R chains
and cross-linked â chains. In the absence of 2-mercaptoethanol,
the same system gives a band at 64 kDa, the combined mass of
four â globin chains, consistent with a disulfide-linked bis-
tetramer (shown schematically in Scheme 4; the rigidity of the
reagent keeps the pairs of reaction sites far apart and in a linear
relationship: the two ends must react with different protein
molecules.)
SDS-PAGE analysis of the coupling reaction between cross-
linked hemoglobin and cross-linked SOD shows the presence
of the protein-protein conjugate as well as excess cross-linked
SOD. Size exclusion chromatography was used to separate the
intact proteins under conditions that do not separate subunits.
The 96 kDa conjugate was separated from the 32 and 16 kDa
species (in the presence of TCEP). Fractions were pooled,
concentrated, and reloaded onto the column until analysis by
gel filtration column showed that the sample was homogeneous
(Figure 4). The sample was then dialyzed using a membrane
(cutoff ∼ 100 kDa).
The material collected by size exclusion chromatography
binds and releases oxygen and also has SOD activity. The
oxygen binding curve of the conjugate (Figure 5) indicates that
the conjugate has a lower oxygen affinity (P50 ) 6.1 Torr) than
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A R T I C L E S
Alagic et al.
Scheme 2. Synthesis of the Cross-Linking Reagent (5) for SOD
Scheme 3. Formation of Hb-SOD
Scheme 4. Formation of Cross-Linked Hemoglobin with a Free
Thiola
hemoglobin that is modified only with NEM (P50 ) 3.8 Torr).
This suggests that the cross-link may restrict movement neces-
sary to go from the T to the R state upon addition of oxygen,
leading to a lower energy requirement to release oxygen. Native
hemoglobin binds oxygen with P50 ) 5.0 Torr under the same
conditions. The Hill coefficient of the conjugate, n50, is 1.55
(Figure 5), which is lower than the Hill coefficient of NEM-
modified hemoglobin (n50 ) 2.60), native hemoglobin
(n50 ) 2.7), or cross-linked hemoglobin tetramers that are not
conjugated to another protein (n50 ) 2.8).8,10
The SOD activity of the conjugate was determined by
evaluating the ability of the conjugate to inhibit super-
oxide-dependent autoxidation of pyrogallol. The activity
of the conjugate is equal to that of the native protein
(Figure 6).
a â-Cys-93 is blocked with NEM.
is normally associated with the change in the state of ligation
of the hemes in hemoglobin. The development of a negative
peak at 287 nm is indicative of the conformational change of
the aromatic residues trytophan C2(37)â and tyrosine C7(42)â,
which are located at the R1â2 interface.11 These results indicate
that hemoglobin in the conjugate undergoes the R to T transition
upon deoxygenation.
Discussion
The ability of the Hb-SOD conjugate to resist superoxide-
induced heme oxidation (forming methemoglobin, metHb) was
equal to that provided by native SOD in solution with
hemoglobin.
The CD spectrum of the Hb-SOD conjugate decreases near
260 nm upon deoxygenation. This change in the CD spectrum
The ability of the thiol and maleimide to join to create the
double protein is clearly established by our results. By blocking
the accessible native thiols, we can be assured that the
connection has proceeded between the desired groups. Chemo-
selective ligation of two proteins occurs in an addition of the
thiol to the maleimide group (Scheme 5).12
(10) Jones, R. T.; Head, C. G.; Fujita, T. S.; Shih, D. T. B.; Wodzinska, J.;
(11) Perutz, M. F.; Ladner, J. E.; Simon, S. R.; Ho, C. Biochemistry 1974, 13,
Kluger, R. Biochemistry 1993, 32, 215-223.
2163-2173.
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Hemoglobin−Superoxide Dismutase
A R T I C L E S
Figure 1. C4 reverse-phase HPLC of the native SOD (a) and of the products
of the cross-linking reaction of SOD and 5 (b), monitored at 254 nm.
Figure 2. C18 reverse-phase HPLC chromatogram of the tryptic digest of
(a) native SOD and (b) SOD that was treated with 5.
Conjugation of hemoglobin to SOD results in the formation
of a thioether-succinimide bond that permanently links the two
proteins. The succinimide amide linkage can undergo hydrolysis
after conjugation. However, this reaction does not lead to the
cleavage of the bond joining the two proteins.
Evaluation of functional properties of the conjugate indicated
that upon conjugation each protein retained its normal function.
SOD that is modified with 5 in the vicinity of its active site
and conjugated to hemoglobin retains its activity. Thus,
conjugation of the modified SOD to hemoglobin does not block
the active site. The complex structure of the products and the
analysis procedure do not give us a confident quantitative
estimation of the yield.
Since superoxide promotes conversion of hemoglobin to the
ferric heme form (metHb), the presence of SOD reduces the
rate of this destructive process.13 The autoxidation of hemo-
globin occurs through the following reactions:
Figure 3. SDS-PAGE analysis of cross-linked protein species and coupling
reaction between cross-linked Hb and cross-linked SOD: lane 1, molecular
weight standards; lane 2, xlHb + xlSOD; lane 3, 1 + NEM-Hb; lane 4,
NEM-Hb; lane 5, 5 + SOD; lane 6, nSOD.
•-
Hb-Fe(II) + O2 f Hb-Fe(III) + O2
(1)
Hb-Fe(II) + O2•- + 2H+ f Hb-Fe(III) + H2O2 (2)
Hb-Fe(II) + H2O2 f Hb-Fe(III) + HO- + HO• (3)
Hb-Fe(II) + HO• f Hb-Fe(III) + HO-
(4)
Our results show that conjugating SOD to hemoglobin does
improve the chemical stability of hemoglobin, as we observe a
45% decrease in the rate of metHb formation. This level of
protection is likely due to the inhibition of the progress of
reaction 2 above and also prevents dissociation. However, both
spontaneous and enzyme-catalyzed dismutation of superoxide
Figure 4. G75 size exclusion chromatogram of the purified conjugate.
generates hydrogen peroxide, which contributes to reaction 3
above. This would explain why we observe about 50% metHb
formation in the presence of superoxide dismutase alone. When
hemoglobin is incubated with catalase (with no SOD), the rate
of metHb formation is inhibited by over 80%. This suggests
(12) Mattson, G.; Conklin, E.; Desai, S.; Nielander, G.; Savage, M. D.;
Morgensen, S. Mol. Biol. Rep. 1993, 17, 167-183.
(13) Winterbourn, C. C. Methods Enzymol. 1990, 186, 265-72.
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A R T I C L E S
Alagic et al.
anion. Full protection against oxidative damage (over 90%) is
observed when both enzymes are present in the assay mixture,
mimicking the antioxidant system that is present in the red cells.
These findings are consistent with results of Yang and Olsen,
who found that the inclusion of native superoxide dismutase
inhibited metHb formation by about 50%, while the presence
of catalase inhibited metHb formation by about 65%. They
observed that oxidative damage was inhibited by about 80%
when both antioxidant enzymes were present in the assay.14
The hemes derived from hemoglobin within the conjugate
bind and release oxygen with about the same affinity as in the
native protein; cooperativity is diminished (n50 is reduced) but
not abolished. Our spectroscopic data suggest that hemoglobin
in the conjugate undergoes a normal conformational change
(R-T). The alterations of the protein’s structure that arise from
interactions of the heme upon binding of oxygen appear to be
transmitted less efficiently. The general effect of SOD upon
solvation of the attached hemoglobin could account for the
observation. The enzymic activity of SOD is not affected by
operation within the conjugate, indicating that substrate binding
and stabilization of the transition state are not affected by the
presence of the attached hemoglobin. Since the native protein
does not show cooperativity, there is no change that could be
expected to compare to that in hemoglobin. Our results indicate
that protein-protein interactions within the hemoglobin-SOD
conjugate affect the mechanism of allosteric effects without
altering relative energy levels. By extension, transient contacts
within the red cell would reduce the cooperativity of individual
hemoglobins but the longer term and overall allosteric properties
would remain.
Figure 5. (a) Oxygen binding cruve for the hemoglobin-superoxide
dismutase conjugate at 25 °C, pH 7.4, I ) 0.1 (b) Hill plot for data in the
oxygen-binding curve at the P50 region. The slope is the Hill coefficient
(n50).
Conclusions
We described an approach for generating protein-protein
conjugates that relies on site-selective chemical modification
and chemoselective coupling of proteins. The reagents 1 and 5
react selectively with the lysylamines of hemoglobin and
superoxide dismutase, respectively, to generate structurally
stabilized proteins. The thiol group on hemoglobin that is
introduced by chemical modification serves as a point of
attachment for a maleimide-containing superoxide dismutase.
The resulting hemoglobin-superoxide dismutase conjugate
displays both oxygen-carrying and antioxidant properties,
demonstrating that the conjugation process does not abolish the
activity of proteins. Successful ligation of superoxide dismutase
and hemoglobin demonstrates that this approach should be useful
in generating other protein-based materials. Since the material
would be added in small quantities to a circulating system, the
specific oxygen affinity should not affect the efficacy of the
overall system. The need for catalase to be present in order to
remove hydrogen peroxide generated by SOD remains an issue
for further research. On the basis of the work of D’Agnillo and
Chang,5,6 it would appear to be an important goal.
Figure 6. Inhibition of autoxidation of 4.0 × 10-4 M pyrogallol in the
presence of Hb-SOD conjugate. Native SOD (b) and Hb-SOD (9)
samples contain one unit of enzyme dimer (5.3 × 10-7 M). Control samples
of pyrogallol (O) and cross-linked Hb (4) contain no SOD.
Scheme 5. Chemoselective Coupling of Hemoglobin to
Superoxide Dismutase through Thiol-Maleimide Chemistry
Experimental Section
Materials. Syntheses were carried out using commercial reagents
and anhydrous solvents. Buffers were prepared from doubly distilled
deionized water. Synthesized materials were identified using a com-
bination of NMR spectroscopy and high-resolution mass spectrometry.
that catalase inhibited the progress of reaction 3. Under our
conditions, it appears that hydrogen peroxide radicals are more
potent species in causing oxidative damage to hemoglobin than
is the superoxide anion. The rate of metHb formation (about
12%) when catalase is used is probably due to the oxidative
damage caused by prior dismutation to peroxide by superoxide
(14) Yang, T.; Olsen, K. W. Biochem. Biophys. Res. Commun. 1989, 163, 733-
738.
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Hemoglobin−Superoxide Dismutase
A R T I C L E S
Proton NMR spectra were recorded at 200 or 300 MHz. Carbon NMR
spectra were recorded at 75.6 or 100.8 MHz. Phosphorus NMR spectra
were recorded at 120 MHz. Melting points are uncorrected. Protein
concentrations were determined using a protein assay kit. Hemoglobin
A was isolated from red cells donated by the corresponding author
and purified. Superoxide dismutase and reagents were purchased. High
performance liquid chromatography coupled to mass spectroscopy
analysis, MS (ESI), was performed at the Mass Spectroscopy Laboratory
at the Molecular Medicine Research Centre, University of Toronto.
4,4′-Dithiobisbenzoic Acid (2). This procedure is a modified version
of the procedure described by Evans for preparation of aromatic
disulfides.15 4-Mercaptobenzoic acid (0.53 g, 3.4 × 10-3 mol) and iodine
(0.44 g, 1.7 × 10-3 mol) were dissolved in 10 mL of absolute ethanol.
Triethylamine (1.5 mL, 10.2 × 10-4 mol) was added and the solution
was left for 16 h. Excess iodine was removed by reduction with 10%
sodium thiosulfate. The cloudy solution was concentrated and combined
with 60 mL of 0.01 M hydrochloric acid. The white precipitate was
collected and dried under vacuum. The product was recrystallized from
N,N-dimethylacetamide and water (0.51 g, 98% yield). Mp: >300 °C.
1H NMR (DMSO-d6): δ 8.0 (d, 2H, J ) 8.0 Hz, ArH); 7.7 (d, 2H,
J ) 8.1 Hz, ArH). 13C NMR (DMSO-d6): δ 166.6, 140.8, 130.3, 129.7,
126.1. MS (ESI negative): 306, calculated; 305, found for the singly
deprotonated species.
pentoxide (3.0 g, 2.2 × 10-2 mmol).18 The reaction mixture was then
filtered while hot. The cooled filtrate was poured over ice to precipitate
the product, which was collected by vacuum filtration. A second crop
of crystals was obtained by concentrating the filtrate. The product was
washed with cold water and dried under vacuum (2.9 g, 94% yield).
1
Mp: 193-195 °C. H NMR (DMSO-d6): δ 8.0 (d, 2H, J ) 8.6 Hz,
ArH), 7.5 (d, 2H, J ) 8.5 Hz, ArH), 7.2 (s, 2H, CH). 13C NMR (DMSO-
d6): δ 169.5, 166.7, 135.5, 134.9, 129.9, 129.5, 126.1, MS (ESI
negative): 217 (calculated), 216 (found for singly deprotonated species).
5-(4-(2,5-Dioxo-2H-pyrrol-1(5)-yl)benzamido)isophthalic Acid (6).
4-Maleimidobenzoic acid (0.27 g, 1.2 × 10-3 mol) was converted to
the acid chloride (Scheme 4) by refluxing in excess thionyl chloride
for 12 h.19 Excess thionyl chloride was removed in vacuo to give
4-maleimidobenzoyl chloride (0.28 g, 1.2 × 10-3 mol). The acid
chloride (0.28 g, 1.2 × 10-3 mol) was dissolved in 5 mL of
N,N-dimethylacetamide with 4-(dimethylamino)pyridine (0.02 g,
2.0 × 10-4 mol) and mixed with 5-aminoisophthalic acid (0.22 g,
1.4 × 10-3 mol) dissolved in 10 mL of N,N-dimethylacetamide. The
reaction proceeded for 48 h under nitrogen. The reaction was terminated
by addition of 100 mL of water. The white precipitate, 6, was collected
by vacuum filtration, washed with cold acetone and 95% ethanol
(3 × 10 mL), and dried under vacuum (0.36 g, 90% yield). Mp:
1
>250 °C. H NMR (DMSO-d6): δ 10.7 (s, 1H, CONH), 8.7 (s, 3H,
ArH), 8.2 (s, 1H, NH), 8.1 (d, 2H, J ) 7.9 Hz, ArH), 7.6 (d, 2H, J )
7.2 Hz, ArH), 7.2 (s, 2H, CH). 13C NMR (DMSO-d6): δ 169.6, 166.6,
165.2, 139.8, 134.8, 134.6, 133.3, 131.7, 128.4, 126.2, 125.0, 124.7.
MS (ESI negative): 380 (calculated), 379 (singly deprotonated).
Compound 7. The dichloride of 6 (0.21 g, 5.1 × 10-4 mol) was
combined with 2 equiv of aminoisophthalic acid (0.17 g, 1.1 ×
10-3 mol) and 4-(dimethylamino)pyridine (0.04 g, 3.3 × 10-4 mol) in
10 mL of N,N-dimethylacetamide under nitrogen for 48 h. The triamide
product, 7, was precipitated by addition of 250 mL of water. The
mixture was kept at 4 °C for 2 h, and the product was collected by
vacuum filtration. This was washed with 2-propanol, diethyl ether, and
hot methanol (3 × 30 mL, each) and dried under vacuum (0.27 g, 76%
N,N′-Diisophthalyl-4,4′-disulfanlydibenzamide (4). 2 (0.43 g,
1.6 × 10-3 mol) was refluxed in excess thionyl chloride to generate
4,4′-dithiobisibenzoyl dichloride (3). This was combined with
5-aminoisophthalic acid (0.72 g, 4.0 × 10-3 mol) in 20 mL of
N,N-dimethylacetamide and mixed with 4-(dimethylamino)pyridine
(0.05 g, 0.4 × 10-4 mol) according to the general method of Aharoni
and Edwards.16 The reaction proceeded for 16 h under nitrogen and
was quenched with water (50 mL), producing a white precipitate. The
flask was placed in an ice bath for 2 h and the solid was collected by
vacuum filtration. The solid was washed with hot methanol then with
hot water and dried under vacuum. This was recrystallized from hot
N,N-dimethylformamide and water, followed by washing with diethyl
ether to give the product (0.85 g, 85% yield), 4. Mp: 295 °C (dec).
1H NMR (DMSO-d6): δ 10.6 (s, 2H, CONH), 8.6 (s, 4H, ArH), 8.2
(s, 2H, ArH), 8.0 (d, 4H, J ) 8.4 Hz, ArH), 7.7 (d, 4H, J ) 8.4 Hz,
ArH). 13C NMR (DMSO-d6): δ 166.5, 165.0, 139.7, 133.3, 131.7,
128.9, 126.4, 125.1, 124.7. MS (ESI nagative): 634 (calculated), 632
(for the doubly deprotonated species).
1
yield). Mp: >250 °C. H NMR (DMSO-d6): δ 10.8 (s, 3H, CONH),
8.7 (s, 4H, ArH), 8.6 (s, 2H, ArH), 8.4 (s, 1H, ArH), 8.2 (s, 2H, ArH),
8.1 (d, 2H, J ) 7.7 Hz, ArH), 7.6 (d, 2H, J ) 7.7 Hz, ArH), 7.2
(s, 2H, CH). 13C NMR (DMSO-d6): δ 170.3, 167.2, 166.2, 166.0, 140.4,
140.3, 136.0, 135.6, 135.4, 134.0, 132.5, 129.1, 126.9, 125.9, 125.4,
123.7, 122.7. MS (ESI): 706 (calculated), 705 (singly deprotonated
species).
N,N′-Bis[bis(sodium methyl phosphate)isophthalyl)-4,4′-disul-
fanlydibenzamide (1). The tetraacid chloride of 4 was prepared by
refluxing 4 (0.27 g, 3.8 × 10-4 mol) in excess thionyl chloride for
4 h. This was converted to the tetrakis dimethyl phosphate. The solution
was stirred for 16 h in dry tetrahydrofuran under nitrogen with 0.28 g
(1.9 × 10-3 mol) of sodium dimethyl phosphate (prepared in dry
acetone from sodium iodide and trimethyl phosphate17). The solvent
was removed in vacuo, leaving a yellow oil (0.46 g, 4.3 × 10-4 mol).
This was dissolved in dry acetone and mixed with 0.39 g (2.6 × 10-3
mol) of sodium iodide in dry acetone to remove one methyl group
from each phosphate. The yellow solution was left overnight. A pale
yellow precipitate was collected and washed with dry acetone to give
Compound 8. The dichloride of 7 (0.04 g, 5.0 × 10-5 mol) was
prepared by refluxing in excess thionyl chloride. This was combined
with sodium dimethyl phosphate (0.06 g, 4.0 × 10-4 mol, prepared in
dry acetone from sodium iodide and trimethyl phosphate) in dry
tetrhydrofuran and mixed for 48 h under nitrogen. The mixture was
filtered and solvent removed from the filtrate, producing the bis
dimethyl phosphate in Scheme 4 as a brown oil (0.1 g, 1.4 ×
10-4 mol). This was dissolved in dry acetone and mixed with sodium
iodide (0.13 g, 8.7 × 10-4 mol) to give bis methyl phosphate, 5. The
reaction proceeded under nitrogen for 18 h, and the pale yellow
precipitate was collected by vacuum filtration. The product was washed
with cold dry acetone and dried (0.1 g, 60% yield). Mp: >250 °C.
1H NMR (DMSO-d6): δ 11.12 (s, 3H, NH), 8.8 (s, 4H, ArH), 8.7
(s, 2H, ArH), 8.4 (s, 1H, ArH), 8.2 (s, 2H, ArH), 8.0 (d, 2H, J )
6.6 Hz, ArH), 7.6 (d, 2H, J ) 7.0 Hz, ArH), 7.2 (s, 2H, CH), 3.4
(d, 12H, JP ) 10.8 Hz, OCH3). 13C NMR (DMSO-d6): δ 169.6, 166.4,
165.1, 139.9, 139.7, 134.8, 134.6, 133.2, 131.8, 131.7, 128.5, 126.1,
125.1, 124.7, 123.2, 122.3, 52.6. 31P NMR (DMSO-d6): δ -5.7
(decoupled).
1 (0.23 g, 56% yield). Mp: 250 °C (dec); IR (KBr): CdO 1717 cm-1
.
1H NMR (DMSO-d6): δ 10.7 (s, 2H, CONH), 8.7 (s, 4H, ArH), 8.2
(s, 2H, ArH), 8.0 (d, 4H, J ) 6.8 Hz, ArH), 7.7 (d, 4H, J ) 8.4 Hz,
ArH), 3.4 (d, 12H, JP ) 7.0 Hz, OCH3). 13C NMR (DMSO-d6):
δ 166.5, 165.1, 139.8, 131.7, 130.1, 128.9, 126.3, 125.0, 124.6, 52.6
(OCH3). 31P NMR (DMSO-d6): δ -5.9 (decoupled).
4-Maleimidobenzoic Acid. 4-Aminobenzoic acid (2.0 g, 1.5 ×
10-2 mol) and maleic acid (1.7 g, 1.5 × 10-2 mol) were dissolved in
30 mL of dioxane and refluxed for 18 h in the presence of phosphorus
Reaction of Hemoglobin with N-Ethyl Maleimide (NEM). Car-
bonmonoxy hemoglobin (2.0 mL, 3.0 × 10-6 mol) in 0.02 M phosphate,
(15) Evans, B. J.; Doi, J. T.; Musker, W. K. Phosphorus, Sulfur, Silicon 1992,
73, 5-13.
(16) Aharoni, S. M.; Edwards, S. F. Macromolecules 1989, 22, 3361-3374.
(17) Zervas, L.; Dilaris, I. J. Am. Chem. Soc. 1953, 77, 5354-5365.
(18) Barakat, M. Z.; Shehab, S. K.; El-Sadr, M. M. J. Chem. Soc. J. 1957, 4133-
4135.
(19) Meek, J. S.; Argabright, P. J. Org. Chem. 1957, 22, 1708-1710.
9
J. AM. CHEM. SOC. VOL. 127, NO. 22, 2005 8041

A R T I C L E S
Alagic et al.
0.15 M NaCl, 0.01 M EDTA, pH 7.0 was combined with 5 mol equiv
of N-ethyl maleimide (0.002 g). The reaction proceeded at room
temperature for 1 h with stirring. The solution was then passed through
a column of Sephadex G25 (equilibrated with 0.05 M sodium borate
buffer, pH 8.0) to remove residual reagent. The NEM-modifed
hemoglobin (NEM-Hb) was kept on ice.
Cross-Linking NEM-Hemoglobin with 1. Carbonmonoxy NEM-
Hb (2.0 mL, 3.0 × 10-6 mol) was converted to the oxy form by light
from a tungsten lamp under a stream of humidified oxygen (2 h). The
sample was then placed under a stream of humidified nitrogen for 2 h,
converting it to the deoxy form. Cross-linking reagent 1 (6.6 mg, 6 ×
10-6 mol) was added to the deoxy NEM-Hb solution as a solid with
solution under nitrogen. The reaction proceeded for 16 h at 37 °C. The
reaction mixture was then placed under a stream of humidified carbon
monoxide for 10 min. Excess cross-linking reagent was removed by
passing the sample through a Sephadex G-25 column that had been
equilibrated with 0.l M, pH 7.2 MOPS.
Chromatographic Analysis of Hemoglobin Species. The structures
of modified hemoglobins were analyzed by the method of Jones.25
Analytical reverse-phase HPLC using a 330 Å pore C4 Vydac column
(250 × 4.6 mm) was used to monitor the extent of globin chain
modification in the reaction with 1. Modified and native globin chains
were separated and isolated by reverse-phase HPLC using a preparative
330-Å pore C4 Vydac column (250 × 12 mm). The solvent gradients
were 49% A (20% acetonitrile in water, 0.1% TFA) and 51% B (60%
acetonitrile in water, 0.1% TFA) from 0.5 to 60.5 min, 35% A and
65% B from 60.5 to 80.5 min, 14% A and 86% B from 80.5 to 90.5,
and 100% B from 95 to 100 min. The effluent was monitored at
220 nm. The isolated species were analyzed by size exclusion
chromatography on Superdex G75 HR (gel filtration). The column was
calibrated using molecular weight standards: blue dextran (MW
2 000 000), â-amylase (MW 206 000), yeast alcohol dehydrogenase
(MW 141 000), BSA (MW 66 000), and carbonic anhydrase (MW
32 000). The effluent was monitored at 280 and 414 nm.
The modification of SOD was analyzed by HPLC using a C4 Vydac
reverse-phase column26 with 330-Å pore size (250 × 4.6 mm) and
solvent gradients starting with 75% A (0.1% H3PO4 in water) and ending
with 100% B (95% acetonitrile, 0.1% H3PO4 in water) in 15 min.
Chemically modified SOD was isolated by C4 reversed phase or by
analytical anion exchange AX300 HPLC using solvent gradients starting
with 100% A (15 × 10-3 M Tris, pH 8.0) and ending with 100% B
(15 × 10-3 M Tris, 0.15 M NaOAc, pH 8.0) in 30 min.
Reaction of Cross-Linked Hemoglobin Products with TCEP.
Cross-linked carbonmonoxy NEM-hemoglobin (3.0 × 10-6 mol) was
passed through a Sephadex G25 column equilibrated with 25 × 10-3
M Tris-HCl, 0.5 M magnesium chloride pH 7.4 buffer. The sample
was kept at 4 °C for 18 h and then passed through a column of Sephadex
G100, equilibrated with 2.5 × 10-2 M, pH 7.4 Tris buffer containing
0.5 M magnesium chloride. Fractions containing cross-linked hemo-
globin tetramers were collected, pooled, and passed through a Sephadex
G25 column equilibrated with 20 × 10-3 M, pH 7.2 MOPS. The sample
was then passed through a deionizing resin to remove magnesium
chloride. Following the elution of the sample with water, the sample
Molecular Weight Determination of Hemoglobin Species. SDS-
PAGE analysis was used for the reaction of hemoglobin with 1 and
SOD with 5, as well as with the coupling reaction between cross-linked
NEM-SOD dismutase and cross-linked (SH) hemoglobin. Samples
were prepared for SDS-PAGE analysis by a procedure modified from
that of Malinowski and Fridovich.27 A sample of protein (∼(2-5) ×
10-6 g/mL) was prepared in a loading buffer of 0.01 M, pH 7.1
phosphate, 6.0 M urea, 1.0% SDS, 0.05 M EDTA, 1.0% glycerol, 1.0%
â-mercaptoethanol, and 0.5% bromophenol blue. The samples were
placed in boiling water for 10 min and then loaded onto polyacrylamide
gels (separating gel, 12.5% acrylamide; stacking gel, 5% acrylamide
in Tris-HCl) followed by electrophoresis in a dual-slab cell apparatus
at 200 mV. The gels were stained with 0.1% Coomassie Blue R-250,
scanned, and analyzed.
was passed through the column equilibrated with TCEP to cleave the
20-22
disulfide bond of 1
(Sephadex G25 column equilibrated with
20 × 10-3 M MOPS, 2 × 10-3 M TCEP pH 6.8 buffer). The sample
was kept at 4 °C for 18 h and then passed through a Sephadex G100
column equilibrated with 2 × 10-2 M, pH 6.8 MOPS containing
2 × 10-3 M TCEP (tris(2-carboxyethyl)phosphine). Fractions containing
cross-linked hemoglobin activated for conjugation (HbSH species) were
collected and concentrated. The purity of the sample was assessed by
HPLC. The sample was stored at -80 °C.
Cross-Linking Superoxide Dismutase with 5. Superoxide dis-
mutase from bovine erythrocytes (3.0 mg, 9.6 × 10-8 mol) was
dissolved in 1.0 mL of 0.01 M, pH 7.2 phosphate. Cross-linking reagent
5 (0.5 mg, 3.8 × 10-7 mol) was added to the enzyme solution as a
solid. Reaction proceeded for 1 h at room temperature with stirring.
Excess reagent was removed by selective precipitation of the enzyme
with 2 mL of cold acetone.23 The precipitated enzyme was collected
by centrifugation at 5000g for 15 min at 4 °C. The pellet was
resuspended in 0.01 M phosphate buffer, pH 7.0.
Enzymatic Hydrolysis of Native and Cross-Linked Hemoglobin
â Chains. Native and modified â globin chains were separated using
a preparative 330-Å Vydac C4 reverse-phase column (250 × 12 mm)
and lyophilized. Approximately 0.5 mg of globin chains was dissolved
in 0.1 mL of 8 M urea and mixed for 4 h at room temperature. TPCK-
treated trypsin solution (4% of globin weight used) in 0.8 ammonium
bicarbonate buffer (pH 8.5) was then added. The resulting sample was
diluted with the bicarbonate buffer to a final concentration of 2 M urea.
The sample was kept at room temperature for 24 h. It was then placed
in a boiling water bath for 2 min and cooled to room temperature.
This was mixed with a solution of endoproteinase Glu-C from
Staphylococcus aureus (2% of globin weight used), followed by addition
of 0.8 M ammonium bicarbonate to 0.8 mL total volume. The sample
was then left at room temperature for 72 h. The hydrolysates were
filtered through an 0.45 µm filter before injecting onto the C18 HPLC
column.
Coupling Cross-Linked Hb to Cross-Linked SOD. Cross-linked
SOD (9.6 × 10-8 mol) and 0.5 equiv of CO-HbSH (4.8 × 10-8 mol),
prepared as described above, were mixed in 2 × 10-2 M MOPS,
2 × 10-3 M TCEP pH 6.8 and kept for 2 h at room temperature and
then at 4 °C for 18 h. The solution was passed through a column of
Sephadex G100 that had been equilibrated with the same buffer. The
first-eluting fractions were collected, pooled, and concentrated until
the sample eluted as a single peak on a G75 Superdex HR 10 × 30 gel
filtration column under partially dissociating conditions for hemoglobin
(25 × 10-2 M Tris-HCl, 0.5 M magnesium chloride, pH 7.4).24 The
sample was then dialyzed against phosphate buffer, I ) 0.1, pH 7.4,
using a 100 kDa membrane.
Peptide fragments were separated by reverse-phase HPLC using a
Vydac C18 column (93 × 4.7 mm) and eluted using a gradient starting
with 100% in water with 0.1% TFA, and ending with 100% acetonitrile
with 0.1% TFA, in a modification of the method described by Shelton.28
The effluent was monitored at 214 and 280 nm.
(20) Burns, J. A.; Butler, J. C.; Moran, J.; Whitesides, G. M. J. Org. Chem.
1991, 56, 2648-2650.
(21) Han, J. C.; Han, G. Y. Anal. Biochem. 1995, 220, 5-10.
(22) Getz, E. B.; Xiao, M.; Chakrabarty, T.; Cooke, R.; Selvin, P. R. Anal.
Biochem. 1999, 273, 73-80.
(25) Jones, R. T. Methods Enzymol. 1994, 231, 322-343.
(26) McGoff, P.; Baziotis, A. C.; Maskiewicz, R. Chem. Pharm. Bull. 1998,
36, 3079-3091.
(27) Malinowski, D. P.; Fridovich, I. Biochemistry 1979, 18, 5055-5060.
(28) Shelton, J. B.; Shelton, J. R.; Schroder, W. A.; Powars, D. R. Hemoglobin
1985, 9, 325-332.
(23) Bensinger, R.; Crabb, J. W.; Johnson, C. M. Exp. Eye Res. 1982, 34, 623-
634.
(24) Guidotti, G. J. Biol. Chem. 1967, 242, 3685-3693.
9
8042 J. AM. CHEM. SOC. VOL. 127, NO. 22, 2005

Hemoglobin−Superoxide Dismutase
A R T I C L E S
Enzymatic Hydrolysis of Native and Cross-Linked SOD. Lyoph-
ilized protein (approximately 3.4 mg) was dissolved in 8 M urea. The
sample was placed in a boiling water bath for 10 min and then cooled
to room temperature, followed by the dilution with 0.8 M ammonium
bicarbonate buffer (pH 8.5) to the final urea concentration of 2 M.
TPCK-treated trypsin solution (4% of protein weight used) was then
added. This was incubated at room temperature for 24 h. The sample
was then heated in a boiling water bath for 2 min and then allowed to
cool to room temperature. The hydrolysates were filtered through a
0.45 µm filter before injecting onto the C18 column.
simultaneously. Gas mixtures were varied steadily by adjusting the flow
valve and tank pressures.
Assay of SOD Activity and Its Protection of Hemoglobin. This
was done by the method described by Marklund.30 The assay measures
the ability of enzyme to inhibit autoxidation of 1,2,3-trihydroxybenzene
(pyrogallol), which can be followed at 420 nm. Solutions of superoxide
dismutase and pyrogallol were prepared in a pH 8.2 buffer of 0.05 M
Tris-cacodylic acid containing 1.0 × 10-3 M diethylenetriamine-
pentaacetic acid. The enzymatic reaction was initiated by addition of
pyrogallol.
Analysis of Oxygen-Binding/Releasing Properties of Hemoglobin-
Containing Species. Oxygen-binding/releasing measurements were
conducted using a modified version of the method described by Imai.29
A sample of a hemoglobin-containing species in phosphate buffer
(I ) 0.1 M ionic strength, pH 7.4) was irradiated with visible light
under humidified oxygen on ice for 2 h, followed by addition of catalase
solution (0.8 × 10-3 g/mL) and equilibration of the sample at room
temperature for 1 h. Absorbance was recorded at 560 nm. The sample
was deoxygenated or oxygenated at 25 °C under humidified nitrogen
or air, respectively. Data analysis was performed by converting the
observed absorbance to a partial saturation (Y):
The autoxidation of pyrogallol was used as a source of superoxide
anion radical, and its effects upon hemoglobin within the conjugate
were monitored. The final concentrations of protein and pyrogallol in
the assay mixture were 5 × 10-5 and 2 × 10-4 M, respectively. All
measurements were conducted in air-equilibrated, pH 8.2, 5.0 ×
10-2 M Tris-cacodylic acid, 1.0 × 10-3 M diethylenetriaminepentaacetic
acid buffer at 20 °C. The reaction was initiated by the addition of
pyrogallol to the hemoglobin solution. The formation of metHb from
oxyHb was followed at 630 nm.
Circular Dichroism Analysis. The deoxygenated conjugate was
prepared in pH 7.2, 1.0 × 10-2 M phosphate buffer by passing
humidified nitrogen over the oxyhemoglobin-SOD conjugate for 4 h
at 37 °C. The oxygenated conjugate was prepared from the deoxygen-
ated conjugate by exposing the sample to a stream of humidified oxygen
for 15 min. Spectra were recorded with a computer-interfaced spectro-
polarimeter in a quartz cuvette.
Y ) (Aobsd - Aoxy)/(Aoxy - Adeoxy
)
The apparatus for determining oxygen-binding properties was contained
in the sample compartment of a computer-interfaced UV-vis spectro-
photometer. The apparatus also contains an amperometric oxygen probe
connected to an oxygen monitor whose output is digitized and connected
to the same computer. The flow of pure gases from reagent tanks was
controlled by a high-accuracy flow meter. The computer was pro-
grammed to display and record absorbance and oxygen concentration
Acknowledgment. We thank the Natural Sciences and
Research Council of Canada for support.
Supporting Information Available: A table of peptides of
the digest of native superoxide dismutase. This material is
available free of charge via the Internet at http://pubs.acs.org.
(29) Imai, K. Methods Enzymol. 1981, 76, 438-449.
(30) Marklund, S.; Marklund, G. Eur. J. Biochem. 1974, 47, 469-474.
JA050339R
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