Cross-linked bis-hemoglobins: Connections and oxygen binding

Article abstract of DOI:10.1021/ja036596i

Covalently linked pairs of cross-linked hemoglobin tetramers ("bis-tetramers", shown schematically as 6-8) were prepared by reacting hemoglobin A with tetrakis acyl phosphate esters (3-5). The effects of the link between tetramers are observed in the oxygen-binding properties of the bis-tetramers: they bind oxygen cooperatively but with Hill coefficients (n ₅₀) lower than that of the native protein and with a high average affinity. The bis-tetramers with longer connections between tetramers show a higher n₅₀, suggesting that steric interactions between the tetramers affect cooperativity. These results correlate to the observed reduced vasoactivity of heterogeneous solutions of oligomeric cross-linked hemoglobin tetramers.

Full text of DOI:10.1021/ja036596i

Published on Web 08/15/2003
Cross-Linked Bis-hemoglobins: Connections and
Oxygen Binding
Nikolai Gourianov and Ronald Kluger*
Contribution from the DaVenport Chemical Laboratories, Department of Chemistry,
UniVersity of Toronto, Toronto, Ontario, Canada M5S 3H6
Received June 10, 2003; E-mail: rkluger@chem.utoronto.ca
Abstract: Covalently linked pairs of cross-linked hemoglobin tetramers (“bis-tetramers”, shown schematically
as 6-8) were prepared by reacting hemoglobin A with tetrakis acyl phosphate esters (3-5). The effects of
the link between tetramers are observed in the oxygen-binding properties of the bis-tetramers: they bind
oxygen cooperatively but with Hill coefficients (n50) lower than that of the native protein and with a high
average affinity. The bis-tetramers with longer connections between tetramers show a higher n50, suggesting
that steric interactions between the tetramers affect cooperativity. These results correlate to the observed
reduced vasoactivity of heterogeneous solutions of oligomeric cross-linked hemoglobin tetramers.
Bifunctional and trifunctional anionic acylating agents have
been used to introduce cross-links selectively between amino
groups in cationic regions of hemoglobin. The resulting
stabilized tetramers bind oxygen reversibly with altered oxygen
affinity and with significant cooperativity.1-3 Systematic varia-
tion of the span of these rigid links within the cross-linked
tetramers leads to a predictable variation in the free energy of
binding of oxygen.4,5 Crystallographic analysis revealed that the
resulting structures are closely related to the transitional
structures proposed by Perutz to explain the R to T transition
that hemoglobin undergoes upon release of oxygen.6,7 We have
been interested in extending the concept of systematic cross-
linking to produce structurally defined connections between two
tetramers. Since the tetramers are subject to separation into
dimers, we sought reagents that also introduce intratetramer
cross-links. Such entities can report changes in physical proper-
ties that result from interactions between proteins that are
defined by the chemical linkage.
these results come from studies of heterogeneous cross-linked
hemoglobin materials that are thought to contain bis-tetramers
and other variously linked oligomers in addition to tetramers.13-17
It has also been reported that vasoactivity decreases in cross-
linked hemoglobin that is connected to poly(ethylene glycol)
(PEG) compared to unconjugated cross-linked hemoglobin
species, with the size of the material being an important
consideration.10,18,19 Thus, we prepared cross-linked bis-tetram-
ers as structurally defined hemoglobin oligomers to determine
the biophysical effects of total size and connecting linkages.
The first example of a specific intramolecular-intermolecular
cross-linking reagent was N,N′-5,5′-bis[bis-(3,5-dibromosalicyl)-
isophthalyl]terephthalamide, 1.20 This tetrafunctional reagent
contains two spanned pairs of reaction sites. Reaction with
amino groups of lysyl (â-82) residues of human hemoglobin
produces a number of separable products, including the cross-
linked bis-tetramer of hemoglobin, shown schematically as 2.
The product (2) binds and releases oxygen without the normal
cooperative interactions of hemoglobin (n50 ) 1.3). Materials
with the same internal cross-link but lacking the intertetramer
link bind oxygen with n50 close to that of the native protein.
Because the span of the bridge between the tetramers in 2 is
Another motivation for the design of such reagents is the
suggestion that connected hemoglobins should have improved
characteristics as circulating oxygen carriers (red cell substitutes)
compared to cross-linked tetramers, which in clinical studies
have shown significant undesirable vasoactivity.8-12 Many of
(11) Erhart, S. M.; Cole, D. J.; Patel, P. M.; Drummond, J. C.; Burhop, K. E.
Artif. Cell Blood Substitutes 2000, 28, 385-396.
(1) Schumacher, M. A.; Dixon, M. M.; Kluger, R.; Jones, R. T.; Brennan, R.
G. Nature (London) 1995, 375, 84-87.
(12) Freilich, E.; Freilich, D.; Hacker, M.; Leach, L.; Patel, S.; Hebert, J. Artif.
Cells, Blood Substitutes, Immobilization Biotechnol. 2002, 30, 83-98.
(13) Winslow, R. M. Vox Sang. 2000, 79, 1-20.
(2) Kluger, R.; Jones, R. T.; Shih, D. T. Artif. Cells, Blood Substitutes,
Immobilization Biotechnol. 1994, 22, 415-428.
(3) Jones, R. T.; Head, C. G.; Fujita, T. S.; Shih, D. T. B.; Wodzinska, J.;
Kluger, R. Biochemistry 1993, 32, 215-223.
(4) Bucci, E.; Gryczynski, Z.; Razynska, A.; Kwansa, H. Biophys. J. 1998,
74, 2638-2648.
(5) Ji, X.; Braxenthaler, M.; Moult, J.; Fronticelli, C.; Bucci, E.; Gilliland, G.
L. Proteins: Struct., Funct., Genet. 1998, 30, 309-320.
(6) Perutz, M. F. Q. ReV. Biophys. 1989, 22, 139-237, 138 plates.
(7) Perutz, M. F.; Fermi, G.; Luisi, B.; Shaanan, B.; Liddington, R. C. Acc.
Chem. Res. 1987, 20, 309-321.
(8) Schultz, S. C.; Grady, B.; Cole, F.; Hamilton, I.; Burhop, K.; Malcolm, D.
S. J. Lab. Clin. Med. 1993, 122, 301-308.
(9) Krishnamurti, C.; Alving, B. M. Blood Sub. 1996, 99-111.
(10) Abassi, Z.; Kotob, S.; Pieruzzi, F.; Abouassali, M.; Keiser, H. R.; Fratantoni,
J. C.; Alayash, A. I. J. Lab. Clin. Med. 1997, 129, 603-610.
(14) Yu, Z.; Friso, G.; Miranda, J. J.; Patel, M. J.; Lo-Tseng, T.; Moore, E. G.;
Burlingame, A. L. Protein Sci. 1997, 6, 2568-2577.
(15) Kavanaugh, M. P.; Shih, D. T. B.; Jones, R. T. Acta Haematol. 1987, 78,
99-104.
(16) Looker, D.; Abbott-Brown, D.; Cozart, P.; Durfee, S.; Hoffman, S.;
Mathews, A. J.; Miller-Roehrich, J.; Shoemaker, S.; Trimble, S. Nature
(London) 1992, 356, 258-260.
(17) Ogden, J. E. Trends Biotechnol. 1992, 10, 91-96.
(18) McCarthy, M. R.; Vandegriff, K. D.; Winslow, R. M. Biophys. Chem. 2001,
92, 103-117.
(19) Winslow, R. M.; Gonzales, A.; Gonzales, M. L.; Magde, M.; McCarthy,
M.; Rohlfs, R. J.; Vandegriff, K. D. J. Appl. Physiol. 1998, 85, 993-1003.
(20) Kluger, R.; Lock-O’Brien, J.; Teytelboym, A. J. Am. Chem. Soc. 1999,
121, 6780-6785.
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J. AM. CHEM. SOC. 2003, 125, 10885-10892
10885

A R T I C L E S
Gourianov and Kluger
Experimental Section
Materials and Methods. Newly synthesized materials were char-
acterized using a combination of NMR spectroscopy, mass spectros-
copy, and infrared spectroscopy. Proton NMR spectra were recorded
at 300 or 400 MHz. Carbon NMR spectra were obtained at 75 or 100.5
MHz. Phosphorus NMR spectra were obtained at 121.5 MHz. Molecular
modeling was performed using HyperChem 5 (Hypercube, Inc.).
Solutions of human hemoglobin were provided by Hemosol, Inc. or
were prepared from donated red cells. Oxygen-binding curves for
modified hemoglobins were measured using an apparatus based on that
of Imai25 as modified by Shih and Jones.26
N,N′-Bis(isophthalyl)fumarate. 5-Aminoisophthalic acid (7.9 g, 4.4
× 10-2 mol) and 4-(dimethylamino)-pyridine (0.6 g, 5 × 10-3 mol)
were dissolved in anhydrous N,N-dimethylacetamide (100 mL) under
argon. Fumaryl chloride (3.06 g, 2.2 mL, 2.0 × 10-2 mol) was added
(via syringe). The mixture was stirred for 48 h (dark yellow), then
transferred to a 1 L beaker to which 500 mL water was added. Fluffy
white crystals were isolated after centrifugation at 9000g for 40 min.
The crystals were washed 3 times with water and collected. The crystals
were lyophilized (6.0 g, 68% yield). mp: 210 °C (dec). 1H NMR
(DMSO-d6): δ 10.92 (s, 2H, CONH), 8.51 (s, 4H, ArH), 8.18 (s, 2H,
ArH), 7.19 (s, 2H, CH-CdO); 13C NMR (DMSO-d6): δ 165, 162,
135, 132, 123, 124, 120; MS (ESI): 441 (found), 441 (M - H-
calculated for C20H14N2O10).
relatively short, the bis-tetramer will have significant internal
interactions that can affect its conformation and solvation,
perhaps leading to reduced cooperativity. Thus, we sought to
vary the nature of the connection between the tetramers to
determine the effects of the chemical nature of the linkage on
the protein’s oxygen-binding properties.
Reagent 1 utilized Klotz’s dibromosalicyl ester group to react
selectively with amino groups in Hb.21,22 However, we found
that reagents that combine this leaving group with larger
hydrophobic cores were not soluble in water. Since it had
previously been demonstrated that acyl phosphate monoesters
have similar reaction and site specificity,23 we reasoned that
this ionic functional group would be better at maintaining water
solubility. Therefore, we designed reagents that are tetrakis acyl
phosphate monoesters, coupled in pairs for cross-linking and
connected by a relatively rigid linker. The rigidity of the
connection avoids the problem of the reagents folding onto
themselves as seen with completely flexible reagents,24 while
defining structural relationships. The acyl phosphate monoesters
are hydrophilic enough to induce water solubility of the reagents,
even those with highly hydrophobic cores.15
N,N′-Bis[bis(sodium methyl phosphate)isophthalyl]fumarate (3).
N,N′-Bis(isophthalyl)fumarate (0.50 g, 1.13 × 10-3 mol) was combined
with an excess of thionyl chloride (16 mL) under nitrogen and refluxed
for 16 h (brown solution). Thionyl chloride was removed by vacuum
distillation, leaving the acid chloride as a dark solid (0.50 g, 9.7 ×
10-4 mol, 86% yield) that was not purified. This was combined with
sodium dimethyl phosphate (0.645 g, 4.36 × 10-3 mol) (prepared in
dry acetone from trimethyl phosphate and sodium iodide)27 in dry THF
(50 mL). The mixture was stirred for 20 h (dark brown). THF was
evaporated, giving an oil that was pumped under vacuum for 3 h.
Sodium iodide (0.46 g, 3.1 × 10-3 mol) was combined with the crude
product in dry acetone (50 mL) under nitrogen and the solution was
stirred for 24 h. Off-white crystals were collected by vacuum filtration
and washed with dry acetone (0.65 g, 74% yield). mp: >250 °C (dec).
1H NMR (DMSO-d6): δ 11.1 (s, 2H, CONH), 8.5 (s, 4H, ArH), 8.2
(s, 2H, ArH), 7.25 (s, 2H, CH), 3.5 (s, OCH3); 13C NMR (DMSO-d6):
δ 162, 160, 136, 130, 124, 123, 120, 57; 31P NMR (DMSO-d6): δ
-5.82 (decoupled); MS (ESI): 205 (found), 204 (M - 4H4- calculated
as m/z ) 814/4 for species without Na+).
N,N′-Bis(isophthalyl)trans,trans-muconate. trans,trans-Muconic
acid (0.30 g, 2.11 × 10-3 mol) was combined with an excess of thionyl
chloride (20 mL) in a round-bottom flask containing a stirring bar and
a condenser. The mixture was refluxed for 96 h, producing a dark brown
solution of the acid chloride. Thionyl chloride was removed by
distillation. 5-Amino-isophthalic acid (1.22 g, 6.75 × 10-3 mol) and
4-(dimethylamino)-pyridine (0.11 g, 0.88 × 10-3 mol) were dissolved
in anhydrous DMF (20 mL) and cooled to 0 °C. Trans,trans-muconate
chloride was cooled to 0 °C and combined with the solution of 5-amino-
isophthalic acid and 4-(dimethylamino)-pyridine (dark brown solution).
The mixture was stirred overnight at room temperature. The solvent
was removed by vacuum distillation, and water (100 mL) was added
to the product, giving a suspension of light brown crystals. The crystals
were precipitated by centrifugation (4500g, 30 min), the supernatant
was removed, and the crystals were washed with water (3 × 100 mL).
The crystals were lyophilized overnight and rinsed with dry acetone
We have prepared the tetrafunctional (doubly paired) cross-
linking reagents 3, 4, and 5 and studied their reaction with
hemoglobin. From those reactions we have isolated cross-linked
bis-tetramers and assessed the oxygen-binding properties of
these novel species.
(21) Walder, J. A.; Zaugg, R. H.; Walder, R. Y.; Steele, J. M.; Klotz, I. M.
Biochemistry 1979, 18, 4265-4270.
(22) Walder, J. A.; Zaugg, R. H.; Iwaoka, R. S.; Watkin, W. G.; Klotz, I. M.
Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5499-5503.
(23) Kluger, R.; Grant, A. S.; Bearne, S. L.; Trachsel, M. R. J. Org. Chem.
1990, 55, 2864-2868.
(25) Imai, K. Methods Enzymol. 1981, 76, 438-449.
(26) Shih, D. T.; Jones, R. T. Methods Enzymol. 1986, 15, 124-141.
(27) Zervas, L.; Dilaris, I. J. Am. Chem. Soc. 1953, 77, 5354-5365.
(24) Kluger, R.; Paal, K.; Adamson, J. G. Can. J. Chem. 1999, 77, 271-279.
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Oxygen Binding and Cross-Linked Bis-hemoglobins
A R T I C L E S
1
(3 × 20 mL), (0.42 g, 42% yield). mp: >200 °C. H NMR (DMSO-
d6): δ 13.2 (COOH), 10.64 (s, 2H, CONH), 8.5 (s, 4H, ArH, C6,4), 8.2
(s, 2H, ArH, C2), 7.4 (m, 2H, R-CH), 6.6 (m, 2H, â-CH); 13C NMR
(DMSO-d6): 167, 163, 140, 138, 132, 131, 125, 124.
humidified nitrogen at 37 °C for 2 h to give deoxyhemoglobin
(deoxyHb). One equivalent of cross-linking reagent 3, 4, or 5 (3 ×
10-6 mol) was added to the solution of hemoglobin (3 × 10-6 mol). In
some cases, the reagent was initially dissolved in a small volume of
sodium borate buffer (0.05 M, pH 8.5) and then added to the solution
of Hb via syringe. The reaction was allowed to proceed for 18 h at 37
°C under a stream of humidified nitrogen. Carbon monoxide was passed
over the mixture for 15 min before the sample was placed into a column
of Sephadex G-25 and eluted with 0.1 M MOPS (pH 7.2). The resulting
modified HbCO was stored at 4 °C.
HPLC Analysis of Cross-Linked Bis-tetramers. Modified hemo-
globins were analyzed according to the procedure of Jones.28 Analytical
reversed-phase HPLC was employed using a 330 Å pore size C-4 Vydac
column (4.6 × 250 mm) to monitor globin chain modifications.
Modified and unmodified globin chains were separated using developers
containing 0.1% trifluoroacetic acid and a gradient beginning with 20%
and ending at 60% of acetonitrile in water. The effluent was monitored
at 220 nm. The R-chains, â-chains, and modified â-chains were
collected and recovered by lyophilization.
Purification of Bis-tetramers. Modified hemoglobins were sepa-
rated using preparative size-exclusion FPLC, Superdex G-75 HR (10
× 300 mm). The method allows separation of globular proteins based
on their molecular weights. The samples (50-100 µL) were eluted
under conditions that dissociate the Hb tetramer into dimers (25 × 10-3
M Tris-HCl, 0.5 M MgCl2, pH 7.4).29 The effluent was monitored at
280 and 414 nm. The first eluting peak was collected separately, and
multiple injection fractions were pooled. The collected samples were
concentrated in Millipore Centriprep 50 concentrators. The samples
were then added into a column Sephadex G-25 and eluted with 0.1 M
MOPS (pH 7.2). The collected samples were placed under a stream of
humidified carbon monoxide for 10 min and stored at 4 °C. The fraction
corresponding to 128 kDa containing a cross-linked bis-tetramer was
exchanged for sodium phosphate buffer (I ) 0.1 M, pH ) 7.4) and
oxygenated for 2 h under tungsten light. The sample concentration was
adjusted to about 5 × 10-3 M of heme, and its oxygen-binding
properties were determined (Figure 6).
SDS-PAGE Analysis of Modified Hb. The peaks eluting from the
G-75 gel filtration chromatography from the reactions of Hb with cross-
linkers 3, 4, or 5 were analyzed by SDS-PAGE. Protein standards,
modified reaction samples, and native Hb were prepared by mixing a
protein sample (2-15 µL) with loading buffer consisting of 0.0625 M
Tris-HCl, pH 6.8, 1.3 M glycerol, 2% SDS, 0.0125 (w/v) bromophenol
blue, and 0.7 M â-mercaptoethanol. The samples were heated to 100
°C for 15 min, and 15-25 µL was loaded onto a polyacrylamide gel
(12% Tris-HCl). The gels were processed in a mini-PROTEAN 7 dual-
slab cell apparatus at 200 mV in 0.12 M Tris, 1 M glycine, and 0.017
M SDS buffer. The gels were stained with either Coomassie Blue (R-
250) or silver nitrate for at least 1 h. Silver nitrate was used for more
dilute samples.30-33
N,N′-Bis[bis(sodium methyl phosphate)isophthalyl]trans,trans-
muconate (4). N,N′-Bis(isophthalyl)trans,trans-muconate (0.18 g, 3.9
× 10-4 mol) was combined with thionyl chloride (15 mL) under argon
and refluxed for 24 h (red solution). Thionyl chloride was removed by
vacuum distillation and the crude solid acid chloride dissolved in dry
THF (20 mL, ice-cooled). This was combined with sodium dimethyl
phosphate (0.23 g, 1.55 × 10-3 mol) that had been freshly prepared in
dry acetone from trimethyl phosphate and sodium iodide.27 The mixture
stood at room temperature for 18 h. The precipitate was collected by
filtration. The solvent was removed, and the product was dissolved in
dry acetone (20 mL) to which sodium iodide (0.26 g, 1.7 × 10-3 mol)
was added. The mixture was stirred for 24 h at room temperature. Light
yellow crystals were collected by vacuum filtration and washed with
1
dry acetone (3 × 10 mL), (0.25 g, 68% yield). mp: 245 °C (dec). H
NMR (DMSO-d6): δ 10.8 (s, 2H, CONH), 8.5 (s, 4H, ArH), 8.15 (s,
2H, ArH), 7.4 (s, 2H, R-CH), 6.6 (s, 2H, â-CH), 3.5 (m, 12H, OCH3);
13C NMR (DMSO-d6): 163, 161, 146, 128, 125, 122, 121, 119, 59;
31P NMR (DMSO-d6): δ -5.55 (decoupled); 31P NMR (methyl alcohol-
d4): δ -4.61 (decoupled); MS (ESI): 210 found, 210 (M - 4H4-
calculated as m/z ) 840/4 without sodium).
N,N′-Bis(isophthalyl)2,6-naphthalenedicarboxylate. 2,6-Naphtha-
lenedicarboxylic acid (0.5 g, 2.3 × 10-3 mol) was refluxed in excess
thionyl chloride (15 mL) overnight under nitrogen. Residual thionyl
chloride was removed by vacuum distillation. The dark yellow oil was
pumped in vacuo for 2 h. 5-Aminoisophthalic acid (0.92 g, 5.1 × 10-3
mol) and 4-(dimethylamino)-pyridine (0.07 g, 5.6 × 10-4 mol) were
added to the flask containing the acid chloride under nitrogen.
Anhydrous N,N-dimethylacetamide (40 mL) was added via syringe,
and the solution was stirred for 18 h. Water (200 mL) was added to
the solution to induce precipitation. The suspension was centrifuged
(4500g, 45 min). The supernatant was removed, and the solid pellet
was washed with distilled water (3 × 50 mL). The product was
separated by centrifugation. The wet crystals were lyophilized to give
a white crystalline product (0.93 g, 75% yield). mp: >250 °C (dec).
1H NMR (DMSO-d6): δ 10.85 (s, 2H, CONH), 8.73 (s, 2H, ArH) 8.71
(s, 4H, ArH), 8.25 (d, 2H, J ) 6.8 Hz, ArH), 8.22 (s, 2H, ArH), 8.13
(d, 2H, J ) 6.8 Hz, ArH); MS (ESI): 540 (found), 542 (M - H-
calculated for C28H18N2O10).
N,N′-Bis[bis(sodium methyl phosphate)isophthalyl]2,6-naphtha-
lenedicarboxylate (5). N,N′-Bis(isophthalyl)2,6-naphthalenedicarbox-
ylate (0.3 g, 5.5 × 10-4 mol) was refluxed with excess thionyl chloride
(20 mL) for 24 h. Residual thionyl chloride was removed by vacuum
distillation. Sodium dimethyl phosphate (0.35 g, 2.3 × 10-3 mol) and
THF (30 mL) were added to the acid chloride. The reaction was stirred
for 3 days at room temperature. The solvent was removed in vacuo
leaving a brown oil, which was immediately dissolved in acetone and
mixed with sodium iodide (0.20 g, 1.35 × 10-3 mol) (brown solution).
This solution was stirred for 24 h at room temperature, giving a yellow
precipitate. The precipitate was collected by vacuum filtration. The
resulting crystals were washed with acetone (3 × 5 mL), (0.47 g, 85%
yield). mp: 230 °C (dec). 1H NMR (DMSO-d6): δ 10.95 (m, 2H,
CONH), 8.75 (m, 6H, ArH), 8.25 (d, 2H, J ) 6.5 Hz, ArH), 8.20 (d,
2H, J ) 6.0 Hz, ArH), 8.15 (d, 2H, J ) 6.5 Hz, ArH), 3.5 (m, 12H,
OCH3); 31P NMR (DMSO-d6): δ -5.77 (decoupled); MS (ESI): 227
found, 228 (M - 4H4- calculated as m/z ) 914/4 without 4Na+).
Cross-Linking. Carbonmonoxyhemoglobin (HbCO) (2.0 mL, 3 ×
10-6 mol in 0.05 M Bis-Tris, pH 6.5) was passed through a Sephadex
G-25 column equilibrated with 0.05 M sodium borate buffer, pH 8.5,
at 4 °C. The collected hemoglobin sample (∼1.5 × 10-4 M) was
oxygenated and photolyzed under a stream of humidified oxygen at 0
°C and adjacent to an illuminated tungsten lamp for 2 h to give
oxyhemoglobin (HbO). This was deoxygenated under a stream of
Stained gels were placed in a series of destaining cycles with 40%
methanol and 10% acetic acid solution for 12 h. When gels were stained
with silver nitrate, they were fixed in 50% methanol and 10% acetic
acid solution, followed by washing in 50% ethanol (3 × 20 min). The
gels were then treated with sodium thiosulfate (0.05 g/L) for 1 min
and washed with water (3 times). The resulting gels were stained with
a solution of silver nitrate (0.5 g/L) and formaldehyde (0.028%). The
development of the gels was done using a solution of sodium carbonate
(0.57 M), formaldehyde (0.019%), and sodium thiosulfate (0.001 g/L).
(28) Jones, R. T. Methods Enzymol. 1994, 231, 322-343.
(29) Guidotti, G. J. Biol. Chem. 1967, 242, 3685-3693.
(30) Switzer, R. C., III.; Merril, C. R.; Shifrin, S. Anal. Biochem. 1979, 98,
231-237.
(31) Ochs, D. C.; McConkey, E. H.; Sammons, D. W. Electrophoresis 1981, 2,
304-307.
(32) Oakley, B. R.; Kirsch, D. R.; Morris, N. R. Anal. Biochem. 1980, 105,
361-363.
(33) Sammons, D. W.; Adams, L. D.; Nishizawa, E. E. Electrophoresis 1981,
2, 135-141.
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A R T I C L E S
Gourianov and Kluger
Figure 1. G-75 Superdex size-exclusion HPLC chromatograms of the reaction mixtures of Hb with reagent 3 (A), reagent 4 (B), and reagent 5 (C).
Scheme 1
The stains were fixed with 20% ethanol and 5% glycerol. Stained gels
were scanned, and the results digitized using a computer.
The cross-linked bis-tetramers were identified and isolated
using gel filtration chromatography with conditions that facilitate
the dissociation of native tetrameric hemoglobin into the 32 kDa
R,â-dimers.29 Under these conditions (high concentrations of
magnesium chloride), only dimers that have been covalently
linked to other dimers maintain their quaternary structure. Three
species (128 kDa, 64 kDa, and 32 kDa) were collected from
the reaction mixture of each cross-linking reagent with deoxy
Hb and analyzed (Figure 1). Each species was tested for
coelution with native Hb and mono-tetrameric cross-linked Hb.
Reagent 5, with the longest span, gave the highest yield of bis-
tetramer while reagent 3, with the shortest span, gave the lowest
yield of bis-tetramer. The first eluting peak was collected
separately, and fractions from multiple injections were pooled.
Tryptic Peptide Digest Followed by MALDI-MS Analysis.
Modified â-chains from cross-linked Hb and unmodified â-chains from
native Hb were separately collected from a reversed-phase C4 prepara-
tive column using the HPLC technique described above. The samples
were lyophilized, leaving the dry, denatured protein chains. The chains
were dissolved in 8 M urea to which fresh trypsin solution was added
(4% of total mass of Hb). The solution was diluted with 0.080 M
ammonium bicarbonate (pH ) 8.5) to give a final urea concentration
of 2 M and allowed to reacted for 24 h. The tryptic hydrolysate then
was heated to 95 °C for 10 min and stored at -10 °C before MALDI
mass spectroscopy analysis.
The molecular weight fragments from the digest were analyzed by
MALDI-TOF mass spectroscopy using a 2 µL sample of the above
mixture by evaporation on an ionization tray after addition of
3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) to each sample.
SDS-PAGE separates the constituent protein chains by mass.
Those that are covalently connected remain combined and have
a higher mass. Native Hb produces a single band at about 16
kDa, corresponding to each of its four subunits. Reagents 3-5
produced species with masses of 64, 48, 32, and 16 kDa,
confirming that each of the cross-linkers reacts with four, three,
and two â-chains respectively (Figure 2). The 16 kDa species
are unmodified R- or â-chains. Isolation of the 128 kDa peaks
from gel chromatography and SDS-PAGE analysis showed that
the major products are 64 kDa and 16 kDa species, with trace
quantities of 48 kDa species in some cases.
Results
Reagents 3, 4, and 5 have two pairs of methyl isophthalyl
phosphates joined by unsaturated amides. These were prepared
from the corresponding dicarboxylic acids as described in the
Experimental Section (Scheme 1). The highest yield of cross-
linked bis-tetramers resulted from the reaction of 1 equiv of
each reagent with 1 equiv of Hb at pH 8.5.
Modified subunits were identified in the reaction mixtures
using reversed-phase HPLC. This revealed that modifications
occur selectively on the side chains of the â-subunits of
hemoglobin. However, the chromatograms did not give resolved
separations of the species that had â-chain modifications.
This is consistent with linkage between all four â-subunits
(64 kDa) and no modification to the R-subunits. The 48 kDa
species comes from reagents that have reacted with three
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Oxygen Binding and Cross-Linked Bis-hemoglobins
A R T I C L E S
Figure 2. SDS-PAGE analysis of cross-linked bis-tetramers 6, 7, and 8.
were absent in the modified â-chain digest (Figure 5). The
fragment corresponding to T1 (m/z ) 953) was substantially
reduced in modified â-chain tryptic digest. The absence of T9
and T8 + T9 peptides establishes that 4 reacted with the ꢀ-amino
group of lysyl residue 82 of â-chains through one of the
isophthalic phosphate esters since that is the normal site of
hydrolysis between these peptides. The reduced amount of the
T1 peptide in the modified â-chain digest indicates that 4 cross-
linked Hb through the N-terminal amino group of valyl residue
1 of â′-chains on the second arm of the isophthalic phosphate
ester. The presence of these amide linkages prevents trypsin
from hydrolyzing the peptide bond between T9 and T10 chains.
This is exactly the same pattern seen with acyl phosphate cross-
linking reagents in the formation of monotetramers.
The oxygen-binding properties of the bis-tetrameric hemo-
globin species are summarized in Table 1. Oxygen affinity (P50)
was obtained from fitting the data to the Adair equation34 (Figure
6). The oxygen affinities of the bis-tetramers (6, P50 ) 5.0; 7,
P50 ) 4.2; 8, P50 ) 4.3) are somewhat higher (lower P50) than
those we measured for native Hb itself (P50 ) 5.0). The Hill
coefficient (n50), a measure of cooperativity,34 was obtained from
the slope of a Hill plot, where Y is the saturation factor of
oxygen-occupied sites versus the total number of the sites
available for oxygen binding (Figure 7). The bis-tetrameric
hemoglobins in the present series retain some cooperativity (6,
n50 ) 1.8; 7, n50 ) 2.1; 8, n50 ) 2.0), but it is reduced compared
to native Hb (n50 ) 3.0) or cross-linked tetramers produced from
isophthalyl reagents (n50 ) 2.6, P50 ) 18.1).35
Figure 3. G-75 Superdex size exclusion HPLC of crude mixture (dashed
line) and purified bis-tetramer 7 (solid line).
â-subunits (presumably one of the acyl phosphates has hydro-
lyzed prior to reaction). The schematic structures are shown as
6-8.
Discussion
The material collected from the bis-tetrameric peaks was
passed through the gel filtration column. Only species corre-
sponding to 128 kDa were observed, which is the expected
nominal mass of cross-linked bis-tetramers (Figure 3).
The site of the reaction of each reagent with hemoglobin was
analyzed by using a tryptic digest and peptide map. The
modified â-chains collected from the reversed-phase C4 column
were digested. The resulting fragment mixture was assayed
employing MALDI-MS. The spectrum from digested â-chains
modified with reagent 4 (Figure 5) was compared to unmodified
digested â-chains spectrum (Figure 4). The results indicated that
molecular fragments corresponding to the T9 peptide chain (m/z
) 1670) of Hb and uncut T8 + T9 peptide chains (m/z ) 1799)
Reagents 3, 4, and 5 were prepared efficiently and react at
concentrations comparable to that of the dissolved protein. The
acyl phosphate ester permitted reactions to be carried out in
water. Their reaction with Hb introduced the desired internal
and external links in a single reaction. The relatively rigid core
structures resulting from extended conjugation and the amide
linkage to the isophthalyl phosphate esters result in the link
having a predictable span while the reagent does not fold onto
itself.24 The isophthalyl relationship of the reacting sites on each
(34) Bell, J. E.; Bell, E. T. Proteins and Enzymes; Prentice-Hall: Englewood
Cliffs, NJ, 1988.
(35) Kluger, R.; Wodzinska, J.; Jones, R. T.; Head, C.; Fujita, T. S.; Shih, D.
T. Biochemistry 1992, 31, 7551-7559.
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A R T I C L E S
Gourianov and Kluger
Figure 4. MALDI-MS of tryptic digest mixture from native â-chain of human hemoglobin.
Figure 5. MALDI-MS of tryptic digest mixture from modified â-chain of human hemoglobin with reagent 4.
Table 1. Oxygen-Binding Properties of Purified Bis-tetrameric
Hemoglobin Species
bis-tetramer
P50 (torr)
n50
spana (Å)
6
7
8
5.0 ((0.1)
4.2 ((0.1)
4.3 ((0.1)
5.0 ((0.1)
9.1
1.8 ((0.1)
2.1 ((0.1)
2.0 ((0.1)
3.0 ((0.1)
1.29 ((0.03)
14.5
16.8
18.4
n/a
native Hb
220
16.2
a
Distance is measured between two CdO groups on opposite ends of
the link between tetramers.
end facilitates intratetramer cross-linking of two â-subunits at
ꢀ-amino groups of â-lys-82 and the amino group of the
N-terminal â′-Val-1 residues.3,35-37 When two of the isophthalyl
moieties are separated by a sufficient distance, the bis-tetrameric
hemoglobin species is generated.
Our results clearly show that linking the cross-linked tetramers
affects the oxygen-binding properties of the constituent heme
sites. These materials retain cooperative properties, unlike the
previously reported cross-linked bis-tetramer (2), whose binding
oxygen-binding curve is hyperbolic.20
Figure 6. Oxygenation of native Hb (a) and cross-linked bis-tetramers: 8
(b), 7 (c), 6 (d), and calculated 220 (e). Oxygen affinity (P50) is derived at
Y ) 0.5.
linked bis-tetramer being less than that of smaller species that
result from reaction with reagent that has undergone partial
hydrolysis. In producing bis-tetramers, simply increasing the
concentration of the reagent will not improve the yield because
the coupling process requires a combination of the two protein
molecules and one tetrafunctional reagent molecule. However,
Acyl phosphate ester groups undergo hydrolysis in competi-
tion with protein modification. This leads to the yield of cross-
(36) Kluger, R.; Shen, L.; Xiao, H.; Jones, R. T. J. Am. Chem. Soc. 1996, 118,
8782-8786.
(37) Paal, K.; Jones, R. T.; Kluger, R. J. Am. Chem. Soc. 1996, 118, 10380-
10383.
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Oxygen Binding and Cross-Linked Bis-hemoglobins
A R T I C L E S
Figure 7. Hill plot of oxygen binding of native hemoglobin (a), bis-tetramer 8 (b), bis-tetramer 7 (c), and bis-tetramer 6 (d). For comparison, a calculated
curve for bis-tetramer 220 is also shown. The slopes at Y ) 0.5 are the Hill coefficients (n50) and are shown on the inset.
yields can be improved by the procedures used in producing
hemoglobin-dendrimer conjugates,38 especially variation in the
timing of addition of the reagent to the protein solution. In the
present study, we were interested in establishing the existence
and properties of cross-linked bis-tetramers rather than develop-
ing a practical route for their production.
of osmotic stress indicate that 25-72 water molecules bind a
tetramer in the course of oxygenation.40 A decrease in acces-
sibility of the connected tetramers to the solvent will result in
a change in hydration of the protein. Such a change in hydration
level has been observed to be associated with cooperative
binding of oxygen to Hb.41-44 Thus, reduction of cooperativity
of the bis-tetramers may result from alteration of the hydration
level as well as from restricted allosteric movement.
We varied the span of the intertetramer link to test the specific
effect of length on cooperativity. Reagents 3 and 4 contain
amides with linear conjugated hydrocarbon cores. There is a
small increase in cooperativity (∆n50 ) 0.2) where the separation
involves an additional allylic unit (2.3 Å), suggesting that
interactions of the tetramers decrease cooperative interactions
within tetramers that lead to higher Hill coefficients.39
The separation between constituent tetramers in 2, which has
a terephthalyl core, is about the same as in the linear dienyl
core (trans,trans-muconate) of 7: about 16 Å (estimated from
molecular modeling). Thus, the difference in cooperativity must
be due to the chemical properties of the linking structure. To
investigate the separation-cooperativity relationship, we pre-
pared 5, a reagent with a larger aromatic core, producing 8,
which can be compared to 2. Here n50 ) 2.0, indicating that
cooperativity is retained with the longer link, even with a more
hydrophobic core. This suggests that cooperativity requires
movements that cannot be accommodated in the shorter, more
rigid structure of the terephthalyl linkage while hydrophobicity
in the link itself does not prevent cooperative interactions.
The oxygenation of the bis-tetramers in principle could
involve interactions caused by the binding of up to eight
molecules of oxygen on the eight hemes of the connected
tetramers. If there were positive cooperativity between units in
different tetramers, we would expect an increase in the observed
Hill coefficient, but this is not the case. Thus, cooperativity
remains the result of interactions within each tetramer, following
the Perutz model.
The bis-tetramers are possible alternatives to nonspecifically
linked oligomers in red cell substitutes. Research with PEG-
Hb’s has shown that an increase in the overall size of the species
results in prolonged circulation life and minimizes increases in
mean arterial blood pressure.18,19 In addition, the bis-tetramers
are good candidates for studying the relationship of structure
to function in oxygen carriers.20,45 The idea that cross-linked
bis-tetramers might be less vasoactive in circulation than cross-
linked tetramers derives from analyses of heterogeneously cross-
linked hemoglobins that have been used in clinical trials.8-12
The oxygen-binding properties of the individual species in such
mixtures have not been determined, whereas those of the purified
cross-linked tetramers in other trials have been measured.3 Our
studies reveal that cross-linked bis-tetramers have higher oxygen
affinities and decreased cooperativity compared to the clinically
tested mono-tetramers. It has been assumed that vasoactivity is
associated with scavenging of nitric oxide from endothelia by
the hemes in these species.46,47 Our results show oxygen will
bind more tightly to cross-linked bis-tetramers and produce a
high net occupancy in these species relative to cross-linked
tetramers. To the extent that the site is occupied by oxygen there
is less of an opportunity to acquire nitric oxide, which could
cause vasoconstriction and increased blood pressure.
(40) Zhou, Y.; Zhou, H.; Karplus, M. J. Mol. Biol. 2003, 326, 593-606.
(41) Salvay, A. G.; Grigera, J. R.; Colombo, M. F. Biophys. J. 2003, 84, 564-
570.
The intertetramer links should also generate interfacial
interactions. The close interaction leads to reduction of the
exposure to water of each tetramer, resulting in the change of
the hydration level within each connected tetramer.18 Studies
(42) Colombo, M. F.; Seixas, F. A. V. Biochemistry 1999, 38, 11741-11748.
(43) Colombo, M. F.; Sanches, R. Biophys. Chem. 1990, 36, 33-39.
(44) Colombo, M. F.; Rau, D. C.; Parsegian, V. A. Science (Washington, D.C.)
1992, 256, 655-659.
(45) Kluger, R. Can. J. Chem. 2002, 80, 217-221.
(46) Wicks, D.; Wong, L. T.; Sandhu, R.; Stewart, R. K.; Biro, G. P. Artif.
Cells, Blood Substitutes, Immobilization Biotechnol. 2003, 31, 1-17.
(47) Baldwin, A. L.; Wiley, E. B.; Alayash, A. I. Am. J. Physiol. 2002, 283,
H1292-H1301.
(38) Kluger, R.; Zhang, J. J. Am. Chem. Soc. 2003, 125, 6070-6071.
(39) Jayaraman, V.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Science
(Washington, D.C.) 1995, 269, 1843-1848.
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J. AM. CHEM. SOC. VOL. 125, NO. 36, 2003 10891

A R T I C L E S
Gourianov and Kluger
Conclusions
In cases where oxygen-binding properties of red cell substi-
tutes had been rationally produced, cooperative, low oxygen
affinity species were sought to maximize oxygen delivery.45
However, such species would also be most able to acquire nitric
oxide. Clinical trials have been conducted for situations where
a small fraction of the total blood volume is replaced. In such
situations, oxygen comes from the normal red cells: the altered
hemoglobin serves as a sink of additional oxygen. Where the
added oxygen carrier has a high oxygen affinity, it would serve
as a source when the red cells are largely depleted but would
probably not be vasoactive, since it would not selectively bind
nitric oxide.
Cross-linked bis-tetramers with a variety of connecting cores
can be generated from reagents that contain paired reaction sites.
These materials bind oxygen reversibly and cooperatively with
smaller Hill coefficients than do similarly cross-linked tetramers.
These results suggest that there are significant interactions
between the connected tetramers.
Acknowledgment. We thank the Natural Sciences and
Engineering Research Council of Canada for support through
a Strategic Project.
JA036596I
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