Systematically cross-linked human hemoglobin: Functional effects of 10 ? spans between beta subunits at lysine-82

Article abstract of DOI:10.1021/ja961443z

The structure and properties of hemoglobin are altered by the introduction of cross-links of defined structure between specific residues. The bis methyl phosphates and bis 3,5-dibromosalicylates of 4-carboxy-trans-cinnamic acid as well as the bis methyl phosphate of 2,6-naphthalenedicarboxylic acid produce a 10 ? cross-link between the ε-amino groups of each β-lys-82 of human hemoglobin. The oxygen affinity of the modified proteins fits the correlation interpolated from those for shorter and longer cross-links. The oxygen binding curve shows a high degree of cooperativity. These results support the idea that the length of the semirigid cross-link in a structurally homogeneous series constrains the relaxation of the protein upon oxygen binding by a mechanism that is specifically reflected in the oxygen affinity, while interactions between hemes that affect cooperativity are not diminished.

Full text of DOI:10.1021/ja961443z

8782
J. Am. Chem. Soc. 1996, 118, 8782-8786
Systematically Cross-Linked Human Hemoglobin: Functional
Effects of 10 Å Spans between Beta Subunits at Lysine-82
Ronald Kluger,*,† Lixin Shen,† Hong Xiao,‡ and Richard T. Jones*,‡
Contribution from the Lash Miller Chemical Laboratories, Department of Chemistry,
UniVersity of Toronto, Toronto, Canada M5S 3H6, and Department of Biochemistry and
Molecular Biology, School of Medicine, Oregon Health Sciences UniVersity,
Portland, Oregon 97201
ReceiVed May 1, 1996X
Abstract: The structure and properties of hemoglobin are altered by the introduction of cross-links of defined structure
between specific residues. The bis methyl phosphates and bis 3,5-dibromosalicylates of 4-carboxy-trans-cinnamic
acid as well as the bis methyl phosphate of 2,6-naphthalenedicarboxylic acid produce a 10 Å cross-link between the
ꢀ-amino groups of each â-lys-82 of human hemoglobin. The oxygen affinity of the modified proteins fits the correlation
interpolated from those for shorter and longer cross-links. The oxygen binding curve shows a high degree of
cooperativity. These results support the idea that the length of the semirigid cross-link in a structurally homogeneous
series constrains the relaxation of the protein upon oxygen binding by a mechanism that is specifically reflected in
the oxygen affinity, while interactions between hemes that affect cooperativity are not diminished.
Proteins assume defined shapes that are perturbed in the
The selection of spans in the set of cross-linkers used in those
studies was based on a limited set of aromatic and unsaturated
dicarboxylic acids: fumaric acid, isophthalic acid, terephthalic
acid, and (3,3′)-stilbene dicarboxylic acid. Molecular modeling
of the cross-links between nitrogen atoms of the resulting
modified protein gives distances of 6.1, 7.3, 7.5 and 13.2 Å
respectively.2
The two types of cross-linked hemoglobin (RRâ-lysine-82-
X-â′lys-82) and (RRâ-lysine 82-X-â′-valine-1) yield separate
oxygen-affinity correlation. The slope are of opposite sign, and
they intersect at a point corresponding to a span of about 10 Å.
The 10 Å distance is not close to the data points for the cross-
linked proteins but is in the middle of both correlations. Thus,
in order to test one of the correlations as well as to provide
materials for other studies we used molecular modeling to
consider materials that would give a 10 Å cross-link. Our
selection was based on a consistency with the structures in the
correlation. In this paper we report the preparation of cross-
linkers that produce materials in this ∼10 Å range and the
properties of some of the materials resulting from their reaction
with hemoglobin.
course of their function. Site-directed chemical modification
of a protein presents the possibility of controlling the structure
in order to alter function.1-3 Such specifically altered proteins
provide a basis for detailed studies of protein structure and
function. Bifunctional and trifunctional semirigid anionic
acylating agents react regioselectively to cross-link proteins,
introducing the molecular equivalent of a clamp. This is
exemplified by the reactions of bis(acyl methyl phosphates)
derived from a series of unsaturated dicarboxylic acids that
selectively cross-link human deoxy hemoglobin A between its
two â subunits: at the ꢀ-amino groups of lysine-82 of each
subunit or at one of the lysine-82 amino groups and the
N-terminal valine R amino group.1,4 Some of these products
have been observed in reactions of 3,5-dibromosalicylate esters
of the same dicarboxylic acids.5-7 Our analysis of the oxygen
affinities of these cross-linked proteins reveals a correlation
between the free energy of oxygen binding to hemoglobin and
the molecular length of the cross-link.2 We proposed that this
is the result of differential translational restriction in the modified
proteins. Subsequent X-ray crystallographic analysis of several
of the structures substantiated this proposal.8
Experimental Section
Human hemoglobin A was obtained as the highly purified material
in the deoxy state from Hemosol, Inc. The material was stored as the
tetracarbonomonoxy form at 4 °C and converted to the deoxy form
prior to reaction by photodissocation under wet nitrogen.
We calculated the approximate distance between amino groups of
amides resulting from cross-linking between ꢀ-amino groups of lysine
residues of a protein using the bis methyl amide derivative of the core
dicarboxyl compound as a model. The calculations were done under
MS-DOS with the program Alchemy 3 (TRIPOS Associates) which
uses the TRIPOS 5.2 force field2 and the Alchemy minimizer (as
described in the program manual).
Syntheses. General Methods. The reagents developed for these
studies are methyl acyl phosphates and dibromosalicylates of known
dicarboxylic acids. These derivatives are generally reactive and
hygroscopic. They are principally characterized by their nmr spectra
and by their hydrolysis products. The resulting cross-linked hemo-
globins define the structure of interest.
† University of Toronto.
‡ Oregon Health Sciences University.
X Abstract published in AdVance ACS Abstracts, September 1, 1996.
(1) Kluger, R. Can. J. Chem. 1994, 72, 2193-2197.
(2) Jones, R. T.; Head, C. G.; Fujita, T. S.; Shih, D. T.; Wodzinska, J.;
Kluger, R. Biochemistry 1993, 32, 215-223. The computed distances use
the TRIPOS 5.2 force field (Clark, M.; Cramer III, R. D.; Van Opendosch,
J. Comput. Chem. 1989, 10, 982-1012.
(3) Jones, R. T.; Shih, D. T.; Fujita, T. S.; Song, Y.; Xiao, H.; Head, C.;
Kluger, R. J. Biol. Chem 1996, 271, 675-680.
(4) Kluger, R.; Grant, A. S.; Bearne, S. L.; Trachsel, M. R. J. Org. Chem.
1990, 55, 2864-2868.
(5) Walder, J. A.; Zaugg, R. H.; Walder, R. Y.; Steele Biochemistry 1979,
18, 4265-70.
(6) Delaney, E. J.; Massil, S. E.; Shi, G. Y.; Klotz, I. M. Arch. Biochem.
Biophys. 1984, 228, 627-638.
(7) Klotz, I. M.; Haney, D. N.; Wood, L. E. J. Biol. Chem. 1985, 260,
16215-16223.
(8) Schumacher, M. A.; Dixon, M. M.; Kluger, R.; Jones, R. T.; Brennan,
R. G. Nature 1995, 375, 84-87.
(9) Titley, A. F. J. Chem. Soc. 1928, 2581.
S0002-7863(96)01443-6 CCC: $12.00 © 1996 American Chemical Society

Cross-Linked Human Hemoglobin
J. Am. Chem. Soc., Vol. 118, No. 37, 1996 8783
(E)-4-(Carboxylethenyl)benzoic Acid.9 [56148-65-3] (4-Carboxy-
trans-cinnamic Acid). We used the method of Rappoport10 with minor
changes. The stereochemical assignment is based on more recent work
by Cervinka.11 A mixture of 4-carboxybenzaldehyde (5.6 g, 37 mmol),
malonic acid (5.7 g, 55 mmol), and piperidine (0.6 mL) in dry pyridine
(30 mL) was stirred and heated for 90 min at 80 °C, and 2 hours at
100 °C, and then refluxed for 90 min. The solution was then poured
into 3 N HCl (250 mL). The resulting solid was collected by filtration
and washed with 250 mL of water and then with 250 mL of 95%
ethanol. The product was dried under vacuum (6.1 g, 86%): mp 362-
363 °C (decomposed), lit. 358 °C; IR (KBr) CdO 1688 cm-1; 1H NMR
(DMSO-d6) δ 7.97 (ArH, 2 H, d, J ) 8.1 Hz), 7.80 (ArH, 2 H, d, J )
8.4 Hz), 7.65 (HCdC, 1 H, d, J ) 16.1 Hz), 6.65 (HCdC, 1 H, d, J
) 16.1 Hz); 13C NMR (DMSO-d6) δ 167.47, 167.00, 142.79, 138.49,
131.99, 129.90, 128.39, 121.72.
Hz), 134.90, 132.27, 130.29, 127.97 (d, JP-C ) 8.5 Hz), 126.41, 55.56
(d, JP-C ) 5.3 Hz); 31P NMR (CHCl3) δ -4.6 (s, decoupled).
4-Carbonyl-trans-cinnamoyl Bis(sodium methyl phosphate)
(CCMP). A solution of sodium iodide (1.65 g, 11 mmol) in dry
acetonitrile (8 mL) was added to an acetonitrile (8 mL) solution of
4-carbonyl-trans-cinnamoyl bis(dimethyl phosphate) (1.9 g, 4.7 mmol)
in a 25-mL round-bottom flask at room temperature under nitrogen.
The solution was shaken, and the flask was left for 12 h, during which
time the product precipitated as a pale yellow powder. Filtration
followed by washes with dry acetonitrile (2 × 8 mL) and methylene
chloride (2 × 8 mL) resulted in an off-white powder that was dried
under reduced pressure. The powder was recrystallized by dissolving
in 40 mL of methanol with gentle heating. Then 60 mL of 1:1 (v/v)
ethanol:propan-2-ol was added, and the solution was allowed to stand
for 30 min. The resulting white crystals were collected by vacuum
filtration and dried in Vacuo (1.8 g, 90%); mp 278-280 °C; IR (KBr)
CdO 1700, 1730 cm-1; 1H NMR (D2O) δ 7.98 (ArH, 2 H, d, J ) 8.4
Hz), 7.71 (HCdC, 1 H, d, J ) 15.9 Hz), 7.68 (ArH, 2 H, d, J ) 8.1
Hz), 6.55 (HCdC, 1 H, dd, J ) 16.0 Hz, JP-H ) 1.7 Hz), 3.60 (OCH3,
3 H, d, JP-H ) 11.5 Hz), 3.57 (OCH3, 3 H, d, JP-H ) 11.5 Hz). 13C
NMR (D2O) δ 165.22 (d, JP-C ) 7.7 Hz), 164.71(d, JP-C ) 7.1 Hz),
147.30, 140.58, 132.22, 131.58, 130.25, 121.96 (d, JP-C ) 6.0 Hz),
55.11 (d, JP-C ) 2.6 Hz), 54.95; 31P NMR (D2O) δ -5.0 (s, decoupled),
-5.2 (s, decoupled). Anal calc for C14H12O10P2Na2: C, 33.98; H, 2.85.
Found: C, 34.07; H, 2.85.
(E)-4-(Carbonylethenyl)benzoyl Dichloride (4-Carboxy-trans-
cinnamic Acid Dichloride). To a stirred suspension of 4-carboxy-
trans-cinnamic acid (1.0 g) and a catalytic amount of dry dimethyl-
formamide (2 drops) in dry dichloromethane (15 mL), oxalyl chloride
(1.0 mL, 11.4 mmol) was added dropwise at room temperature under
nitrogen. After addition, the reaction mixture was refluxed under
nitrogen for 2 h. Solvent was removed under reduced pressure. The
resulting yellow needles were recrystallized from dry ether (10 mL)
giving 1.1 g (92%) of pale yellow crystals: mp 125-126 °C; IR (KBr)
1
CdO 1740, 1770 cm-1; H NMR (CDCl3) δ 8.19 (ArH, 2 H, d, J )
2,6-Naphthalenedicarbonyl bis(sodium methyl phosphate) (NMP)
was prepared from 2,6-naphthalenedicarbonyl bis(dimethyl phosphate)
(0.9 g, 2.1 mmol) and sodium iodide (0.7 g, 4.7 mmol) in acetonitrile
(10 mL) as above. The crude product was recrystallized by dissolving
in 20 mL of methanol with gently heating. Then 40 mL of 1:1 (v/v)
ethanol:propan-2-ol was added, and the solution was allowed to stand
for 30 min. The resulting off-white crystals were collected by vacuum
filtration and dried in Vacuo (0.9 g, 96%): mp 290-292 °C; IR (KBr)
8.4 Hz), 7.87 (HCdC, 1 H, d, J ) 15.6 Hz), 7.74 (ArH, 2 H, d, J )
8.5 Hz), 6.80 (HCdC, 1 H, d, J ) 15.6 Hz); 13C NMR (CDCl3) δ
167.50, 165.63, 147.53, 139.16, 135.21, 131.86, 129.12, 126.03.
2,6-Naphthalenedicarbonyl Dichloride. To a stirred suspension
of 2,6-naphthalenedicarboxylic acid (0.5 g) containing one drop of
dimethylformamide in dry dichloromethane (10 mL) was added oxalyl
chloride (0.44 mL) slowly under nitrogen at room temperature. After
addition, the reaction mixture was refluxed under nitrogen for 4 h.
Solvent was removed under reduced pressure. The resulting solid
(yellow needles) was recrystallized from dry ether (8 mL (0.55 g,
CdO 1710 cm-1
.
1H NMR (D2O) δ 8.46 (2 H, s), 7.91 (4 H, s), 3.67
(OCH3, 6 H, d, JP-H ) 11.4 Hz).13C NMR (D2O) δ 164.95 (d, JP-C
)
7.7 Hz), 135.93, 133.02, 131.65, 130.39 (d, JP-C ) 7.0 Hz), 127.56,
55.29 (d, JP-C ) 4.7 Hz). 31P NMR (D2O) δ -4.6 (s, decoupled).
tert-Butyl-3,5-dibromosalicylate was prepared as described pre-
viously.6,12-14
94%): mp 189-190 °C; IR (KBr) CdO 1750 cm-1 1H NMR (CDCl3)
.
δ 8.79 (2 H, s), 8.20 (2 H, d, J ) 8.7 Hz), 8.12 (2 H, d, J ) 8.4 Hz).
13C NMR (CDCl3) δ 168.02, 135.19, 133.70, 133.60, 130.82, 126.69.
4-Carbonyl-trans-cinnamoyl Bis(dimethyl phosphate). This fol-
4-Carbonyl-trans-cinnamoyl Bis(tert-butyl-3,5-dibromosalicylate).
To a solution of tert-butyl-3,5-dibromosaliylate (1.34 g, 3.8 mmol) in
dry tetrahydrofuran (5 mL) was added 3.8 mL of 1.0 M potassium
tert-butoxide in tetrahydrofuran. The mixture was stirred for 15 min
at room temperature. 4-Carbonyl-trans-cinnamoyl dichloride (0.41 g,
1.8 mmol) in tetrahydrofuran (5 mL) was added dropwise over 15 min.
Stirring was continued for 2 h at room temperature. The solution was
filtered and concentrated. The residue was recrystallized from ethanol
(15 mL) to give the product (1.32 g, 85%): mp 208-210 °C; IR (KBr)
CdO 1710, 1720, 1740 cm-1; 1H NMR (CDCl3) δ 8.30 (ArH, 2 H, d,
J ) 8.3 Hz), 7.92-8.04 (ArH, 4 H, m), 7.97 (HCdC, 1 H, d, J ) 15.8
Hz), 7.77 (ArH, 2 H, d, J ) 8.4 Hz), 6.83 (HCdC, 1 H, d, J ) 16.0
Hz), 1.52 (C(CH3)3, 9 H, s), 1.39 (C(CH3)3, 9 H, s). 13C NMR (CDCl3)
δ 163.15, 162.85, 162.01, 161.87, 146.58, 146.53, 145.86, 139.10,
138.73, 133.59, 133.53, 131.06, 130.50, 129.11, 128.84, 128.55, 119.44,
119.34, 119.24, 119.18, 83.10, 28.02, 27.86.
lows the general procedure for other acyl dimethyl phosphates.4
A
suspension of sodium dimethyl phosphate (1.58 g, 10.7 mmol) and
4-carbonyl-trans-cinnamoyl dichloride (1.2 g, 5.2 mmol) was stirred
in dry tetrahydrofuran (15 mL) under nitrogen at room temperature
for 2 h. The solution was filtered, and solvent was removed, leaving
the product as a solid. Recrystallization from dry benzene and ether
gives the product as white flakes (1.9 g, 90%): mp 78-79 °C; IR
(KBr) CdO 1700, 1730 cm-1; 1H NMR (CDCl3) δ 8.11 (ArH, 2 H, d,
J ) 8.1 Hz), 7.83 (HCdC, 1 H, d, J ) 16.1 Hz), 7.67 (ArH, 2 H, d,
J ) 8.4 Hz), 6.55 (HCdC, 1 H, dd, J ) 15.8 Hz, JP-H ) 1.8 Hz), 4.01
(OCH3, 6 H, d, JP-H ) 11.7 Hz), 3.97 (OCH3, 6 H, d, JP-H ) 11.6
Hz). 13C NMR (CDCl3) δ 160.19 (d, JP-C ) 7.9 Hz), 160.00 (d, JP-C
) 8.0 Hz), 146.98, 139.06, 131.25, 129.80 (d, JP-C ) 8.6 Hz), 128.63,
119.20 (d, JP-C ) 9.3 Hz), 55.62 (d, JP-C ) 3.0 Hz), 55.57 (d, JP-C
)
2.8 Hz); 31P NMR (CHCl3) δ -4.6 (s, decoupled), -4.9 (s, decoupled).
2,6-Naphthalenedicarbonyl Bis(dimethyl phosphate). A suspen-
sion of sodium dimethyl phosphate (0.7 g, 4.7 mmol) and 2,6-
naphthalenedicarbonyl dichloride (0.55 g, 2.2 mmol) was stirred in dry
tetrahydrofuran (10 mL) under nitrogen at room temperature for 3 h.
Solvent was removed under reduced pressure. The resulting white
powder was dissolved in dry benzene (10 mL). The mixture was
filtered through a sintered-glass funnel and washed with dry benzene
(10 mL). The solvent was removed under reduced pressure, leaving
the product as a solid. Recrystallization from benzene and ether yielded
the product as white flakes (0.9 g, 95%): mp 97-99 °C; IR (KBr)
4-Carbonyl-trans-cinnamoyl Bis(3,5-dibromosalicylate)(CCDS).
4-Carbonyl-trans-cinnamoyl bis(tert-butoxy-3,5-dibromosalicylate) (0.5
g, 0.6 mmol) was dissolved in anhydrous trifluoroacetic acid (10 mL)
at room temperature. After 10 min, the mixture was diluted with ether
(2 mL) and then placed in ice bath for 20 min. The crude product was
collected by vacuum filtration and recrystallized from tetrahydrofuran/
trifluoroacetic acid to give the product (0.36 g, 84%): mp 237-239
°C; IR (KBr) CdO 1700, 1740 cm-1
.
1H NMR (DMSO-d6) δ 8.03-
8.33 (ArH, 8 H, m), 8.02 (HCdC, 1 H, d, J ) 16.0 Hz), 7.16 (HCdC,
1 H, d, J ) 16.1 Hz). 13C NMR (DMSO-d6) δ 163.62, 163.35, 163.26,
162.97, 146.81, 146.72, 145.90, 139.17, 138.82, 133.44, 133.34, 130.60,
130.43, 129.81, 129.75, 129.38, 128.08, 127.93, 119.56, 119.48, 119.30,
1
CdO 1740 cm-1; H NMR (CDCl3) δ 8.68 (2 H, s), 8.15 (2 H, dd, J
) 8.5 Hz, JP-H ) 1.4 Hz), 8.09 (2 H, d, J ) 8.6 Hz), 4.06 (OCH3, 12
H, d, JP-H ) 11.7 Hz); 13C NMR (CDCl3) δ 160.52 (d, JP-C ) 8.3
(12) Razynska, A.; Rak, J.; Fronticelli, C.; Bucci, E. J. Chem. Soc., Perkin
Trans. 2 1991, 1531-40.
(10) Rapoport, H.; Williams, A. R.; Lowe, O. G.; William, W. S. J. Am.
Chem. Soc 1953, 75, 1125.
(11) Cervinka, O.; Krisz, O. Coll. Czech. Chem. Commun. 1983, 48,
2952-64.
(13) Kluger, R.; Song, Y.; Wodzinska, J.; Head, C.; Fujita, T. S.; Jones,
R. T. J. Am. Chem. Soc. 1992, 114, 9275-9279.
(14) Kluger, R.; Song, Y. J. Org. Chem. 1994, 59, 733-736.

8784 J. Am. Chem. Soc., Vol. 118, No. 37, 1996
Kluger et al.
119.13. Anal calc for C24H12O8Br4: C, 38.54; H, 1.62. Found: C,
38.33; H, 1.77.
Biochemical Methods. Biochemical preparations and materials
follow earlier descriptions.2,15,16
4-carboxy-trans-cinnamic bis carboxamide. These were con-
verted to the corresponding bis(methyl acyl phosphates) (1 and
2). We also prepared 4-carboxy-trans-cinnamic acid bis(3,5-
dibromosalicylate) (3).
Cross-Linking Reactions. A solution of 1.4 g of carbonmonoxy
hemoglobin in 20 mL of water was added to 20 mL of 0.1 M borate,
pH 9.0. Oxygen was passed over the rotating solution to remove carbon
monoxide. Nitrogen was flushed over the solution in three evacuation-
fill cycles to produce deoxy hemoglobin. The solution was kept under
nitrogen. The reagent (2 or 5 equiv) was placed into a total volume of
7 mL of 0.05 M pH 9.0 borate. Dioxane was substituted for 2 mL of
buffer if needed to dissolve the reagent. The solution was flushed with
nitrogen and kept under nitrogen. The preparation of cross-linker
solution was done within 10 min preceding reaction with hemoglobin.
The reagent solution was injected slowly into the deoxyhemoglobin
solution at 35 °C under humidified nitrogen. Two equivalents of
reagent was used in order to assure completion of the reaction and to
compensate for competing hydrolysis. The rotating flask was left for
2 h at this temperature. At the end of the reaction period, the flask
was flushed with carbon monoxide to stabilize the product and placed
on ice. Reagents and low molecular weight byproducts were then
removed by gel filtration.
Separation and Characterization of Modified Hemoglobins. The
procedures described by Jones15 were used. Modified hemoglobins
were separated by ion exchange chromatography. The various native
and modified globin chains were separated by HPLC on reversed-phase
C-4 columns. The modifications of the major product were identified
by digestion of the globin chains with trypsin and endoproteinase Glu-
C, peptide mapping, and amino acid analysis. Ion spray mass
spectrometry of the HPLC-separated globin chains in the major product
was used to identify the nature of chemical modifications by changes
in molecular mass. Each peak gave a characteristic mass.
Measurement of Functional Properties of Isolated Hemoglobins.
The hemoglobin-oxygen equilibrium properties of modified hemo-
globin were measured as previously reported.3 The separated major
component was isolated by ion-exchange chromatography, and its
oxygen binding properties was measured. The oxygen pressure at
which the modified hemoglobin has half its total oxygen capacity
utilized, P50, and the Hill coefficient at oxygen pressure ) P50 were
determined in 50 mM Bis-Tris (pH 7.4) at 25 °C and 55 µM heme, 0.1
M chloride. The effect of pH on oxygen affinity (Bohr effect) was
measured between pH 6.0 and pH 9.0 and then calculated for the change
associated with a change from pH 7 to pH 8. The effect of chloride
on oxygen affinity was measured over a concentration range of sodium
chloride from 0.007 to 0.5 M.
Reactions of Deoxyhemoglobin with Reagents. Deoxyhe-
moglobin was combined with the cross-linking reagents, and
the products were analyzed by ion-exchange separation of
modified proteins as well as by reversed-phase HPLC under
conditions that separate hemes and globin chains.2,15 Reaction
with 2 equiv of each cross-linker (35 °C, 2 h, pH 9 borate)
leaves <10% unmodified hemoglobin. Increasing the concen-
tration of cross-linker to 5 eq gives little more conversion and
a more complex collection of products. The reactions produce
a diverse collection of modified hemoglobins. Analysis by ion
exchange and reversed-phase HPLC reveals two major products
resulting from reaction with CCDS, seven with CCMP, and
five with NMP. The structures of the major products were
determined by tryptic-Glu-C digestion of the globin chains along
with amino acid analysis.15
Peptide Patterns of Modified Globin Chains. Figure 1
shows the results of reversed-phase HPLC analysis of the major
â chain product obtained from the reaction of hemoglobin with
CCDS. For comparison, Figure 2 is the digest pattern of
unmodified hemoglobin. The peptides in Figure 2 designated
as âT-9 contains the â-lysine 82 residue. Peptide âT-9 and
the next peptide in the â chain sequence, âT-10a′, are not
present in the modified peptide pattern in Figure 1. The new
peptide in Figure 1 corresponds to the cross-linked residues as
determined by amino acid analysis. When the ꢀ-amino group
of lysine residue is acylated by the 4-carbonyl-trans-cinnamoyl
group, the residue will no longer serve as a substrate for trypsin
catalyzed hydrolysis. The â82 lysyl residue is normally
hydrolyzed by trypsin to form the âT-9 and âT-10a peptides
or âT-10a′ peptide in the case of additional treatment with Glu-C
endoproteinase. Thus, the absence of the peaks arising from
âT-9 and âT-10a′ in Figure 1 indicates that the ꢀ-amino groups
of the â82 lysyl residues have been blocked. This is consistent
with a bis-amide cross-link between the ꢀ-amino group of the
lysyl residue 82 of one â chain and the ꢀ-amino group of the
Results
In our earlier studies we used conjugated and aromatic
bifunctional anionic acylating agents to introduce length-defined
cross-links into the BPG-binding site of hemoglobin between
amino groups of different subunits.2-4,7,13 The reagents were
bis methyl acyl phosphates4 and bis(3,5-dibromosalicylates).7,12,13
The examples we prepared spanned the following distances
(Å): 5.3 (fumaryl), 6.6 (isophthalyl), 7.6 (terephthalyl), and 11.6
(3,3′-stilbene dicarboxylic) between reacting amino groups. The
correlation of oxygen affinity and cross-link spans was linear
for two series of cross-links but with opposite slopes. The data
plots cross at 10 Å, where the properties coincide with those of
native hemoglobin. Since that point was not experimentally
accessible, we sought a route to cross-linkers for the 10 Å span.
To be consistent with the types of compounds we used for other
cross-linkers we chose 2,6-naphthyl dicarboxylic acid and
4-carboxy-trans-cinnamic acid as our basis. Calculations with
molecular mechanics minimization give maximum spans of 9.9
Å between the amino groups of N,N′-dimethyl-2,6-naphthyl bis
dicarboxamide and 9.8 Å for the amino groups in N,N′-dimethyl-
(15) Jones, R. T. Methods Enzymol. 1994, 231, 322-343.
(16) Kluger, R.; Wodzinska, J.; Jones, R. T.; Head, C.; Fujita, T. S.;
Shih, D. T. Biochemistry 1992, 31, 7551-9.

Cross-Linked Human Hemoglobin
J. Am. Chem. Soc., Vol. 118, No. 37, 1996 8785
Figure 1. Tryptic-Glu-C peptides of â chains obtained from the major
modified hemoglobin formed from the reaction of deoxy hemoglobin
with CCDS.
Figure 3. Oxygen affinity of cross-linked hemoglobins plotted as a
function of the span of the cross-link between â subunits.2 The data
points for the two (â-82-Lys-X-Lys-â-82 cross-linked) species in the
present study are at 9.8 and 9.9 Å (Table 1).
hydrophobicity of the cross-linker in both cases is similar to
those in the other cases in Figure 3, as is the degree of
unsaturation but the oxygen affinities are very different. We
had observed that the oxygen affinity is related to the length of
the cross link span. The properties of the two hemoglobins
with 10 Å spans fits into the correlation at the predicted location.
Most of the structure within hemoglobin associated with the
Bohr effect is linked to that portion of the structure to which
chloride ion binds to affect oxygen affinity.17,18 This corre-
sponds to the cationic core of the protein which includes the
protonated amino group of â lysine 82.19,20 Acylation of this
residue on both subunits by cross-linking reduces the availability
of this residue to interact with chloride.
Figure 2. Tryptic-Glu-C peptides of unmodified â chains of hemo-
globin.
Table 1. Oxygen Binding Properties of Cross-Linked Hemoglobins
P50,
The Hill coefficients for both species are close to that of
unmodified hemoglobin (Table 1). Although the Hill coefficient
is a macroscopic property, it originates from the interactive
effects of oxygen binding to each heme.21 If the communication
between sites is blocked or reduced by the cross-link, then
cooperativity should be affected. We see in the present case
that cooperativity as measured by the Hill coefficient is
maintained or possibly enhanced by the cross-link. The cross-
link necessarily restricts movement of the globin chains.
Clearly, the interactions responsible for changes in oxygen
affinity are not simply results of free motions of the protein
chains but are distinctly channeled. The cross-links permit the
channels of communication to be maintained while affecting
the overall position of the total equilibrium. Since oxygen
affinity is reduced in the cross-linked protein, the full coopera-
tive effect occurs over a smaller range of energy differential.
Hb structures
cross-link
Torr n50 % Bohr eff. % Cl- eff.
native Hb
none
5.0 3.0 100 (-0.54) 100 (-0.44)
RRâ-82-N-82â naphthyldicarbonyl 6.7 3.0 83
RRâ-82-C-82â carboxycinnamyl 7.2 3.3 85
43
43
a Conditions for P50 and n50 are pH 7.4, 50 mM bis-Tris, 0.1 M Cl-,
55 µM heme 25 °C.
lysyl residue 82 of the other â chain. The new peptide confirms
the assignment. The chemical formula of the hemoglobin from
which this modified globin was obtained is designated RR(â82-
C-â82) where “C” represents the 4-carbonyl-trans-cinnamoyl
group. The product containing the naphthalene dicarboxylic
cross-link between the same residues is designated RR(â82-N-
â82).
The modified proteins that were isolated and analyzed contain
cross-links between the lysyl 82 residues of each of the â
subunits. Both products have the same span in the cross-link,
and both have the same oxygen affinity (P50 ∼ 7 Torr, Table
1). These values fit the correlation we observed for longer and
shorter cross-links (Figure 3).
Both species show a high degree of cooperativity in binding
oxygen. The Hill coefficients (n50) are about the same as for
the unmodified protein. The effect of protons (Bohr effect) and
the effect of chloride ion on oxygen affinity are reduced in the
modified hemoglobins. However, the Bohr effect is reduced
to a lesser extent than is the chloride effect.
The structural basis of the physical effects of cross-links in
hemoglobin can be understood in terms of insights from by
X-ray crystallography.8 The general restriction of motion
permits higher energy states to be maintained upon binding of
oxygen. The two species produced in the present study have
been crystallized, and their detailed structures have been
determined.22 The location of the cross-linkers is consistent
with the results we have presented. Detailed analysis of the
complete structures places these hemoglobin derivatives in
(17) Kilmartin, J. V.; Fogg, J. H.; Perutz, M. F. Biochemistry 1980, 19,
3189-3193.
(18) Shih, D. T.-b.; Luisi, B. F.; Miyazaki, G.; Perutz, M. F.; Nagai, K.
J. Mol. Biol. 1993, 230, 1291-1296.
(19) Ueno, H.; Manning, J. M. J. Protein Chem. 1992, 11, 177-85.
(20) Perutz, M. F.; Shih, D. T.-b.; Williamson, D. J. Mol. Biol. 1994,
239, 555-560.
Discussion
The two modified hemoglobins contain cross-links that are
chemically different between â subunits at lysine-82. Yet, the
oxygen affinities are very close to each other and significantly
different from those containing homologous cross-links. The
(21) Jayaraman, V.; Rodgers, K. R.; Mukerji, I.; Spiro, T. G. Science
1995, 269, 1843-1848.
(22) Schumacher, M. A.; Zhelznova, E.; Kluger, R.; Jones, R. T.;
Brennan, R. G. to be submitted for publication.

8786 J. Am. Chem. Soc., Vol. 118, No. 37, 1996
Kluger et al.
context with respect to conformational pathways through which
hemoglobin undergoes structural changes upon oxygenation.8,22
change to a protein’s structure can be used to introduce
systematic changes in a measurable property.
Acknowledgment. We thank Hemosol, Inc., for supplying
us with purified hemoglobin. The Natural Sciences and
Engineering Research Council of Canada provided support for
research at the University of Toronto. Work at the Oregon
Health Sciences University was supported by Grant HL20142
from the National Institutes of Health. We thank Professor
Richard Brennan for communication of preliminary X-ray
crystallographic results.
Conclusion
Derivatives of naphthalenedicarboxylic acid and 4-carboxy-
cinnamic acid produce cross-links within hemoglobin that span
approximately 10 Å. The resulting modified proteins have
oxygen-binding properties that fit a linear free energy relation-
ship to the length of the cross-link. This suggests that a single
coordinate along the cross-link can be analyzed for its relation-
ship to the overall free energy change in the protein associated
with oxygen binding. It also shows that a simple systematic
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