1 Department of Physiology and Biophysics and 3 Division of Hematology, Albert Einstein College of Medicine, Bronx, NY 10461 and 4 Structural BioInformatics, San Diego, CA 92127, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: -fumaryl cross-linking/chloride binding/Hb-Presbyterian/low oxygen affinity
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bis(sulfosuccinimidyl) suberate, an affinity-directed bifunctional reagent, cross-links Va11(ß) and Lys82(ß) within the DPG binding pocket of oxy-HbA and lowers of its O2 affinity (Manjula et al., 1994, 1995
). Like HbA, cross-linking of the ß-chains of Hb-Presbyterian by Bis(sulfosuccinimidyl) suberate also results in a lowering of its O2 affinity in chloride-free buffers (O'Donnel et al., 1994
). However, the enhanced chloride effect seen with uncross-linked Hb-Presbyterian is lost upon this ßß intramolecular cross-bridging.
The bifunctional reagent bis(3,5-dibromosalicyl) fumarate (DBBF) introduces an intramolecular fumaryl cross-bridge into deoxy HbA between the -amino groups of the two
99 lysines in the middle of the central cavity. The
-fumaryl cross-linking of HbA also results in a marked lowering of its O2 affinity (Chatterjee et al., 1986
; Snyder et al., 1987
; Walder et al., 1994
). If the altered electrostatic potential in the middle of the central cavity as a result of the positive charge of Lys108(ß) in Hb-Presbyterian is the primary molecular factor responsible for the increased chloride sensitivity of its O2 affinity, a loss of the positive charge of Lys99(
) by fumaryl cross-bridging could result in the loss of the latent O2 affinity-lowering potential of Lys108(ß). On the other hand, if the low O2 affinity of Hb-Presbyterian is a consequence of a site-specific effect at Lys108(ß), an additivity or synergy may be seen with the introduction of an
-fumaryl cross-bridge into Hb-Presbyterian.
In the present study, intramolecular cross-bridging of Hb-Presbyterian by DBBF was undertaken to determine whether the site selectivity of cross-bridging is conserved even in the presence of Lys108(ß) and, if it does, whether this
-cross-bridging would result in an additivity or synergy between the functional consequences of the two structural perturbations of the Hb molecule, i.e. the Asn108(ß)
Lys mutation and the
-cross-linking, both of which are in the middle of the central cavity and independently reduce the O2 affinity of HbA.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of hemoglobins
Human HbA was purified from erythrocyte lysates by chromatography on Whatman DE-52 cellulose, followed by a second chromatography on Whatman CM-52 cellulose, by methods previously described (Acharya et al., 1983). Production of transgenic swine expressing the human Hb variant Hb-Presbyterian has been described earlier (O'Donnel et al., 1994
). Hb-Presbyterian from the erythrocyte lysates of transgenic swine was purified by two successive chromatographies on Whatman DE-52. Purity of the chromatographed Hb-Presbyterian was confirmed by RP-HPLC and isoelectric focusing (IEF) analyses.
Cross-linking of Hb-Presbyterian with DBBF
Hb-Presbyterian was reacted with DBBF under deoxy conditions and in the presence of STP at 37°C essentially by procedures previously described for HbA (Highsmith et al., 1997). Briefly, Hb-Presbyterian (1 mM) in 50 mM BisTris-Ac, pH 6.5, was incubated overnight at 4°C with an 8-fold molar excess of STP to saturate its DPG binding site and thus prevent the subsequent reaction of DBBF at this site. The following morning, the STP-treated Hb-Presbyterian was deoxygenated with argon, a 2-fold molar excess of a freshly prepared solution of DBBF was added and the reaction was carried out with stirring under an atmosphere of argon for 4 h at 37°C. The reaction was terminated by the addition of a 10-fold molar excess of glycylglycine over DBBF. The efficiency of cross-linking was evaluated by analyzing the reaction product by size exclusion chromatography (SEC) on a Superose 12 column (Amersham Pharmacia Biotech) in 50 mM BisTris0.9 M MgCl2, pH 7.4 (i.e. dissociating conditions) and the stoichiometry of
-cross-linking was evaluated by analyzing the reaction product by RP-HPLC. Control
-HbA was prepared under the same conditions as described above for Hb-Presbyterian.
Purification of DBBF cross-linked Hb-Presbyterian and HbA
The DBBF-cross-linked Hb-Presbyterian was purified on a Whatman DE-52 column in 50 mM Tris-acetate, pH 8.5. A decreasing pH gradient from 8.5 to 7.0 was used for the elution of the protein. The purity of the chromatographically isolated cross-linked Hb-Presbyterian was further verified by SEC and RP-HPLC analyses. Cross-linked HbA was also similarly purified.
Analytical methods
The globin chains of HbA and Hb-Presbyterian were analyzed by RP-HPLC on a Vydac C4 column (300 Å, 4.6x250 mm), using an acetonitrile0.1% TFA solvent system, by methods previously described (Rao et al., 1994). Stabilization of the tetrameric structure against dissociation into dimers, as a consequence of intramolecular cross-linking, was evaluated by size exclusion chromatography on Superose 12 columns under dissociating conditions.
Oxygen affinity measurements
Oxygen equilibrium curves were measured at 37°C using a Hem-O-Scan (Aminco) either in 50 mM BisTris50 mM Tris-acetate, pH 7.4, or in 50 mM BisTris-acetate, pH 7.4, at a hemoglobin tetramer concentration of 1 mM. Oxygen equilibrium curves were measured in the presence and absence of effectors.
Molecular modeling
The hemoglobin model was prepared on a Silicon Graphics workstation using the modeling software InsightII from Biosym. The starting model was the refined structure of deoxyhemoglobin (Fermi et al., 1984). The coordinates were obtained from the Protein Data Bank. The biopolymer module of the InsightII software package was used to model the central cavity. Interactive graphics of InsightII were used to check the stereochemistry. The residue Asn108(ß) was mutated to Lys using the Ponder and Richards rotamer library (Ponder and Richards, 1987
) as implemented in Proteomine (Structural Informatics, San Diego, CA) mutant module. The best rotamer side chains were chosen in terms of space and conformational energy considerations. The fumaryl cross-bridge was constructed using the InsightII (MSI) fragment builder. The side chain dihedrals of Lys99(
) were modified to make a covalent bond with the fumaryl fragment. The two alpha chains were bonded through the central cavity. The modeling was carried out with minimal perturbation to the backbone of the hemoglobin. This modification is only a local effect. The four chloride ions were placed such that the interactions of chloride atoms were optimum with hemoglobin.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hb-Presbyterian pretreated with STP was reacted with DBBF under deoxy conditions at a tetramer to reagent molar ratio of 1:2. HbA was also reacted with DBBF at the same time, to serve as a control. The results of the globin chain analysis of the DBBF-reacted Hb-Presbyterian and HbA by RP-HPLC is shown in Figure 1. As can be seen, the RP-HPLC pattern of DBBF-reacted Hb-Presbyterian (Figure 1A
) revealed the presence of two major peaks. The first peak eluted in the position of unmodified ß-globin. The ß-globin peak was completely absent. Instead, a new peak eluting at a retention time of ~65 min was present. A similar pattern was obtained for the DBBF-reacted HbA (Figure 1B
). The peak eluting at ~38 min is unmodified
-globin and the peak eluting at ~65 min is the cross-linked
-globin. The retention time of the modified
-globin in the RP-HPLC map of the DBBF-reacted Hb-Presbyterian corresponded to that of the cross-linked
-globin obtained by the DBBF cross-linking of HbA. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis of this modified
-globin peak yielded a mass of 30 336 Da, a value that is in good agreement with the theoretical value of 30 332.85 Da for two
-subunits joined by a fumaryl cross-bridge. Thus, the RP-HPLC pattern of DBBF-reacted Hb-Presbyterian reflects the high selectivity of the fumaryl cross-linking to the
-chains of Hb-Presbyterian, even in the presence of a lysine at ß108, proximal to Lys99(
) in the middle of the central cavity.
|
|
The -cross-linked Hb-Presbyterian was purified by ion-exchange chromatography on a Whatman DE-52 column (Figure 3
) and the protein fractions were pooled as indicated. The results of the IEF analysis of the purified cross-linked Hb-Presbyterian is shown in Figure 3
(inset), along with those of HbA and
-fumaryl-HbA. As can be seen, the
-cross-linked Hb-Presbyterian exhibits an isoelectric point that is lower than that of the unmodified Hb-Presbyterian. Furthermore, the lowering of the isoelectric pH of Hb-Presbyterian on
-fumaryl cross-bridging is noticeably more significant than that observed with HbA. The net result is that the isoelectric point of
-fumaryl Hb-Presbyterian is close to that of
-fumaryl HbA, in spite of the fact that the isoelectric points of unmodified Hb-Presbyterian and unmodified HbA are very distinct.
|
The oxygen equilibrium curves of Hb-Presbyterian and -fumaryl Hb-Presbyterian in 50 mM BisTris50 mM Tris acetate, pH 7.4, are shown in Figure 4
. As can be seen,
-cross-linking of Hb-Presbyterian results in a significant right shifting of its oxygen equilibrium curve. Nevertheless, calculation of the Hill coefficient values from the data presented in Figure 4
indicated that the cross-linking of Hb-Presbyterian does not alter its cooperativity in oxygen binding (n values being 2.7 and 2.6, respectively for cross-linked and uncross-linked Hb-Presbyterian). A comparison of the consequences of
-fumaryl cross-bridging on the O2 affinity of Hb-Presbyterian relative to that of HbA is presented in Table I
. As can be seen, cross-linking of HbA with DBBF results in a marked reduction in its O2 affinity, a result that is consistent with that reported previously in the literature (Walder et al., 1994
). It may also be seen from Table I
that, unlike in the HEPES and BisTris-acetate buffers at pH 7.4, the intrinsic O2 affinity of Hb-Presbyterian in 50 mM Bistris50 mM Tris acetate buffer, pH 7.4, is slightly lower than that of HbA even in the absence of chloride. Cross-linking of the
-chains of Hb-Presbyterian with DBBF resulted in a further reduction of its O2 affinity (Figure 4
and Table I
). More strikingly, the cross-link-induced reduction in the O2 affinity of Hb-Presbyterian is even greater than that observed with HbA (4.1- vs 2.7-fold, respectively). Thus, in addition to its intrinsic potential to lower the O2 affinity of Hb, the
-fumaryl cross-bridging of Hb-Presbyterian activates the low O2 affinity-inducing potential of Lys108(ß).
|
|
The data on the influence of allosteric effectors on the O2 affinity of -fumaryl Hb-Presbyterian relative to uncross-linked Hb-Presbyterian, and of
-fumaryl HbA relative to uncross-linked HbA, are presented in Table I
. As can be seen, as with HbA, introduction of the
-fumaryl cross-bridge into Hb-Presbyterian resulted in a reduction of the DPG modulated lowering of its O2 affinity. However, this reduction in DPG-mediated lowering of the O2 affinity of
-fumaryl Hb-Presbyterian was more pronounced than that observed with
-fumaryl HbA. The 4.1-fold increase in P50 observed with HbA in the presence of DPG was decreased to 3.2-fold on
-cross-linking. More strikingly, the 4.7-fold increase in P50 observed for Hb-Presbyterian in the presence of DPG was decreased to 2.3-fold on
-cross-linking. Hence it is apparent that the communication of the structural consequences of the ß108 Asn
Lys mutation to the ßß-cleft is conserved even after the
-cross-linking, but to a lesser degree compared with uncross-linked Hb-Presbyterian.
As can be seen from the data in Table I, the
-fumaryl cross-linking also results in a reduction of the propensity of chloride to activate the latent low O2 affinity-inducing potential of Lys108(ß). The 5.4-fold increase in the P50 of uncross-linked Hb-Presbyterian observed in the presence of 1 M chloride is decreased to 2.3-fold on
-fumaryl cross-bridging. Nevertheless, unlike with DPG, the chloride sensitivity of Hb-Presbyterian is conserved relative to HbA (2.3- vs 1.6-fold), even after
-fumaryl cross-linking. Thus, the propensity of chloride to activate the low O2 affinity-inducing potential of Lys108(ß) of Hb-Presbyterian appears to be a site-specific phenomenon rather than just a direct consequence of the increased positive charge density in the middle of the central cavity. Furthermore, both in the presence of chloride and in the presence of DPG, the O2 affinity of
-fumaryl Hb-Presbyterian reaches the limiting value that is attained by both HbA and Hb-Presbyterian in the presence IHP. Thus, the presence of the
-fumaryl cross-bridge permits Hb- Presbyterian to access a T-state-like quaternary structure in the presence of DPG and chloride that is accessed by HbA in the presence of IHP. However, in the presence of IHP, the
-fumaryl cross-bridge has no further low O2 affinity- inducing potential either with Hb-Presbyterian or with HbA. Both cross-linked proteins exhibit the same limiting value of P50 as the uncross-linked proteins under similar conditions.
Influence of the allosteric effectors on the degree of synergy between the two chemical perturbations
A comparison of the relative increases in the P50 of HbA and Hb-Presbyterian as a consequence of -fumaryl cross-linking is shown in Table II
. Here, the data from Table I
are analyzed as ratios of the P50s of the two samples under comparison. The maximum influence of the
-fumaryl cross-bridging on the O2 affinity of HbA and Hb-Presbyterian is seen in the absence of the allosteric effectors chloride, DPG and IHP. The
-fumaryl cross-linking increased the P50 of HbA by 2.7-fold whereas that of Hb-Presbyterian increased by 4.1-fold, clearly reflecting a synergy between the molecular consequences of
-fumaryl cross-bridging and Asn108(ß)
Lys mutation. The
-fumaryl cross-bridging and Asn108(ß)
Lys mutation exhibit a synergy even in the presence of chloride (1.1- vs 1.8-fold, respectively). On the other hand, the influence of
-fumaryl cross-bridging on the DPG-mediated O2 affinity is nearly the same (2.1- vs 2.0-fold, respectively) for both HbA and Hb-Presbyterian, reflecting the absence of synergy between the
-fumaryl cross-bridging and Asn108(ß)
Lys mutation with this effector. The cross-linking had very little influence, if any, on the IHP-modulated O2 affinity of HbA as well as Hb-Presbyterian. Thus, the
-fumaryl cross-bridge appears to elicit elements of the same or similar structural modulations of Hb-Presbyterian that are achieved through interactions with allosteric effectors. However, the extent to which the cross-bridge can facilitate the access of the T-state-like features is distinct with each of these effectors.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The decrease in the isoelectric pH of Hb-Presbyterian resulting from its -fumaryl cross-bridging is consistent with a reduction in the positive charges of the protein due to the modification of the
-amino groups of the protein. This is in contrast to the lack of any appreciable change in isoelectric point on the
-fumaryl cross-bridging of HbA (Walder et al., 1994
). This anomalous behavior of
-fumaryl HbA has been attributed to an increase in the pKa of the neighboring Glu101(ß), leading to an uptake of protons at that site. If Hb-Presbyterian also experiences the same chemical perturbations as HbA on cross-linking at Lys99(
), then the isoelectric points of
-fumaryl HbA and
-fumaryl Hb-Presbyterian would conserve the same difference that existed between the two parent proteins. The observed lack of such conservation in the isoelectric point of
-fumaryl Hb-Presbyterian suggests that the perturbations in the ionization behavior of the neighboring residues induced by the
-fumaryl cross-bridging of Hb-Presbyterian are distinct from those of HbA.
The overall influence of -fumaryl cross-bridging on the O2 affinity of HbA and Hb-Presbyterian and its modulation by allosteric effectors is the same. However, the greater reduction in the intrinsic O2 affinity of Hb-Presbyterian on
-cross-linking suggested a synergy between the molecular consequences of
-fumaryl cross-bridging and Asn108(ß)
Lys mutation. Apparently, in chloride-free buffer, the structural modulations resulting from the ß108 Asn
Lys mutation and the
-fumaryl cross-bridging act in concert with one another in reducing the O2 affinity of Hb. While a synergy between the two molecular consequences was still apparent in the presence of chloride, the effect was only additive, at best, in the presence of DPG.
The results of the present study, in conjunction with the previously reported results, further indicate that the nature and location of the intramolecular cross-link within Hb-Presbyterian has distinct influences on its O2 binding properties. As described in the present study, intramolecular cross-bridging at the middle of the central cavity via the -chain (fumaryl cross-bridging) preserves the chloride-mediated lowering of the O2 affinity of Hb-Presbyterian to a large degree and exhibits synergy with the Asn108(ß)
Lys mutation. In contrast, rHb1.1, the recombinant
-Hb-Presbyterian wherein the two
-chains are linked genetically C- to N-termini by a glycine bridge, i.e. the cross-link is at the
-end of the central cavity, exhibits nearly the same O2 affinity as the uncross-linked Hb-Presbyterian (Looker et al., 1992
). However, the O2 affinity of the recombinant
-Hb was measured in the presence of 100 mM chloride. Hence, whether there is a synergy between the low O2 affinity-inducing potential of Lys108(ß) and this genetic
-cross-bridging in the absence of chloride is not apparent. It may be further noted that unlike the
-cross-linking at the middle of the central cavity, intramolecular cross-linking of Hb-Presbyterian via the ß-chains at the ßß-cleft (suberate cross-bridging) results in a significant loss of its chloride sensitivity (O'Donnel et al., 1994
). Thus, the influence of the Asn108(ß)
Lys mutation on the O2 affinity of Hb appears to be primarily dictated through the ßß-cleft.
Titration studies with a fluorescent DPG analog (Gottfried et al., 1999) as well as resonance Raman spectroscopic analyses (A.S.Acharya et al., unpublished results) point to the presence of T-state-like signatures in liganded Hb-Presbyterian. Crystallographic analysis of rHb1.1 (Hb-Presbyterian with a genetic
-glycine cross-bridge) has revealed that the structure of the liganded protein is distinct from those of the other known structures of liganded Hb. This new structure, referred to as the `B-structure', has T-state-like signatures at its ßß-cleft (Kroeger and Kundrot, 1997
). It is likely that the T-state-like structural signatures observed from the solution studies of the uncross-linked oxy Hb-Presbyterian (Gottfried et al., 1999; A.S.Acharya et al., unpublished results) may also be indicative of a `B' or `B-like' structure for this protein. It may be further noted that resonance Raman spectroscopic studies have also suggested a T-state-like structure for the ßß-cleft of ligated
-fumaryl HbA (Larsen et al., 1990
). In addition, our recent proton NMR studies (C.Ho et al., unpublished results) indicate that in the presence of IHP both Hb-Presbyterian and
-fumaryl HbA exhibit T-state-like signatures at their
1ß2 interface even in their liganded state. Thus, the two low O2 affinity-inducing structural modifications in the middle of the central cavity of Hb, namely Asn108(ß)
Lys mutation and
-fumaryl cross-bridging, appear to induce similar T-state-like signatures into liganded-HbA. The results of the present study have suggested that when present together, these two structural perturbations of HbA complement with one another and exhibit synergy.
The synergy between the low O2 affinity-inducing propensities of the Presbyterian mutation and the -fumaryl cross-bridging observed in this study is analogous to the synergy reported recently (Tsai et al., 1999
) for a recombinant double mutant of HbA, namely rHb(ßN108K,
V96W). It may be noted that the
V96W mutation is also located within the same region of the central cavity as the Presbyterian (ßN108K) mutation and the
-fumaryl cross-bridging. However, whereas the Presbyterian mutation is located at the
1ß1 interface and results in an increase in the positive charge within the central cavity, the Val96(
)
Trp mutation is located at the
1ß2 interface and is a neutral substitution. Nevertheless, like the Presbyterian mutation, the
V96W mutation also induces low O2 affinity into Hb (Kim et al., 1995
). This mutant protein also generates `T-state-like' signatures in the liganded-state in the presence of IHP. Tsai et al. observed that when these two mutations are present together in the double mutant rHb(ßN108 K,
V96W), they exhibited a synergistic effect in lowering the O2 affinity of Hb (Tsai et al., 1999
).
A comparison of the low O2 affinity-inducing propensity of the Asn108(ß)Lys mutation in conjunction with
-fumaryl cross-bridging and with the Val96(
)
Trp mutation is shown in Table II
. Also included in this table is a comparison of the influence of
-fumaryl cross-linking vs introduction of the
96V
W mutation on the O2 affinity of HbA. The low O2 affinity-inducing propensity is represented as a ratio of the P50s of the two samples under comparison. The data on the mutant Hbs rHb(
V96W) and rHb(ßN08K) and the double mutant rHb(
V96W, ßN108K) are taken from Tsai et al.'s paper (Tsai et al., 1999
). As can be seen from Table II
, the O2 affinity-lowering propensity induced into HbA by both the Asn108(ß)
Lys mutation and the Val96(
)
Trp mutation is nearly the same. On the other hand, the lowering of the intrinsic O2 affinity of HbA by
-fumaryl cross-linking is considerably greater than that induced by either the Asn108(ß)
Lys mutation or the Val96(
)
Trp mutation. In contrast, the O2 affinity-lowering propensity of the Asn108(ß)
Lys mutation in conjunction with either the
-fumaryl cross-bridging or with the Val96(
)
Trp mutation is nearly the same. As with the
-fumaryl cross-linking, the maximum synergy between the Asn108(ß)
Lys and the Val96(
)
Trp mutations is seen in the absence of effectors. Furthermore, the influence of the double mutation on the O2 affinity is also synergistic in the presence of chloride (1.4- vs 2.2-fold), whereas the DPG-mediated modulation is nearly the same.
The similarity of some of the synergistic activity of the -fumaryl cross-bridge and the Val96(
)
Trp mutation with the Asn108(ß)
Lys mutation suggests either an overlap or similarity in the molecular consequences of the
-fumaryl cross-bridging and the Val96(
)
Trp mutation. Conceivably, both the
-fumaryl cross-bridging and the Val96(
)
Trp mutation partially mimic the structural consequences of the interaction of Hb-Presbyterian with chloride, phosphate, DPG or protons. The data of the present study also reveal that the low O2 affinity-inducing propensity of the
-fumaryl cross-bridge also overlaps partially with the chloride- and/or DPG-mediated lowering of the O2 affinity of HbA. It may therefore be argued that both the
-fumaryl cross-bridge and the Val96(
)
Trp mutation are able partially to activate the latent low O2 affinity-inducing propensity of Lys108(ß) that is normally expressed in the presence of allosteric effectors. In this regard, it is of interest that in the presence of chloride the synergy of the Asn108(ß)
Lys mutation with the
-fumaryl cross-bridging and with the Val96(
)
Trp mutation is nearly the same, suggesting that these chemical/molecular manipulations appear to facilitate the access to the same low O2 affinity structure. It may also be added that the
-fumaryl cross-bridging of HbA has been suggested to influence the pKa of Glu101(ß) (Walder et al., 1994
). It is conceivable that the Val96(
)
Trp mutation could also result in a similar effect since
96(W) is located at the
1ß2 interface and in the T-state it forms a water-mediated hydrogen bond with Glu101(ß) (Puius et al., 1998
). Thus, the Val96(
)
Trp mutation appears to mimic protonation or the charge neutralization effect similar to that seen with the
-fumaryl cross-bridging of HbA.
In view of the differential influence of chloride on the O2 affinity of -fumaryl-HbA and
-fumaryl-Hb-Presbyterian, we have modeled the binding of chloride in the middle of the central cavity of these two proteins (Figure 5
). The chloride binding within the central cavity can neutralize the excess positive charge of the central cavity and this has been advanced as a molecular mechanism for the induction of low O2 affinity into Hb (Perutz et al., 1994
). However, a site-specific binding of the chloride ions within the central cavity is not an essential element of this hypothesis. The present molecular modeling studies of
-fumaryl-HbA and
-fumaryl-Hb-Presbyterian has shown that four chloride ions could bind in the middle of the central cavity involving three amino acid residues: two basic residues His103(
) and Arg104(ß) and the residue at ß108 (either Asn or Lys). In
-fumaryl-HbA, Asn108(ß), located between the two basic residues, could stabilize the interaction of the chloride ions with the positively charged residues through hydrogen bonds. This interaction is strengthened in
-fumaryl-Hb-Presbyterian owing to the replacement of the hydrogen bonds of Asn by the ionic interactions with Lys108(ß). Direct electrostatic calculations (with dielectric constant 80) carried out to determine the energy difference between the mutated and regular models in the presence of chloride ions indicated that each lysine residue was energetically more stable (1.07 kcal/mol) due to the presence of chloride ions in an aqueous environment. A comparative biophysical investigation of
-fumaryl-HbA and
-fumaryl-Hb-Presbyterian could provide new insights into potential new approaches for engineering of HbA to generate low O2 affinity hemoglobins that could be used as building blocks for the generation of Hb-based oxygen carriers.
|
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brucker,E.A. (2000) Acta Crystallogr., 56D, 812816.[ISI]
Chatterjee,R., Welty,E.V., Walder,R.Y., Pruitt,S.L., Rogers,P.H., Arnone,A. and Walder,J.A. (1986) J. Biol. Chem., 261, 99299937.
Fermi,G, Perutz,M.F., Shaanan,B. and Fourme,R. (1984) J. Mol. Biol., 175, 159174.[ISI][Medline]
Gottfried,D.S., Manjula,B.N., Malavalli,A., Acharya,A.S. and Friedman,J.M. (1999) Biochemistry, 38, 1130711315.[ISI][Medline]
Highsmith,F.A., Driscoll,C.M., Chung,C., Chavez,M.D., Macdonald,V.W., Manning,J.M., Lippert,L.E., Berger,R.L. and Hess,J.R. (1997) Biologicals, 25, 257268.[ISI][Medline]
Ho,C., Sun,D.P., Shen,T.-J, Ho,N.T., Zou,M., Hu,C.-K., Sun,Z.Y. and Lukin,J.A. (1998) In Tushida,E. (ed.), Present and Future Perspectives in Blood Substitutes. Elsevier, Lausanne, pp. 281296.
Kim,H.-W., Shen,T.-J., Sun D.P, Ho,N.T., Madrid,M. and Ho,C. (1995) J. Mol. Biol., 248, 867882.[ISI][Medline]
Kroeger,K.S. and Kundrot,C.E. (1997) Structure, 5, 227237.[ISI][Medline]
Larsen,R.W., Chavez,M.D., Ondrias,M.R., Courtney,S.H., Friedman,J.M., Lin,M.J. and Hirsch,R.E. (1990) J. Biol. Chem., 265, 44494454.
Looker,D.L. et al. (1992) Nature, 356, 258260.[ISI][Medline]
Macleod,R.M. and Hill,R.J. (1970) J. Biol. Chem., 245, 48754879.
Manjula,B.N., Roy,R.P., Smith,P.K. and Acharya,A.S. (1994) Artif. Cells Blood Subst. Immob. Biotechnol., 22, 747752.[ISI][Medline]
Manjula,B.N., Smith,P.K., Malavalli,A. and Acharya,A.S. (1995) Artif. Cells Blood Subst. Immob. Biotechnol., 23, 311318.[ISI][Medline]
Moo-Penn,W.F., Wolff,J.A., Simon,G., Vacek,M., Jue,D.L. and Johnson,M.H. (1978) FEBS Lett., 92, 5356.[ISI][Medline]
O'Donnel,J.K. et al. (1994) J. Biol. Chem., 269, 2769227699.
Perutz,M.F., Shih,D.T.B. and Williamson,D. (1994) J. Mol. Biol., 239, 555560.[ISI][Medline]
Ponder,J.W. and Richards,F.M. (1987) J. Mol. Biol., 193, 775791.[ISI][Medline]
Puius,Y.A., Zou,M, Ho,N.T., Ho,C. and Almo,S.C. (1998) Biochemistry, 37, 92589265.[ISI][Medline]
Rao,M.J., Schneider,K., Chait,B.C., Chao,T.L., Keller,H.L., Anderson,S.M., Manjula,B.N., Kumar,R.A. and Acharya,A.S. (1994) Artif. Cells Blood Subst. Immob. Biotechnol., 22, 695700.[ISI][Medline]
Snyder,S., Welty,E.V., Walder,R.Y., Williams,L.A. and Walder,J.A. (1987) Proc. Natl Acad. Sci. USA, 84, 72807284.[Abstract]
Tsai,C.-H., Shen T-J, Ho,N.T. and Ho,C. (1999) Biochemistry, 38, 87518761.[ISI][Medline]
Walder,R.Y., Andracki,M.E. and Walder,J.A. (1994) Methods Enzymol., 274280.
Received October 23, 2000; revised January 8, 2001; accepted February 22, 2001.