Hb Kempsey Research Articles

Fourier transform infrared evidence against Asp beta 99 protonation in hemoglobin: nature of the Tyr alpha 42-Asp beta 99 quaternary H-bond.

The Tyr alpha 42-Asp beta 99 intersubunit H-bond stabilizes the T quaternary structure in hemoglobin (Hb) tetramers. We had proposed that Tyr alpha 42 acts as an acceptor in this H-bond, because the tyrosine Y8a/8b and Y7a' UVRR (ultraviolet resonance Raman) bands shift in directions opposite to those expected if tyrosine is an H-bond donor. If Asp beta 99 is the H-bond donor, then it must be protonated in the T state, and would be a previously unrecognized contributor to the Bohr effect. This implication was strengthened by the discovery that an R-minus-T difference FTIR (Fourier transform infrared) band at 1693 cm-1, which might be a signal from protonated carboxylate, is missing in Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. However, we now find that this FTIR signal is insensitive to 13C-labeling of the aspartate residues in Hb, and cannot arise from protonated Asp beta 99. There are no other difference signals in the 1700 cm-1 region at a sensitivity of one COOH group. We conclude that Asp beta 99 is not protonated, and that the anomalous UVRR shifts must arise from compensating polarization of the Tyr alpha 42 OH. Candidates for this compensation are the H-bond donated by the Asp beta 94 backbone NH, and the nearby positive charge of Arg beta 40.

FT-IR Difference Spectroscopy of Hemoglobins A and Kempsey: Evidence That a Key Quaternary Interaction Induces Protonation of Asp .beta.99

Fourier transform infrared difference spectra are reported for the CO adduct of human hemoglobin versus deoxyHb, in H2O and D2O. In addition to the well-known CO stretching and S-H(D) stretching bands, the difference spectra reveal numerous bands in the 1200-1700 cm-1 region, a number of which are assigned. Several amide modes are identified via their frequencies and D2O sensitivities. Bands arising from histidine protonation have also been found via comparison of the difference spectra at different pH(D) values, with the aid of aqueous histidine spectra. Of particular interest is the observation of a negative band at 1697 cm-1, which is assigned to the C = O stretch of carboxylic acid. This carboxylic acid is tentatively identified as the side chain of Asp beta 99, because it is missing in the difference spectrum of Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. Asp beta 99 forms a critical contact with Tyr alpha 42 across the alpha 1 beta 2 interface in deoxyHb, which is broken upon ligation. Protonation of Asp beta 99 in deoxyHb is consistent with UV resonance Raman evidence that Tyr alpha 42 is the acceptor rather than the donor of the quaternary H-bond.

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Evidence for a kinetic heterogeneity in ligand binding to R-state haemoglobin Kempsey [Asp-G1(99) beta----Asn

Thermodynamic and kinetic properties of O2 and CO binding to haemoglobin (Hb) Kempsey [Asp-G1(99) beta----Asn] were investigated and the activation parameters for the two ligands were determined. At every temperature the O2-binding isotherms display a weak co-operativity, n ranging between 1.1 and 1.2, and dissociation kinetics show a single-exponential behaviour. O2-binding kinetics were studied at 25 degrees C by temperature jump and are characterized at each saturation (from Y = 0.31 to Y = 1.0) by two processes, a fast bimolecular one and a slow monomolecular one (tau -1 = 20 s-1), which contributes to approx. 30% of the whole relaxation amplitude at every Y. CO-binding kinetics to Hb Kempsey were followed at several temperatures by flash photolysis and stopped flow. The process is biphasic, as reported elsewhere [Bunn, Wohl, Bradley, Cooley & Gibson (1974) J. Biol. Chem. 249, 7402-7409], and the relative contributions of the two bimolecular rates to the whole process are only slightly affected by temperature. On taking account for the fraction of dimers at every protein concentration, the slow phase corresponds to approx. 50% of the ligand binding to tetramers. Correlation of these results with previous spectroscopic data leads to the hypothesis that the biphasic time course of CO binding may be attributed to alpha/beta heterogeneity of the R-state of tetrameric Hb Kempsey.

A Comparative Study of the Separation of the Tryptic Peptides of the β-Chain of Normal and Abnormal Hemoglobins by Reversed Phase High Performance Liquid Chromatography

Abstract The separation of the tryptic peptides of the human hemoglobin A β-chain by reversed phase high performance liquid chromatography under different elution conditions on several microparticulate alkylsilica supports is described. Similar methods have been used to separate the tryptic peptides of β-chain hemoglobin variants including HbC, HbE, and Hb (Kempsey). Selectivity differences which can be achieved under the different chromatographic conditions have been exploited to permit the assignment of all the anticipated peptide fragments derived from the tryptic digestion of these β-chain Hb-variants.

Tryptophan fluorescence of human hemoglobin II. Effect of inositol hexaphosphate on the T-R transition

Fluorescence spectra of tryptophan residues of human hemoglobin in the absence and presence of inositol hexaphosphate were measured at room temperature. The tryptophan fluorescence intensity of deoxy HbA was observed to decrease in accordance with the binding with inositol hexaphosphate. The fluorescence intensity of HbA, Hb Kempsey (β99 Asp-Asn), Hb Chesapeake (α92 Arg-Leu) and NES-des-Arg Hb (des-141α Arg and β93 Cys- N-ethylsuccinimide derivative) in the presence of inositol hexaphosphate exhibits a considerable decrease in the deoxy to oxy transition, while no or slight fluorescence intensity change was observed in the deoxy to oxy transition of Hb Kempsey and NES-des-Arg Hb in the absence of inositol hexaphosphate. The tryptophan fluorescence behavior suggests that the inositol hexaphosphate-induced structural change in these hemoglobins is attributable to the formation of a different T type of structure from that of the normal T-R transition.

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.

Absence of heme-localized strain in T state hemoglobin: insensitivity of heme-imidazole resonance Raman frequencies to quaternary structure.

Substitution of pentadeuterated 2-methylimidazole in (2-methylimidazole)-Fe(II)-protoporphyrin IX, a model complex for deoxyHb, shifts three bands in the low-frequency resonance Raman spectrum 380 leads to 373 cm-1, 348 leads to 345 cm-1, and 220 leads to 218 cm-1. The first of these is assigned primarily to Fe-imidazole stretching, and the other two are assigned to porphyrin deformation modes with substantial Fe-pyrrole stretching contributions. The three bands are observed in deoxyHb and Mb. The Fe-pyrrole modes are at essentially the same frequencies in the two proteins, but the Fe-imidazole mode is 6 cm-1 lower in deoxyHb than Mb, implying a slight alteration in the heme-imidazole linkage. No change greater than 2 cm-1 is observed when Hb Kempsey is switched from the R to the T state. This observation places an upper limit on the energy stored in the Fe-imidazole bond of T state deoxyHb, which is estimated to be less than 0.2 kcal/mol (less than 836.8 J/mol).

Structure-function relations in hemoglobin as determined by x-ray absorption spectroscopy.

Conclusions concerning the structure around the iron atom in oxy- and carbonmonoxyhemoglobin have been obtained by fluorescent x-ray absorption studies. The bis-imidazole heme complex was used as a model system of known structure. The ligated forms of hemoglobin, and cytochrome c at high pH, gave spectra which were very similar to the bis-imidazole complex, where the average Fe-N bond distance is known to be 1.98 A. By comparison it was possible to determine that the average Fe-N bond distances were 1.99 A in oxyhemoglobin, 1.98 A in carbonmonoxyhemoglobin, and 1.98 A in cytochrome c at pH less than 10.5, with an experimental accuracy of +/-0.02 A. An experimental comparison between oxy- and deoxyhemoglobin A showed much larger spectral changes than amongst the ligated forms. A comparison was made between the low oxygen affinity form of deoxy HbA and the high affinity form of doexy Hb Kempsey (alpha2beta992 Asp leads to Asn). All the spectral features coincided, allowing us to conclude that the average iron-ligand bond differences must be less than or equal to 0.02 A. Since the strain energy is proportional to the square of this displacement, we show that the strain energy at the iron is less than or equal to 4 X 10(-3) eV. This is negligible compared to the difference of binding energy of the high and low affinity forms, which is 0.15 eV, showing that the energies responsible for the increase of oxygen affinity are not localized at the heme.

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.

Binding of triphosphate spin labels to hemoglobin kempsey

Summary The binding of 1-oxyl-2, 2, 6, 6-tetramethylpiperidine-4-triphosphate (I) to a mutant of human hemoglobin (Hb), Hb Kempsey (β-99 Asp→Asn), has been studied as a function of heme ligation. The (ligand-free Hb Kempsey) — (I) complex has a stoichiometry of 1.0±0.1 moles of I per mole of Hb Kempsey tetramer and a dissociation constant 1.7±0.5 × 10 −4 M at 13° in 0.05 M bisTris buffer, pH 7.3 and 0.1M in Cl − . A second spin label, N 6 --(1-oxyl-2, 2, 6,6-tetramethyl-4-piperidinyl) adenosine triphosphate, was used to probe the structure of the organic phosphate binding site in ligand-free Hb Kempsey. Neither label binds to fully liganded Hb Kempsey under these conditions. The results of these experiments are consistent with a generalized concerted transition model for cooperative ligand binding to hemoglobin.

Nuclear magnetic resonance studies of hemoglobins: VI. Heme proton spectra of human deoxyhemoglobins and their relevance to the nature of co-operative oxygenation of hemoglobin

The proton nuclear magnetic resonance spectra of human adult deoxyhemoglobin and several mutant or modified deoxyhemoglobins taken in 0.1 m-deuterated sodium phosphate at pD 7 and 25 °C show three prominent hyperfine shifted lines at approximately −18, −12 and −7 p.p.m. from HDO. In deoxyhemoglobins with amino acid substitutions or modifications near the heme group of the β chain, such as Hb Zurich (β63 His → Arg), Hb F3 (β → γ), carboxypeptidase A-treated Hb A, and spin-labeled Hb A, the position of the lowest field resonance line is variable but the two high-field lines are unshifted. In those hemoglobins with substitutions in the α 1 − β 2 subunit contact region of the protein, such as Hb Chesapeake (α92 Arg → Leu), Hb J Capetown (α92 Arg → Gln), Hb Yakima (β99 Asp → His), and Hb Kempsey (β99 Asp → Asn), all three resonance lines show simultaneous variations in their positions, regardless of which chain is abnormal. The lowest two field lines are assigned to heme methyl groups, and it is proposed that the heme groups in deoxyhemoglobin are non-equivalent. Since the lowest field line is sensitive to modification in the β chain, it is assigned to a methyl group of the β heme. The invariant line at about −12 p.p.m. is assigned to a homologous methyl of the α heme. The simultaneous variation of the spectral lines in hemoglobins with altered subunit contacts is interpreted to mean that an amino acid substitution in this region of the molecule perturbs not only the heme in the mutant chain but also the heme in the adjacent chain. This result suggests that in normal hemoglobin, the unliganded hemes may be influenced by the state of ligation of the hemes in the contiguous chains.