Hb Chesapeake Research Articles

Hb Chesapeake [alpha 92 (FG 4) Arg replaced by Leu] and Hb J Cape Town [alpha 92 (FG 4) Arg leads to Gln] first discovered in Japanese.

Hb Chesapeake [alpha 92 (FG 4) Arg replaced by Leu] and Hb J Cape Town [alpha 92 (FG 4) Arg leads to Gln] first discovered in Japanese.

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.

Proton nuclear magnetic resonance studies of hemoglobin Malmö: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malmö (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malmö are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malmö do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malmö can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malmö with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.

Hemoglobin Chesapeake (92α, Arginine → Leucine): PRECISE MEASUREMENTS AND ANALYSES OF OXYGEN EQUILIBRIUM

Abstract Precise oxygen equilibrium curves of hemoglobin (Hb) Chesapeake and human adult hemoglobin (Hb A) were determined by the automatic recording method in the presence and absence of NaCl, 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). The Adair constants, k1, k2, k3, and k4, and some oxygenation parameters were obtained from the equilibrium data. Maximum slope of the Hill plot for Hb Chesapeake, which was 1.22 under stripped conditions, was profoundly increased on the addition of 2 mm DPG and still more, beyond 2, on the addition of 2 mm IHP. It has been shown that DPG increases the cooperativity of oxygen binding to Hb Chesapeake by reducing k1, k2, and k3 without significant change of k4, as previously observed in Hb A. Within experimental errors, the k4 value for Hb Chesapeake was equal to that for Hb A whether DPG was present or absent. The ki value for Hb Chesapeake did not monotonously increase with i: k3 was significantly smaller than k2 whether DPG and IHP were present or absent. This resulted in slightly biphasic oxygen equilibrium curves of Hb Chesapeake. The biphasic oxygen binding was tentatively interpreted as a result of significantly cooperative interactions between the α1 and β1 (and α2 and β2) subunits and impaired subunit interactions at the α1β2 (and α2-β1) interface.

Allosteric Transition and Ligand Binding in Hemoglobin Chesapeake

Abstract The human hemoglobin (Hb) mutant Chesapeake (α92 Arg→Leu) is characterized by increased oxygen affinity, a Bohr effect, and a lowered value of n in Hill's equation in the midrange of saturation. The data presented here demonstrate that, in addition, the dissociation of oxygen from oxy Hb Chesapeake proceeds with a rate of 15 per s and that the carbon monoxide binding proceeds with a slightly accelerating time course at a rate of 3.5 x 105 m-1 s-1. The reactions of bromthymol blue and p-hydroxymercuribenzoate with oxy and deoxy Hb Chesapeake demonstrate that the R ⇄ T interconversion does occur in this mutant. Finally, oxygen binding and the oxygen equilibrium curve can be satisfactorily accounted for by a Monod, Wyman, Changeaux model (in its simplest form) with a small value of L (5 to 10) and a normal value of c. These results are in agreement with the analysis of Edelstein (Edelstein, S. (1971) Nature 230, 224–227) and the predictions of Shulman et al. (Shulman, R. G., Ogawa, S., and Hopfield, J. J. (1972) Arch. Biochem. Biophys. 151, 68–74) that the functional properties of Hb Chesapeake can be explained by an early conversion from the T to the R state as ligand binding progresses.

The hemoglobin-haptoglobin reaction as a probe of hemoglobin conformation

The dissociation of hemoglobin to dimers is a prerequisite for the binding of hemoglobin to haptoglobin. This reaction, then can be used as a conformational probe of the state of the α 1β 2 area of contact. With this approach, we find that mixed liganded hybrids ( α 2β 2 ∗ and α 2 ∗β 2 , where ∗ identifies the ferric pair of hemes) can exist in a conformation that resembles deoxyhemoglobin when deoxygenated. Also stripped deoxy Hb A can exist in a form able to dimerize more readily than deoxy Hb A bound to phosphates. Finally, the data shows that deoxy Hb Hiroshima and deoxy Hb Chesapeake exist in a conformation different from that of Hb A, expressed in a shift in the equilibrium between dissociating and nondissociating forms. This equilibrium is strongly affected by phosphates, with deoxy Hiroshima reverting to an almost normal behavior while deoxy Chesapeake still reacting rapidly with haptoglobin.

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.

Ligand binding in hemoglobin J Capetown

Hemoglobin J Capetown (α 92Gln 2β 2) exhibits a two- to threefold increase of oxygen affinity and, in preliminary evaluation, a normal Bohr effect. The combination velocity constant for CO is less than twice as great as that for Hb A, while the dithionite reaction with oxy Hb J Capetown shows a decreased rate as compared with Hb A. These data suggest that the kinetic basis of the high affinity of this hemoglobin variant is a combination of an increased combination velocity and a decreased dissociation velocity for ligands. The most striking finding is the presence of considerable co-operativity ( n in Hill's equation = 2.2) in the reaction of the hemes with ligands. The difference between the rate of the replacement reaction of O 2 by CO and the rate observed with the dissociation of O 2 by dithionite was comparable to Hb A, confirming the existance of co-operativity. Finally, the binding of haptoglobin occurs at a somewhat faster rate than normal Hb A. These findings in Hb J Capetown are in distinct contrast with the ligand binding properties of another mutation of the same site, Hb Chesapeake (α 92Leuβ 2). In this Hb, a much higher oxygen affinity is observed, in addition to a drastically reduced heme-heme interaction ( n = 1.37). Additionally, Hb Chesapeake exhibits a slower binding rate with haptoglobin as compared with Hb A. The differences in the type of perturbation introduced by the polar glutamine as compared with the hydrophobic leucine residue in the α 1 β 2 area of contact must explain the different array of properties in these two hemoglobins.

Nuclear magnetic resonance studies of hemoglobins. V. The heme proton spectra of human deoxyhemoglobins A, F, Zürich, and Chesapeake

The proton nuclear magnetic resonance spectra of human deoxyhemoglobins A, F, Zürich, and Chesapeake indicate that the heme groups in different subunits are magnetically nonequivalent. These results are in accord with earlier work on the cyano- and azido-derivatives of these methemoglobins. In addition, the nuclear magnetic resonance studies of Hb Chesapeake suggest that the altered functional properties of this mutant hemoglobin involve not only changes in the α 1- β 2 subunit contact region but also changes which affect the heme groups in the α-chains.

Nuclear magnetic resonance studies of hemoglobin. IV. The structure-function relationship of human adult hemoglobins a and Chesapeake and its implication to the nature of oxygenation of hemoglobin

Nuclear magnetic resonance spectra of Hb A and Hb Chesapeake in both carboxy- and deoxy-forms suggest that (1) there are differences in the aromatic proton resonances between these two proteins which may be due to the altered interactions among the amino acid residues at α 1-β 2 contacts as a result of the amino acid substitution at α-92 in Hb Chesapeake, and (2) amino acid residues at FG3 (leucine) and/or FG5 (valine) may be responsible for transmitting information from α 1 to β 2 chain or vice versa in the course of oxygenation of hemoglobin.

Abnormal human haemoglobins: Separation and characterization of the α and β chains by chromatography, and the determination of two new variants, Hb chesapeake and Hb J (Bangkok)

A method for the separation of the α and β chains of human haemoglobin by chromatography on carboxymethyl-cellulose in 8 M -urea has been described. Chromatography of a number of abnormal globins showed that the resolution of the system was sufficient to enable charged abnormal α or β chains to be separated from their normal counterparts. The use of this procedure to identify abnormal chains without performing hybridization experiments has been discussed. The aminoethyl derivatives of normal α and β chains obtained by chromatography have been characterized by peptide mapping. The tryptic peptides of aminoethyl-α and aminoethyl-β chains can be resolved by paper electrophoresis at pH 4·7 followed by chromatography in n-butanol—acetic acid—water—pyridine. Amino acid analyses have been performed on peptides eluted from the ninhydrin-stained peptide maps. The procedure has been applied to the characterization of two new haemoglobin mutants. Haemoglobin Chesapeake has been identified as α292Leuβ2 ,and haemoglobin J (Bangkok) has been identified as α2β256Asp.