Solid phase reaction of hemoglobin with spillover hydrogen

The reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between bovine hemoglobin and spillover hydrogen (SH) was studied. It was shown that, in the field of subunit contact, there is a significant decrease in ability for hydrogen exchange by SH. A comparison of the distribution of the isotope label in the hemoglobin α-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free α-subunit. To this end, enzymatic hydrolysis of protein under the action of trypsin was carried out. The separation of tritium-labeled tryptic peptides was achieved by HPLC. Changes in availability of polypeptide chain fragments caused by complex formation were calculated using a molecular model. The formation of the protein complex was shown to lead to a decrease in the ability of fragments of α-subunits MFLSFPTTK (A_32?40) and VDPVNFK (A_93?99) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 Å) twice decreased on the average. The decrease in ability to an exchange of hydrogen by spillover tritium on the formation of hemoglobin complex was shown to be connected with a reduction in availability of polypeptide chain fragments participating in spatial interactions of subunits with each other. Thus, the HSCIE reaction can be used not only for the preparative obtaining of tritium-labeled compounds, but also for determining the contact area in the formation of protein complexes.     

Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.     

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1.

Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α₂^Aδ²² -x- β⁵⁰, a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α₂^Aδ⁸⁷ -x- β¹¹⁶. Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A₁ (α₂β₂) and hemoglobin A₂ (α₂δ₂) have been carried out. A high value of n, the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A₁ and A₂ reflects changes of sequence that are common to both types of hemoglobin Lepore.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

Ligand-dependent aggregation of chicken hemoglobin AI

The hemoglobin A_I component of the white leghorn chicken may potentially provide an animal model for the $\text{in vitro}$ aggregation behavior of human hemoglobin S. In solutions of low ionic strength, it has been found to undergo a striking loss of solubility upon deoxygenation, leading to the formation of macromolecular aggregates. This property is not shared by the other major chicken hemoglobin component, designated A_II. Compositional and NH₂-terminal sequence analysis indicate that extensive primary structural differences reside in the alpha chains of these two hemoglobins. The beta chains appear to be identical. Examination by electron microscopy suggests that the deoxyhemoglobin A_I forms microcrystalline arrays. The A_I component shows diminished reactivity with ¹³CO₂, as judged from ¹³C NMR measurements.     

A proton motive force transducer and its role in proton pumps, proton engines, tobacco mosaic virus assembly and hemoglobin allosterism

In this paper I am proposing a new, conformationally dependent basic site in proteins. The initial formulation of this proposal was based on the following: (1) bacteriorhodopsin is a light-driven proton pump and as such is a prototype for understanding proton-mediated energy transduction in biological systems; (2) current evidence suggests about 2 protons are pumped for each photon absorbed; (3) given the usual role of prolines as helix breakers, it is surprising to find about 2 prolines deeply embedded in the membrane-spanning, probably α-helical, portion of the bacteriorhodopsin molecule; (4) another presumptive proton translocator, the F₀ proteolipid, is also helical and has a critical proline in its structure; (5) workers interested in protein folding have explained the existence of fast and slow folding subgroups of the same protein molecule as being due to cis : trans isomerization about the proline imide group; (6) the cis : trans isomerization is acid catalyzed; (7) simple chemical considerations predict that the proton affinity of the proline nitrogen should increase dramatically as the imide group is distorted away from planarity and should be a maximum midway between the cis and trans forms; thus, stabilization of the intermediate by protonation accounts for the acid catalysis of the proline cis : trans isomerization.Linking these observations together suggests that proline-containing α-helices may play a role in proton motive energy transduction. Due to the absence of a proton on the proline nitrogen, a proline-containing helix has a “proton hole” between the proline nitrogen and the carbonyl oxygen four residues earlier in the sequence. Here I propose a model in which the paramount feature is the change in pK_a associated with a change in geometry of the “proton hole.” Order of magnitude calculations suggest that the proton hole should change its pKa by about 8 units, corresponding to a 10⁸ change in proton affinity, for every 10 kcal of distortion energy, V. Calculations also show that it is energetically feasible to modulate the pK_a of this site over the dynamic range of pK_a = 2–14. Such a large value for $\text{ΔpK}{}_{\mathrm{a}}\text{ΔV}$ and such a dynamic range makes this site an ideal basis for an “integral proton injector,” an abstract model for proton pumping suggested on purely theoretical grounds by Nagle &; Mille (J. chem. Phys.74, 1367–1372, 1981).Finally, two well studied proteins, the α-chain of hemoglobin and tobacco mossaic virus coat protein, both show features in their X-ray determined structures suggesting the possibility of protonation and deprotonation of the proton hole in α-helices containing proline. For TMV coat protein, there is a proline-containing α-helix that is located precisely in the region of the protein which undergoes an acid-induced conformational rearrangement. Structural changes at this locus have been singled out in comparisons of the X-ray structures of the TMV protein in its two conformations. For the α-chain of horse hemoglobin, there are two concurrent sites that are likely protonated and one contrary site that likely becomes deprotonated as hemoglobin converts from the liganded to the deoxy form. The contrary proline is proposed to help maintain co-operative oxygen binding over a wide pH range. The absence of one of the concurrent proline site in marsupial hemoglobin accounts for the small Bohr effect exhibited by these hemoglobins. The absence of the contrary proline site in carp hemoglobin accounts in a very logical way for the large Bohr effect and the lack of cooperative oxygen binding at both low and high pH by this hemoglobin.     

Ontogeny of hemoglobins: Evidence for hemoglobin M

A new autosomal codominant hemoglobin mutation alters hemoglobin M of the primitive red cell line and hemoglobin D found in definitive cells. That Hb M and Hb D are altered by the same gene mutation supports the idea that Hb M shares a polypeptide chain with Hb D. It is concluded that in the switch from primitive hemoglobins to those of the definitive type, there are at least two α chains conserved; α^A of Hb E in Hb A and α^D of Hb M in Hb D.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Thermodynamics of equilibria of hemoglobins M Milwaukee-I and Saskatoon and isolated chains of hemoglobin a with carbon monoxide

The thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=?17 and ?18 kcal/mole heme and ΔS′=?30 and ?29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9^· and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=?15 kcal/mole and ΔS′=?11 e.u.).     

Implication of the alpha 1 beta 1 interface in the hemoglobin affinity changes. A comparative study between normal and San Diego fully ligated hemoglobins

The alpha 1 beta 1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of beta-112 cysteine, by Fourier-transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the alpha 1 beta 1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.     

19 F-NMR studies of oxygen binding to hemoglobin

The binding of oxygen to hemoglobin has been investigated by ¹⁹F-nuclear magnetic resonance spectroscopy. The ¹⁹F-nmr spectrum of hemoglobin trifluoroacetonylated at cysteine β 93 exhibits chemical shift changes on binding of ligands, which differ depending on which chains are undergoing complexation. Comparison of these changes to the fractional ligation of all chains, determined concurrently from the fractional change in the visible spectrum, shows that initial oxygen molecules bind preferentially to α-chains. The ¹⁹F-nmr spectrum of partially oxygenated hemoglobin contains resonances at the normal chemical shift positions of the oxygenated and deoxy species, in addition to two small resonances at intermediate positions. Analysis of the relativ magnitudes of these four peaks as functions of oxygen pressure permits identification of the intermediate species     

The tryptic peptides of coyote (Canis latrans) hemoglobin

The tryptic peptides from α- and β-chains of coyote (Canis latrans) hemoglobin have been isolated and their amino acid compositions determined. The compositions are identical to those previously found for dog hemoglobin in all respects except one: the αT-13 peptide of coyote has only threonine at residue 130 of the chain. This indicates only one α-chain in coyote instead of two as in dog, which has one α-chain with threonine and one α-chain with alanine at this position. The α-chain from wolf (Canis lupus) is like that from coyote in having only threonine at residue 130.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

Sulfhydryl groups and the structure of hemoglobin

1. Addition of 2 moles of mersalyl, mercuric chloride, p-chloromercuribenzoate (PCMB), or methyl mercury hydroxide per mole of hemoglobin greatly reduces heme-heme interactions (n), yet these substances have quite different effects on the oxygen affinity (-log p₅₀). Mersalyl and mercuric chloride at this concentration each increase the oxygen affinity, while PCMB and methyl mercury have little or no effect on the oxygen affinity. These effects are primarily associated with the binding of —SH groups, and are largely reversed on the addition of glutathione. —SH groups do not appear to be responsible for the Bohr effect. 2. Evidence is presented for the belief that the two hemes of each half-molecule of horse hemoglobin are situated on either side of a cluster of—SH groups. 3. The mechanism of interaction between the hemes is discussed. It is concluded that the reorganization of the protein architecture which accompanies oxygenation plays a central role in this interaction, in agreement with the views of Pauling and Wyman.     

The binding of 2,3-diphosphoglycerate as a conformational probe of human hemoglobins

Since 2,3-diphosphoglyeerate preferentially binds to deoxygenated hemoglobin A, this binding reaction can be used to detect the change in quaternary conformation of hemoglobin associated with the change in ligand state of the hemes. We have studied the binding to two M hemoglobins (M_HydePark, M_Milwaukee-1) that have the substituted chains in the ferric state, as well as to the mixed liganded hybrids α1₂β² and α²β1₂ (1 heme in cyanmet form) prepared from hemoglobins A and H. The studies demonstrate that when these hemoglobin variants and derivatives are deoxygenated, they bind the organic phosphate to an extent similar, but not identical, to that for fully deoxygenated hemoglobin A. The results indicate that removal of ligand from only two of the four hemes results in a change in quaternary structure to a deoxy-like conformation.     

The Nature and Significance of the Bohr Effect in Mammalian Hemoglobins

The oxygenation of hemoglobins is accompanied by the dissociation of protons. The number of protons discharged is inversely related to the size of the mammal from which the hemoglobin comes. The number of mercuric ions which are immediately bound by hemoglobins is approximately equal to the number of protons dissociated during oxygenation. Pretreatment of human hemoglobin by N-ethylmaleimide, which appears to bind only sulfhydryl groups prevents the binding of any mercuric ions under conditions when mercuric ions would otherwise be bound. These facts suggest that those mammals with higher metabolic rates will generally possess hemoglobins with a larger number of appropriately placed cysteine residues.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.