Ligand binding kinetics of des arginine haemoglobin

Des arginine 141 a haemoglobin (the haemoglobin in which the C-terminal arginine of the a chain has been removed) has a high affinity for oxygen and a reduced co-operativity in its oxygen equilibrium binding. The kinetic consequences of this modification are investigated in this paper. Deoxy des Arg haemoglobin binds carbon monoxide faster than does haemoglobin A, whilst oxy des Arg haemoglobin loses oxygen more slowly. These results are correlated with the oxygen equilibrium binding properties of des Arg haemoglobin. The carbon monoxide binding kinetics have been interpreted as implying a change in the parameter c (of the allosteric model), as well as L, when this arginine is removed from haemoglobin.     

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

The abnormal human hemoglobin Malm? (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 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? 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 Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? 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 Wood beta97(FG4) His replaced by Leu. A new high-oxygen-affinity hemoglobin associated with familial erythrocytosis.

The characterization of hemoglobin Wood (beta97(FG4) His replaced by Leu), a high oxygen affinity hemoglobin with reduced Hill constant is described. The amino acid substitution occurs at the alpha1beta2 interface, in the same position as in hemoglobin Malm? (beta97(FG4) His replaced by Gln) and in an homologous position when compared with hemoglobins Chesapeake (alpha92(FG4) Arg replaced by Leu) and J. Capetown (alpha92(fg4) arg replaced by Gln).     

Structures of deoxy and carbonmonoxy haemoglobin Kansas in the deoxy quaternary conformation.

Haemoglobin Kansas (Asn102(G4)β → Thr) is the only widely studied mutant or modified haemoglobin having four functional haems and displaying lower than normal oxygen affinity. Two forms of this mutant have been investigated by X-ray diffraction. The deoxy form, whose crystals are isomorphous with the native form, has been examined directly by the difference Fourier technique (3.4 Å). In addition to the replaced residue itself, the difference electron density map shows signs of slight movements throughout the region between α and β haem pockets. However, neither type of chain shows stereochemical evidence of a very abnormal oxygen affinity in the tetramer. The nature of the perturbation is different from that proposed in the earlier, low-resolution study of Greer (1971a). Exposure of deoxy crystals to carbon monoxide produces two new crystal forms similar but not identical to the native deoxy form. One of these structures has been solved by rotation and translation function methods and a difference map between carbonmonoxy haemoglobin Kansas in the deoxy quaternary structure and native deoxy haemoglobin has been calculated at 3.5 Å resolution. Carbon monoxide molecules are observed at three of the four haems, and two unidentified large peaks³ appear next to the sulphydryl groups of Cys93β. None of the interchain salt bridges which stabilize the deoxy quaternary state appears to be broken, but large tertiary structural changes are seen in the liganded chains. It seems that when the molecule is subjected to the lattice constraints of the deoxy crystal, the salt bridges do not break upon ligand binding, even though the pH dependence of the first Adair constant and the linearity of proton release with ligand uptake both imply that this does happen to stripped HbA in solution.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Disruption of a specific molecular interaction with a bound lipid affects the thermal stability of the purple bacterial reaction centre

Relatively little is known about the functions of specific molecular interactions between membrane proteins and membrane lipids. The structural and functional consequences of disrupting a previously identified interaction between a molecule of the diacidic lipid cardiolipin and the purple bacterial reaction centre were examined. Mutagenesis of a highly conserved arginine (M267) that is responsible for binding the head-group of the cardiolipin (to leucine) did not affect the rate of photosynthetic growth, the functional properties of the reaction centre, or the X-ray crystal structure of the complex (determined to a resolution of 2.8 Å). However, the thermal stability of the protein was compromised by this mutation, part of the reaction centre population showing an approximately 5 °C decrease in melting temperature in response to the arginine to leucine mutation. The crystallised mutant reaction centre also no longer bound detectable amounts of cardiolipin at this site. Taken together, these observations suggest that this particular protein-lipid interaction contributes to the thermal stability of the complex, at least when in detergent micelles. These findings are discussed in the light of proposals concerning the unfolding processes that occur when membrane proteins are heated, and we propose that one function of the cardiolipin is to stabilise the interaction between adjacent membrane-spanning α-helices in a region where there are no direct protein-protein interactions.     

Structure of the Fe-heme in the homodimeric hemoglobin from Scapharca inaequivalvis and in the T72I mutant: an X-ray absorption spectroscopic study at low temperature

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.     

Structure of deoxyhaemoglobin Yakima: a high-affinity mutant form exhibiting oxy-like 1 2 subunit interactions

Crystals of deoxyhaemoglobin Yakima (Asp Gl(99)β → His) are isomorphous with those of deoxyhaemoglobin A, even though the mutation produces disturbances in both the tertiary structure of the subunits and the quaternary structure of the tetramer. Asp Gl(99)β₂ lies at the α₁β₂ subunit interface, and in deoxyhaemoglobin A forms a crucial hydrogen bond with Tyr C7(42) α₁. The histidine residue that replaces the aspartate results in the removal of this single important intersubunit bond, and it further acts as a wedge between the α₁ and β₂ subunits, so that they are pushed apart and displaced part of the way towards the oxy structure. These disturbances are accompanied by the formation of a new intersubunit hydrogen bond, which is usually only observed in the oxy quaternary structure of haemoglobin. The disturbances at the α₁β₂ contact affect the stereochemistry of the entire molecule and are transmitted to the α and β haems. The X-ray structure of deoxy Yakima therefore provides a stereochemical explanation for its abnormal function; this being an abnormally high affinity for oxygen and vastly diminished haem-haem interactions.     

Studies on haemoglobin immobilized by cross-linking with glutaraldehyde. Cross-linked soluble polymers and artificial membranes

Human haemoglobin was immobilized by cross-linking with glutaraldehyde as soluble polymers and artificial membranes. Effects of pH and 2,3-diphosphoglycerate on oxygen binding and cross-linking were studied with haemoglobin immobilized in both the oxy and deoxy states. The cooperativity is suppressed and the affinity is increased when compared with native haemoglobin. Haemoglobin immobilized in the oxy state exhibited a higher oxygen affinity than that immobilized in the deoxy state. The alkaline Bohr effect is not significantly different from that of native haemoglobin. The 2,3-diphosphoglycerate influence on oxygen binding was reduced by one third with immobilization. In order to separate the chemical and the "conformation freezing' effects on the properties of immobilized haemoglobin, glutaraldehyde-modified haemoglobin in oxy and deoxy states was produced. Oxygen binding was studied and chemical modifications were checked by electrophoresis and gel filtration. This chemically modified haemoglobin without polymerization and without intra-chain bridging exhibits a behaviour similar to that of cross-linked soluble polymers or membranes of haemoglobin.     

Disruption of a specific molecular interaction with a bound lipid affects the thermal stability of the purple bacterial reaction centre

Relatively little is known about the functions of specific molecular interactions between membrane proteins and membrane lipids. The structural and functional consequences of disrupting a previously identified interaction between a molecule of the diacidic lipid cardiolipin and the purple bacterial reaction centre were examined. Mutagenesis of a highly conserved arginine (M267) that is responsible for binding the head-group of the cardiolipin (to leucine) did not affect the rate of photosynthetic growth, the functional properties of the reaction centre, or the X-ray crystal structure of the complex (determined to a resolution of 2.8 A). However, the thermal stability of the protein was compromised by this mutation, part of the reaction centre population showing an approximately 5 degrees C decrease in melting temperature in response to the arginine to leucine mutation. The crystallised mutant reaction centre also no longer bound detectable amounts of cardiolipin at this site. Taken together, these observations suggest that this particular protein-lipid interaction contributes to the thermal stability of the complex, at least when in detergent micelles. These findings are discussed in the light of proposals concerning the unfolding processes that occur when membrane proteins are heated, and we propose that one function of the cardiolipin is to stabilise the interaction between adjacent membrane-spanning alpha-helices in a region where there are no direct protein-protein interactions.     

Mutational Analysis of Residues in the Coiled-Coil Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263–273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426–430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.     

Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)beta leads to Ser).

A difference Fourier synthesis of deoxyhaemoglobin Tacoma minus deoxyhaemoglobin A at 3.5 Å resolution has been calculated. The map shows a large negative peak due to the removal of the guanidinium group of Arg B12(30)β, surrounded by positive and negative peaks indicative of some atoms moving towards, and others away from, the vacated site. Among the latter, the most important is the carboxylate of Glu B8(26)β which is hydrogen-bonded to the guanidinium group of the arginine in haemoglobin A, but swings round its α-β carbon bond towards the imidazoles of histidines G18(116) and 19(117)β in haemoglobin Tacoma. This movement would raise the pK values of the histidines, so that their positive charges compensated for the loss of the arginine. This may explain why haemoglobin Tacoma has the same electrophoretic mobility as haemoglobin A. It is shown that haemoglobin Tacoma has a lower oxygen equilibrium constant K_T and a larger allosteric constant L than haemoglobin A. The lowering of K_T may be due to the loosening of the T structure in haemoglobin Tacoma consequent upon the removal of the hydrogen bonds made by the guanidinium group of Arg B12(30)β at the α₁β₁ contact. Their removal also accounts for the decreased stability of haemoglobin Tacoma. We cannot yet explain its diminished Bohr effect, nor the increase in L.     

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-l-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-l-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding.     

Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

Oligomeric structure and chaperone-like activity of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.