Titratable Histidine Research Articles

Charactering ionizable residue networks at SH2 interfaces in pH-sensitive proteins.

Intracellular pH (pHi) dynamics (7.0 to 7.8) regulate myriad cell processes but the molecular mechanisms driving these behaviors are largely unknown. While some pH-sensitive proteins use titratable histidine residues to sense physiological changes in pHi, others have unknown or histidine-independent mechanisms. Charged amino acids (Asp, Glu, and Lys) can have pKas that are up- or down-shifted into the physiological range depending on protein environment, but the role for these residues in regulating pH-dependent protein function is understudied. Here, we use computational and biochemical approaches to investigate pH sensors that use networks of cooperative ionizable residues with a cumulative physiological pKa. We performed computational analyses of two pH sensitive signaling proteins with unknown mechanisms: (phosphatidylinositol-3-kinase (PI3K) and SHP-2 tyrosine-protein phosphatase). In both proteins, we identified networks of ionizable residues with upshifted pKas at the inhibitory SH2 domain interface. We next used biochemical mutational analyses to probe the accuracy of our computational approaches by measuring pH sensitive activity of wild-type and mutant proteins in vitro. Importantly, because SH2 domains are structurally conserved, this pH-sensing mechanism could be conserved in a subset of kinases and phosphatases with inhibitory SH2 domains. We tested this hypothesis by identifying a network of ionizable residues in other signaling proteins that contain inhibitory SH2 domains. We analyzed JAK2 kinase, which has not been shown to have pH sensitive function, and identified an ionizable network at the SH2 domain interface. Future work will validate this JACK2 result biochemically and explore whether our computational approaches can be applied to identify pH sensitive proteins across the human proteome. Improved understanding of how ionizable networks respond to pH could reveal paths to better therapeutic targeting of these proteins in diseases with dysregulated pHi.

A unique activation-promotion mechanism of the influenza B M2 proton channel uncovered by multiscale simulations.

The influenza B M2 protein (BM2) forms an acid-activated proton channel that is important for the virus's lifecycle. Despite extensive research efforts, the detailed activation mechanism of the BM2 proton channel is often elusive. Herein a pH-regulated mechanism of the BM2TM domain has been systematically characterized using multiscale computer simulations, including classical molecular dynamics, constant pH molecular dynamics (CpHMD) and quantum mechanics/molecular mechanics (QM/MM) approaches. Our simulations reveal a pH-dependent conformational switch from the C-terminal closed to the C-terminal open conformers, and provide the free energy of conformational activation coupled to the titration of the His19 tetrad. Importantly, our results confirm the coupling titration between the His19 tetrad and His27 tetrad, and identify that the full-cationic state (His2744+) dominates at the low pH (the His19 tetrad at +2, +3 and +4 charge states). Our QM/MM simulations indicate that the second titratable histidine, His27, could further promote the BM2 acid activation and speed up proton dissociation from the HxxxW motif, thus facilitating proton conduction by BM2. Taken together, a unique "activation-promotion mechanism" about the BM2 proton channel is proposed, and these results may be helpful for the understanding of other similar proton channels and the development of BM2 inhibitors.

Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR

The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, 15N NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2 is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical 19F-19F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15-20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.

Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel.

Together with the influenza A virus, influenza B virus causes seasonal flu epidemics. The M2 protein of influenza B (BM2) forms a tetrameric proton-conducting channel that is important for the virus lifecycle. BM2 shares little sequence homology with AM2, except for a conserved HxxxW motif in the transmembrane (TM) domain. Unlike AM2, no antiviral drugs have been developed to block the BM2 channel. To elucidate the proton-conduction mechanism of BM2 and to facilitate the development of BM2 inhibitors, we have employed solid-state NMR spectroscopy to investigate the conformation, dynamics, and hydration of the BM2 TM domain in lipid bilayers. BM2 adopts an α-helical conformation in lipid membranes. At physiological temperature and low pH, the proton-selective residue, His19, shows relatively narrow (15)N chemical exchange peaks for the imidazole nitrogens, indicating fast proton shuttling that interconverts cationic and neutral histidines. Importantly, pH-dependent (15)N chemical shifts indicate that His19 retains the neutral population to much lower pH than His37 in AM2, indicating larger acid-dissociation constants or lower pKa's. We attribute these dynamical and equilibrium differences to the presence of a second titratable histidine, His27, which may increase the proton-dissociation rate of His19. Two-dimensional (1)H-(13)C correlation spectra probing water (1)H polarization transfer to the peptide indicates that the BM2 channel becomes much more hydrated at low pH than at high pH, particularly at Ser12, indicating that the pore-facing serine residues in BM2 mediate proton relay to the proton-selective histidine.

Heterodimerization within the TREK Channel Subfamily

TREK1, TREK2, and TRAAK form the TREK subfamily of two-pore-domain K+ (K2P) channels. Despite sharing up to 78% sequence identity and showing overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli, such as phosopholipids or external pH. For instance, TREK1 is inhibited by external acidification while TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single molecule pull-down and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2 and TRAAK readily co-assemble to form heterodimers. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the different heterodimers and found that all combinations form outwardly-rectifying potassium-selective channels but with variable voltage-sensitivity and regulation. TREK1/TREK2 heterodimers show low levels of activity at physiological pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modelling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel can be regulated independently of each other via titratable histidines and their interaction with nearby charged residues. TREK1/TRAAK heterodimers is also functional and unlike the TRAAK homodimer is able to be regulated by PLD2 which binds the TREK1 moiety. Together, this study greatly expands the number of channels within the TREK subfamily and defines how heterodimerization can alter the physiological properties of K2P channels.

Molecular and physicochemical characterization of hemoglobin from the high-altitude Taiwanese brown-toothed shrew (Episoriculus fumidus)

Red-toothed shrews (subfamily Soricinae) exhibit the highest mass-specific rates of O₂ consumption recorded among eutherian mammals, though surprisingly no data appears to be available on the functional characteristics of their hemoglobin (Hb). As a first step in addressing this shortcoming, we investigated the O₂ binding characteristics of Taiwanese brown-toothed shrew (Episoriculus fumidus) Hb and its temperature and pH dependence in the absence and presence of anionic red blood cell effectors. Although comparative data regarding the intrinsic O₂ affinity of other shrew species are currently unavailable, our data suggest that the sensitivity of this high-elevation endemic species' Hb to allosteric effector molecules is similar to that of the two lowland species of white-toothed (crocidurine) shrews examined to date. The efficient exploitation of blood O₂ reserves by E. fumidus appears to be achieved via synergistic modulation of O₂ affinity by Cl⁻ and organic phosphates that moreover dramatically lowers the overall enthalpy of oxygenation of their Hb. Oxygen unloading is presumably further enhanced by a relatively high Bohr effect (ΔLog P₅₀/ΔpH = -0.69) and marked reduction in the titratable histidine content (predicted low proton buffering value) of the component globin chains relative to human HbA. Notably, however, the limited data available suggest these latter attributes may be widespread among shrews and hence likely are not adaptations to chronic altitudinal hypoxia per se.

Kv1.7 - Interactions with Protons and a blocking Conotoxin

We have examined the interactions of protons and an inhibitory, poly-cationic conotoxin with the human voltage-gated potassium channel, hKv1.7. This channel differs from some members of the Kv1 sub family by having a titratable histidine residue near the N-terminal end of the putative pore-supporting P-helix, suggesting that intrinsic channel functions and pharmacology may depend on the pH of the external solution. Channels were expressed in HEK-293 cells and studied by whole-cell patch clamp. The voltage dependence of channel activation was evaluated using a tail-current protocol in which the voltage was stepped to -40 mV following a variable activating pre-pulse. Lowering of the pH of the external solution from 7.4 to 5.0 produced a positive shift in the half-activation of 36±3 mV (n=4). The lowering of pH also dramatically decreased the ability of the conotoxin to inhibit currents through the channels. Thus, following the largest depolarization, at pH5.0 very little inhibition was observed at a toxin concentration near IC50 for pH7.4. The tail current in the presence of the conotoxin was near the value seen in the absence of the toxin for external pH of 7.4. Our observations are consistent with a two-fold action of the conotoxin. First, they suggest that the positively charged toxin binds close enough to the S4 segment of the voltage sensor to inhibit activation following a depolarizing voltage step. Second, the observed decrease in maximal conductance at pH 7.4 following addition of the toxin is consistent with a block of current through the open channels. Toxin binding appears to be inhibited by protonation of a residue on the external surface of the channel, perhaps the histidine residue near the N-terminal end of the pore helix.

Cationic composition and acid–base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis

Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.

PH-dependent modulation of Kv1.3 inactivation: role of His399.

The Kv1.3 K(+) channel lacks N-type inactivation, but during prolonged depolarized periods it inactivates via the slow (P/C type) mechanism. It bears a titratable histidine residue in position 399 (equivalent of Shaker 449), a site known to influence the rate of slow inactivation. As opposed to several other voltage-gated K(+) channels, slow inactivation of Kv1.3 is slowed when extracellular pH (pH(o)) is lowered under physiological conditions. Our findings are as follows. First, when His399 was mutated to a lysine, arginine, leucine, valine or tyrosine, extracellular acidification (pH 5.5) accelerated inactivation reminiscent of other Kv channels. Second, inactivation of the wild-type channel was accelerated by low pH(o) when the ionic strength of the external solution was raised. Inactivation of the H399K mutant was also accelerated by high ionic strength at pH 7.35 but not the inactivation of H399L. Third, after the external application of blocking barium ions, recovery of the wild-type current during washout was slower in low pH(o). Fourth, the dissociation rate of Ba(2+) was pH insensitive for both H399K and H399L. Furthermore, Ba(2+) dissociation rates were equal for H399K and the wild type at pH 5.5 and were equal for H399L and the wild type at pH 7.35. These observations support a model in which the electric field of the protonated histidines creates a potential barrier for potassium ions just outside the external mouth of the pore that hinders their exit from the binding site controlling inactivation. In Kv1.3, this effect overrides the generally observed speeding of slow inactivation when pH(o) is reduced.

Essential Role of a Single Arginine of Photosystem I in Stabilizing the Electron Transfer Complex with Ferredoxin

PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K(d) of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K(d) value strongly depends on the charge of the residue substituting Arg-39: 0.22 microM for wild type, 1.5 microM for R39K, 56 microM for R39Q, and more than 100 microM for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K(d) value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin.

Hydrogen ion titrations of the anodic and cathodic haemoglobin components of the European eel Anguilla anguilla.

H+ titrations were conducted on the separated haemoglobin components of eel Anguilla anguilla in both the oxygenated and deoxygenated states. In anodic haemoglobin, the addition of GTP, and to a lesser extent C1-, increased the magnitude of the Haldane effect and shifted its maximum value into the in vivo pH range. Of the 22 histidine residues in the anodic component, only approximately seven were titratable, presumably the beta-chain residues at positions 41, 97, 109 and 146 (helical positions C7, FG4, G11 and HC3, respectively). In cathodic haemoglobin, a small negative Haldane effect was observed at pH values between 6.8 and 8.5 which disappeared in the presence of GTP (molar ratio 3:1 GTP:haemoglobin tetramer). GTP had virtually no effect on the buffer value at fixed oxygenation status, and the lowest buffer value was observed at in vivo pH values. No titratable histidine residues were observed in the cathodic component, indicating that all 14 histidines in this component are buried. We conclude that the anodic component, which constitutes two-thirds of the haemoglobin in the eel, plays the predominant role in CO2 transport and pH homeostasis in vivo.

Molecular localization of a titratable histidine residue in the intracellular pH modulation of human Kv1.4 N-type inactivation kinetics

Molecular localization of a titratable histidine residue in the intracellular pH modulation of human Kv1.4 N-type inactivation kinetics

Temperature dependence of histidine ionization constants in myoglobin

The standard enthalpy of ionization of six titratable histidines in horse metaquomyoglobin was determined by repeating proton NMR titrations as a function of temperature and using the van't Hoff relationship. It was found that deltaH degrees varies between 16 and 37 kJ mol(-1) in the protein, compared with a value of 29 kJ mol(-1) in free histidine. The standard entropy change was evaluated by combining the enthalpy and free energy changes derived from the pKa values. Although the entropy change could not be precisely and accurately obtained by this method, it could be established that it spans a wide range, from -60 to 0 J K(-1) mol(-1), about the value of -23 J K(-1) mol(-1) for the free histidine. The entropy change was used within the framework of enthalpy-entropy compensation to partition the solvation component from the standard thermodynamic quantities for each of the titrating residues. It was shown that the partitioning of the values in the protein is not readily understood in terms of solvent accessibility or electrostatic interactions. The contribution of solvation effects to the temperature response appeared to be significant only in the case of His-119 and His-48. The standard quantities were also used to explore the energetics of proton binding in the native state at temperatures below the onset of thermal denaturation.

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization.

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.

Kinetics and mechanism of carbonic anhydrase isoenzymes.

A mechanism model has been presented that can describe most known kinetic properties of carbonic anhydrase isoenzymes I, II, and III. The essential features of this model include: Nucleophilic attack of metal-bound OH- on CO2 to form metal-bound HCO-3. Formation of metal-bound OH- from metal-bound H2O. In isoenzyme II, and probably also in isoenzyme I, this reaction step involves an intramolecular transfer of H+ between the metal site and a titratable histidine residue via a number of hydrogen-bonded H2O molecules. In isoenzyme II, this step limits the maximal rate of catalysis. Also in isoenzyme III, the H2O-splitting step may be rate limiting, but since this isoenzyme has no titratable active-site histidine, H+ transfer may take place directly with components of the solvent. In isoenzymes I and II, rapid H+ transfer between active site and solution proceeds in a reaction between the titratable histidine residue and buffer molecules. The model can also rationalize a variety of observed inhibition patterns.

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonance corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196–9204). This substitution has small but significant effects on the behaviour of the other active-site histidines, His-64 and His-200. However, His-64 has an anomalously low p K a value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469–489).

A comparative study of some physico-chemical properties of human serum albumin samples from different sources—I: Some physico-chemical properties of isoionic human serum albumin solutions

Human serum albumin samples from different sources were investigated. The fatty acid content of the albumin before and after deionization on a mixed bed ion-exchange column varied from sample to sample. When an albumin sample from one source was deionized under standard conditions the amount of fatty acid bound by the albumin was reduced to a reproducible amount. In samples from different sources, however, the amount bound varied considerably. Also the isoionic pH of the albumin varied from sample to sample. This variation could be attributed to the difference in the fatty acid content and the different number of titratable histidines and acid amino-acid residues in the albumin from different sources. It can be concluded from the specific conductance of these isoionic solutions that ions other than H +, OH − and protein are effectively removed by a mixed bed ion-exchange column. The specific conductance of the albumin samples is directly related to the isoionic pH. Therefore, the isoionic pH and the specific conductance of the albumins reflect the heterogeneity of the albumin samples with respect to their primary structure and fatty acid content.

Isomeric incorporation of the Haem into monomeric haemoglobins of Chironomus thummi thummi. 2. The Bohr effect of the component III explained on a molecular basis and functional differences between the two isomeric structures.

The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH-dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C-2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH-dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile-E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His-G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III. Although the change in chemical shift of the His-G2 C-2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range. Considering the tertiary structure obtained from an X-ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His-G2 with the carboxyl group of the C-terminal methionine-H22.

The Bohr proton-binding site in a monomeric haemoglobin. A nuclear-magnetic-resonance study.

Individual haemoglobin III from Chironomus thummi thummi is a monomeric protein (Mr= 15500) which has recently been shown by us to be characterized by a simple Bohr effect. Three of the four histidines, the position of which in the polypeptide chain is known from sequence analysis, are distinguished by their titration characteristics, namely His‐G2, His‐G19 and His‐E5. The chemical shift of the C‐2 proton resonances of the imidazole groups, which varies upon protonation of the histidine, has been used for the determination of the intrinsic pK values. His‐F8, the proximally bound histidine, is not observable because its C‐2 proton resonance is broadened.One of the three titratable histidines reveals a pK value which is varied in the range from 7.5 to 7.1, depending on the 6th ligand at the haem iron. The relatively high pK value of this histidine and its abnormal value of the chemical shift of the C‐2 proton resonance at low pH indicate an interaction with a carboxylate group. This shift of the pK value caused by the binding of CO to reduced haemoglobin is in agreement with the interaction energy, calculated from the amplitude of the Bohr effect curve, and corresponds to 500 cal.In this simple case the assignment of the C‐2 proton resonances to the known histidines is possible considering the atomic model, described by Huber et al. [4]. The results indicate that His‐G2 is the allosteric binding site; the remaining two detectable histidines are not involved in the allosteric interaction. The molecular mechanism of the Bohr effect in this haemoglobin is discussed.

Titration Behavior of Histidines in Human, Horse, and Bovine Hemoglobins

Abstract The titration curves of the human hemoglobins A2 and F, the slow and fast components of horse hemoglobin, and bovine hemoglobin B have been analyzed, mainly to obtain information about the titration behavior of the histidines in these hemoglobins. The results were also compared with earlier data concerning human hemoglobin A and bovine hemoglobin A. In both hemoglobin A2 and F, 18 titratable histidines were found, two less than in hemoglobin A. Assuming that those histidyl residues which in the three-dimensional model occupy the same sites will show similar titration behavior, we reached the conclusion that in human hemoglobin histidine G19 and histidine H21 are titratable and that histidine G18 is not titratable. Both the slow and fast components of horse hemoglobin were found to contain 22 titratable histidines and 70 titratable carboxyl groups. Combining these results with the known number of amides in the α chain we calculated that the β chain should contain 11 amides. It appeared that in bovine hemoglobin B the histidines at B1(18)β, which are absent in bovine hemoglobin A, are titratable. The pK of these histidines, as estimated from the difference titration curve, is about 7.8, both in oxy- and deoxyhemoglobin. This high pK is probably caused by the formation of a saltbridge with the carboxyl group of aspartic acid B3(20)β in the same chain. Both hemoglobins contain 72 titratable carboxyl groups. Comparing the human, horse, and bovine hemoglobins, assuming again similar titration behavior for structurally identical histidines, we were able to correlate the titration results of human and horse hemoglobin. The human and bovine hemoglobins failed to show an equally good correlation, although the discrepancy was small.