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His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s(-1) in α subunits and ∼10-fold faster in β subunits, ∼175,000 s(-1). Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is ∼80-90 μm(-1) s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, ∼0.08 μm(-1) s(-1). A transient intermediate with k'(CO) ≈ 0.7 μm(-1) s(-1) is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.

We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.

In order to investigate the role of the R (relaxed) to T (tense) structural transition in facilitating polymerization of deoxy-Hb S, we have engineered and expressed two Hb S variants which destabilize either T state (Hb S-Kempsey, alpha 2 beta 2 Val-6,Asn-99) or R state structures (Hb S-Kansas, alpha 2 beta 2 Val-6, Thr-102). Polymerization of deoxy-Hb S-Kempsey, which shows high oxygen affinity and increased dimer dissociation, required about 2- and 6-fold higher hemoglobin concentrations than deoxy-Hb S for polymerization in low and high phosphate concentrations, and its kinetic pattern of polymerization was biphasic. In contrast, oxy- or CO Hb S-Kansas, which shows low oxygen affinity and increased dimer dissociation, polymerized at a slightly higher critical concentration than that required for polymerization of deoxy-Hb S in both low and high phosphate buffers. Polymerization of oxy- and CO Hb S-Kansas was linear and showed no delay time, which is similar to oversaturated oxy- or CO Hb S. These results suggest that nuclei formation, which occurs during the delay time prior to deoxy-Hb S polymerization, does not occur in T state oxy-Hb S-Kansas, even though the critical concentration for polymerization of T state oxy-Hb S-Kansas is similar to that of T state deoxy-Hb S.

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.

The CO-binding kinetics and the optical spectra of the NO derivative of the homodimeric hemoglobin from Scapharca inaequivalvis have been investigated over the range between pH 7.0 and 2.0. In the deoxygenated derivative, protonation of the proximal imidazole at very low pH values and the consequent cleavage of the Fe-N epsilon bond result in a approximately 50-fold enhancement of the rate constant for CO binding, as found in other hemoproteins. However, in the case of the hemoglobin from S. inaequivalvis, the pH profile displays a cooperative behavior (n = 1.8 +/- 0.1), a unique feature that differentiates this protein from any other hemoprotein investigated thus far. Cleavage of the proximal bond in the NO derivative of S. inaequivalvis hemoglobin likewise displays a very steep pH transition. The mode of assembly of the homodimer, in which the heme-carrying E and F helices provide the subunit interface and bring the hemes at a much shorter distance (18.4 A) than in vertebrate hemoglobins, is likely to provide the structural basis for this unique behavior.

Binding of CO to the hemoglobin of Tunnus thynnus has been studied by kinetic and static methods in the range pH 6.75-8.0 and at temperatures between 1.5-40 degrees C. The results may be described in terms of the two-state model of cooperativity (Monod. J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) extended to include chain differences. The rate constants for CO binding to alpha and beta chains in the R state, at 20 degrees C, were 8.4 and 1.9 microM-1 s-1 with associated apparent activation energies of 3.5 and 10.5 kcal mol-1. Dissociation from the R state was monophasic with a rate constant, at 20 degrees C, of 0.016 s-1 and Ea of 26.4 kcal mol-1. The difference spectrum for binding of CO to the alpha chain in the T state is displaced 2 nm to the red as compared to the spectra associated with CO binding to the beta chain in the T state and to both chains in the R state. The proportion of T state hemoglobin present at different fractional saturations can thus be measured and compared with the results predicted from the model. When the temperature is raised, the value of L decreases greatly with an enthalpy of +80 kcal mol-1. The effects of the change in L override the decrease in intrinsic CO affinity of T. thynnus hemoglobin in the R and T states and result in a reverse temperature dependence of CO equilibria compared to mammalian hemoglobins.

The results of a series of light scattering experiments of the reaction of inositol hexaphosphate (at pH 7.0) over 6 orders of magnitude of concentration (10(-8) to 10(-2) M) with carboxyhemoglobin indicates that there is a shift in the tetramer-dimer equilibrium towards the tetramer, reaching a maximum effect at 0.1 mM inositol hexaphosphate. Raising the phosphate concentration beyond this latter value promotes dissociation to dimers. However, in this range some of the dissociation of carboxyhemoglobin was undoubtedly due to the increase in ionic strength from the inositol hexaphosphate ion. If the effect of ionic strength is allowed for by classical Debye-Huckel theory, one- and possibly two-phosphate binding sites per dimer can be detected. (Approximate association constant is 8000 M-1 for a single site at 0.1 ionic strength). The location of such sites is considered to lie near the dissociable plane of the hemoglobin tetramer and possibly to include half of the residues that bind inositol hexaphosphate in the tetramer.

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.

The rate of reaction between alpha-amino groups and cyanic acid was followed at 26 degrees and ionic strength 0.2 M as a function of pH of human hemoglobin Ao solutions to determine the pK and the pH-independent second order rate constant, kappa, for these groups in the alpha and beta chains. At a given point in time, the extent of the reaction was determined by employing the Beckmann Sequencer as a quantitative tool in which the yields of leucine and histidine in the second Edman degradation cycle were used to define the rates of reaction of the alpha and beta chains, respectively. From these results, the individual were evaluated (Garner, M.H., Garner, W.H., and Gurd, F. R.N. (1973) J. Biol. Chem. 248, 5451-5455). Values for pK for the alpha and beta chains were, respectively, 6.74 and 6.93 for cyanoferrihemoglobin, 6.95 and 7.05 for carboxyhemoglobin, and 7.79 and 6.84 for deoxyhemoglobin. Values for kappa, M- minus 1 S-minus 1, for the alpha and beta chains were, respectively, 12.5 and 17 for cyanoferrihemoglobin, 12 and 18 for carboxyhemoglobin, and 91 and 24 for deoxyhemoglobin. Limits of significance were estimated for both variables in each case. The pK results for valine 1alpha agree well with the value obtained by Hill and Davis (1967) J. Biol. Chem. 242, 2005-2012) for carboxyhemoglobin and with that of Kilmartin and Rossi-Bernardi ((1971) Biochem. J. 124, 31-45) for deoxyhemoglobin. Values obtained for sperm whale myoglobin were 7.77 for pK and 7.4 for kappa. The results are useful for the interpretation of the allosteric interactions of hemoglobin with hydrogen ions, with CO2, and with phosphate.

These experiments indicate that absorbance changes observed at the 425 nm isosbestic point of the Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb (the immediate product of full photolysis) and slowly reacting normal deoxyhemoglobin. Hb, first observed by Gibson (Gibson, Q.H. (1959) Biochem. J. 71, 293-303), Has been interpreted as deoxyhemoglobin remaining in the liganded quaternary conformation following rapid removal of ligand by a light pulse. In borate buffers between pH 8.4 and 9.6 particularly simple pH-independent results were obtained which allowed the use of a Monod. Wyman, and Changeux model (Monod, J., Wyman, J., and Changeux, J (1965) J. Mol. Biol. 12, 88-118) to fit the data. In this case Hb is taken to be R state deoxyhemoglobin. Partial photolysis experiments at 425 nm show that the rate of the R - T conformational change at 20 degrees decreases by about a factor of 2 for each additional bound ligand. The rate of the ligand-free conformational change is found to be 920 +/- 60s(-1), 6400 +/- 600s(-1), and 15,700 +/- 700(-1) respectively at 3 degrees, 20 degrees, and 30 degrees. The previously uninterpreted effects of flash length and partial photolysis on the CO recombination kinetics can be explained in terms of the present model. Kinetic results obtained below pH 8 are found to be inconsistent with a two-state model. It appears that binding of inositol hexaphosphate produces a new rapidly reacting quaternary conformation of HbCO.

Des-arg iron-cobalt hybrid hemoglobins, des-arg alpha(Co)2 beta (Fe)2, and des-arg alpha (Fe)2 beta (Co)2, in which the alpha-141 arginine residues are digested by carboxypeptidase B, were prepared, and their functional and EPR spectral properties were examined. The overall oxygen affinities of the alpha and beta subunits in both iron and cobalt hemoglobin tetramers were increased by the removal of this arginine residue and that of the alpha subunits was markedly influenced as compared with the beta subunits. In des-arg hemoglobins, the difference in the oxygen affinities between the alpha and beta subunits was smaller than that in the unmodified hemoglobins. The rate of carbon monoxide binding to the ferrous subunits in deoxy iron-cobalt hybrid hemoglobins were increased by 6- and 10-fold for the alpha (Fe)2 and beta (Fe)2 subunits, respectively, by this modification. The deoxy EPR spectrum of des-arg alpha (Co)2 beta (Fe)2 showed that the stripped deoxy des-arg hemoglobins are predominantly in the oxy quaternary structure. Comparison of the functional and EPR spectral data of des-arg hemoglobins with those of the unmodified hemoglobins indicated that the ligand affinity of the beta subunits is higher than that of the alpha subunits in the low affinity, quaternary structure of deoxyhemoglobin, but that this difference is small in the oxy, high affinity, quaternary structure.

The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.

An anti-hemoglobin antiserum obtained from a sheep immunized with human carboxyhemoglobin A0 demonstrated little difference in its reactivity with deoxy- or carboxyhemoglobin A0. However, a subpopulation of this antiserum isolated by synthetic peptide affinity chromatography clearly distinguished between these two hemoglobin species. This subpopulation, designated alpha(129-141) anti-hemoglobin antibodies, represents less than 1% of the total anti-hemoglobin antibodies. They are nonprecipitating by Ouchterlony analysis, and fluorescence-quenching studies demonstrate the interaction of a single antibody binding site per hemoglobin dimer. These antibodies bind preferentially to carboxyhemoglobin with a median affinity constant of 5 X 10(8) M-1 compared to binding to deoxyhemoglobin with a binding affinity of less than 1 X 10(8) M-1. Furthermore, the presence of these antibodies in stoichiometric amounts increases the oxygen affinity of hemoglobin, and thus antibody and oxygen binding to hemoglobin can be considered as a linked function.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

Kinetics of CO combination with and dissociation from isomer III, (alpha 1CO beta 1CO)alpha 2 beta 2 or alpha 1 beta 1 (alpha 2CO beta 2CO), and Hb Rothschild have been studied using the double mixing and microperoxidase methods. Isomer III was prepared in a manner so that it was the only reactive species in the reaction mixture. The biphasic reaction time course in both the "on" and "off" reactions of isomer III and the CO combination reaction of Hb Rothschild are attributed to slow relaxation between the fast and slow CO-reacting species in the two proteins: isomer III: l'f = 6 x 10(6) M-1 s-1, l'dimer = 1.7 x 10(6) M-1 s-1, l's = 2.2 x 10(5) M-1 s-1, lf = 0.15 s-1, ls = 0.01 s-1; Hb Rothschild: l'f = 2.8 x 10(6) M-1 s-1; l's = 2.7 x 10(5) M-1 s-1.

Reviewing the reaction of potassium cyanate, an antisickling agent, with alpha-amino groups of hemoglobin, it was found that the reaction was a slow process and requires a large excess of the reagent. The reason for the slow reaction rate of carbamylation of hemoglobin by cyanate is that cyanate itself does not react with hemoglobin. It is rather isocyanic acid, the reactive species, that reacts with hemoglobin. Since the pK of isocyanic acid is 3.8, only one out of 4,000 cyanate ions is present as isocyanic acid at physiological pH. Therefore, it appears that a large excess of cyanate ions is required to achieve the carbamylation of hemoglobin S, both in vivo and in vitro. Furthermore, the pH optimum for carbamylation of carboxyhemoglobin and deoxyhemoglobin is 5.5 and, at pH 7.4, the reaction velocity drops to one-third for carboxyhemoglobin and one-half for deoxyhemoglobin. To seek an approach to reduce the dosage of cyanate and to increase the reaction velocity, an isocyanate derivative, methylisocyanate which is already in the reactive form, was tested for its antisickling activity and its reaction with hemoglobin S. It was found that methylisocyanate had antisickling activity and that only a stoichiometric amount to 2-fold excess of the reagent over hemoglobin S alpha-amino groups was required to prevent the sickling of erythrocytes. Methylisocyanate-treated sickle erythrocytes showed an increased oxygen affinity compared to untreated methylisocyanate reacted with alpha-amino groups of hemoglobin S and the reaction was complete in less than 1 min. Methylcarbamylated hemoglobin S had a higher minimum gelling concentration than the untreated hemoglobin S. There was no detectable reaction of free sulfhydryl and epsilon-amino groups of hemoglobin S with methylisocyanate. These results indicate that methylisocyanate, and probably other isocyanate derivatives, possesses powerful antisickling activity.

A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.

Hb Suresnes is a human hemoglobin variant in which histidine replaces arginine at the COOH terminus of the alpha chains. The COOH-terminal arginines of the alpha chains play a major role in normal human hemoglobin (HbA), both by electrostatic interactions which constrain deoxygenated hemoglobin in a low affinity quaternary conformation and by involvement in oxygen-linked anion binding knoiwn to give rise to a large part of the alkaline Bohr effect. An x-ray crystallographic analysis of deoxyHb Suresnes reveals the loss of the normal intersubunit salt bridge to lysine 127 alpha and a decrease in the occupancy of inorganic anions at the alpha chain aniom-binding site. Relative to normal HbA, Hb Suresnes has a high affinity for oxygen, a reduced cooperativity in oxygen biding, and greatly reduced pH and chloride sensitivity. similar changes are found in carboxypeptidase B-digested hemoglobin Ao where the COOH-terminal arginine is removed by enzymatic digestion. Both equilibrium and kinetic manifestations of the destabilization of the normal low affinity configuration are observed. Inositol hexaphosphate restores cooperativity and pH sensitivity but, even in the presence of this strong allosteric effector, the low and high affinity conformations of Hb Suresnes appear to differ from those of HbA. In both the unliganded and liganded states, apreciable subunit dissociation of Hb Suresnes is apparent, which suggests that the substitution disrupts bonds which normally stabilize the tetramer. Structural changes at both the quaternary and tertiary level appear to contribute to alterations in the high and low affinity conformations of ths abnormal hemoglobin. Hb Suresnes thus illustrtaes the consequences of the loss of an important oxygen-linked anion-binding site.

The reactions of hemoglobin Kansas (102 β Asn → Thr) have been examined by kinetic and equilibrium methods. The behavior of the T state toward oxygen, derived from oxygen binding and oxygen pulse experiments, appears similar to that of hemoglobin A. The behavior of the R tetramer is difficult to separate from that of the dimer (the dissociation constant, K4,2, for oxyhemoglobin Kansas is about 70 µm as against 1.5 µm for hemoglobin A), but correlation of kinetic and equilibrium data suggests that the R tetramer of hemoglobin Kansas has a lower affinity for oxygen than that of hemoglobin A. Carbon monoxide binding is slower than for hemoglobin A, chiefly because of a lower rate of binding by the β chains. Comparisons between carbon monoxide binding and release of 8-hydroxypyrene-1,3,6-trisulfonic acid, and reaction with p-hydroxymercuribenzoate suggest that a conformation change (T → R transition) occurs on ligand binding as with hemoglobin A. Detailed examination of the dissociation reactions from the liganded dimers of oxygen and of carbon monoxide shows abnormally rapid dissociation from the β chain. The hemoglobin-haptoglobin reaction shows that deoxyhemoglobin Kansas is too slightly dissociated to give a measurable reaction. This result is consistent with an abnormally low ligand affinity of the R form. It is suggested that the differences between hemoglobin Kansas and hemoglobin A are due, at least in part, to differences between the R forms, and that an R → T transition occurs normally in hemoglobin Kansas. The allosteric effector 2,3-diphosphoglycerate lowers the affinity of hemoglobin Kansas for oxygen, but the effect is much smaller than for hemoglobin A when measured at high concentrations of hemoglobin. The data are most consistent with the assumption that the binding constant for 2,3-diphosphoglycerate at the primary site on tetrameric, liganded hemoglobin Kansas is only slightly lower than that for the unliganded form.

Observation of the ^(31)P signal from various intracellular phosphates can provide a convenient, nondestructive technique for determining intracellular conditions such as pH. This procedure has been explored with particular reference to the erythrocyte. Both the chemical shift of intracellular inorganic phosphate relative to that of serum phosphate and the positions of, and more especially the difference between, the chemical shifts of 2,3-diphosphoglycerate have been used to monitor intracellular pH of erythrocytes whose hemoglobin has been liganded with carbon monoxide.

The stoichiometry of carbon monoxide binding to beef heart cytochrome c oxidase has been reinvestigated both by titration of the reduced oxidase with CO and by measuring the amount of carboxyhemoglobin that is formed after adding oxyhemoglobin to a solution of the CO-enzyme complex. In the titration experiments the ratio of CO bounds to total heme a present was always less than 0.50 while in the experiments where oxyhemoglobin was added the results were variable and of lower accuracy. These observations do not agree with the recent conclusion of Volpe, J.A., O'Toole, M.C., and Caughey, W.S. (1975) Biochem. Biophys. Res. Commun. 62, 48-53 that CO is bound in a 1:1 ratio with heme a. An explanation for their results is suggested.