Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

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.     

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.     

Cation Modulation of Hemoglobin Interaction with Sodium n-Dodecyl Sulfate (SDS). III: Calcium Interaction with R- and Mixed Spin States of Hemoglobin S at pH 5.0: The Musical Chair Paradox

We investigate the interaction of Ca²⁺ (0–500 µM) and a membrane mimic (0.60 mM SDS) with both the R- and mixed spin states hemoglobin S (HbS) as a function of time. These interactions were carried out at pH 5.0. We aim at ascertaining if there is or are differences in the UV–Visible spectra of such interactions to account for the dynamics of calcium ion concentrations [Ca²⁺] in initiating structures which may ultimately suggest HbS polymerization and or resistance to Plasmodium attack. From our results, we conclude that (a) simultaneous interaction of 40 µM Ca²⁺ and 0.60 mM SDS with the R state protein would promote structural formations that can “lock up” the protein for nucleation on the membranes and or become cytotoxic to the parasite; (b) simultaneous R state HbS-SDS or R state HbS-Ca²⁺ would lead to enhanced hemin formation and less deoxyHb species. This condition is unlikely to precipitate polymerization in the HbS but the resulting hemin would poison the parasite; (c) the mixed spin state HbS-SDS and 40 µM Ca²⁺ interaction yields more toxic products to that of the interaction of the mixed spin HbS-SDS with 500 µM Ca²⁺ thus suggesting why the 40 µM Ca²⁺ is important in parasite Hb proteolysis; and (d) pronounced structural changes on interaction with SDS and Ca²⁺ are more in the R state to the mixed spin state.     

Structural nonequivalence of the alpha- and beta- heme-pockets in human methemoglobin. A proton magnetic relaxation study in solution.

Solvent-proton longitudinal magnetic relaxation rates as dependent on temperature were measured for human (H)/canine (C) valency hybrids of the type {α^H(III)β^C(II)}₂ and {α^C(II)β^H(III)}₂. The two metheme irons in the human methemoglobin chains induce quite different proton magnetic relaxation (pmr) rates reflecting a tighter β-heme-pocket compared to the α subunit. Both heme-pockets appear to be loosened in the presence of inositol hexaphosphate (IHP) although this allosteric effector binds only to the β chains, the binding assumed to be the same for canine as for human hemoglobin. The subunit nonequivalence is retained also in the T-quaternary state induced by IHP. In the species hybrids the pmr rates due to the metheme iron are sensitive to the valency (ligand) state, which was either CO or H₂O in the partner half of the hybrid. All results show very clearly the interrelationship of the tertiary (protomer) structure with the quaternary (oligomer) structure in hemoglobin.     

Facile synthesis and reactivity study of mixed phosphane-isocyanide Pd(II) and Pd(0) complexes

The reaction between an equimolecular mixture of isocyanide CNR (CNR = di-methylphenyl isocyanide (DIC), tert-butyl isocyanide (TIC), triphenyl phosphane (PPh₃) and a dechlorinated solution of the palladium allyl dimers [Pd(η³-allyl)Cl]₂ (allyl = 2-Meallyl, 1,1-Me₂allyl) in stoichiometric ratio yields the mixed derivative [Pd(η³-allyl)(CNR)(PPh₃)] only. Apparently, the mixed derivative represents the most stable species among all the possible ones that might be formed under those experimental conditions. Theoretical calculations are in agreement with the experimental observation and the energy stabilization of the mixed species with respect to the homoleptic derivatives is traced back to an overall push-pull effect exerted by the isocyanide and the phosphane acting synergically. Similar behavior is observed in the case of the synthesis of the palladacyclopentadienyl complexes [Pd(C₄(COOMe)₄)(CNR)(PPh₃)] and of the palladium(0) olefin complexes whose synthesis invariably yields the mixed [Pd(η²-olefin)(CNR)(PPh₃)] derivatives. The paper includes studies on the reactivity toward allylamination in the case of the palladium(II) allyl complexes. A diffractometric investigation on the solid state structures of four different palladium isocyanide-phosphane complexes is also included.     

Conformational requirements for the polymerization of hemoglobin S: studies of mixed liganded hybrids

Gelation experiments with artificially formed half-liganded hybrid tetramers of hemoglobin S demonstrate that when either the α chains or the β^s chains are fixed in the cyanmet (CNmet) liganded state, gelation occurs upon deoxygenation of the ferrous chains. The minimum concentration of hemoglobin required for gelation is equivalent for both hybrids (α₂^cnmetβ₂^s and α₂β₂^scnmet), is considerably higher than the concentration required to gel deoxy-Hb S (α₂β₂^s), and can be restored to the lower minimum gelling point of α₂β₂^s by reduction of the CNmet chains with dithionite. These results suggest that the most important conformational determinant of the deoxy state for polymerization of Hb S is the quaternary deoxy structure rather than the tertiary structural effect of the ligand state of the α or the β^s chains, and are furthermore consistent with the notion that asymmetric deoxy-CNmet hybrid tetramers assume a conformation which resembles, but is not identical to that of deoxyhemoglobin.The results of gelation experiments with mixtures of hemoglobins S and A in which selected chains of one or both hemoglobins are in the CNmet form support the concept that certain non-S hemoglobins may participate in the sickling process by forming hybrid tetramers with Hb S (such as α₂β^aβ^s). The conformational requirement for participation of these hybrids in polymers also appears to be a quaternary deoxy-like structure.     

The McClure and Weiss models of Fe–O2 bonding for oxyhemes, and the HbO2 + NO reaction

For the Fe–O₂(S = 0) linkages of oxyhemes, valence bond (VB) structures are re-presented for the McClure [Fe^II(S = 1) + O₂(S = 1)], Pauling–Coryell [Fe^II(S = 0) + O₂*(S = 0)], and Weiss [Fe^III(S = ½) + O₂ ^?(S = ½)] models of bonding. The VB structures for the McClure and Weiss models are of the increased-valence type, with more electrons participating in bonding than occur in their component Lewis structures. The Fe–O bond number and O–O bond order for the McClure structure are correlated with measured Fe–O and O–O bond lengths for oxymyoglobin. Back-bonding from O ₂ ^? to Fe^III of the Weiss structure gives a restricted form of the McClure structure. The McClure and Weiss increased-valence structures are used to provide VB formulations of mechanisms for the oxyhemoglobin + NO reaction. The products of these two formulations are Hb⁺ and NO₃ ^? (where Hb is hemoglobin) and Hb⁺ and OONO^?, respectively. Because Hb⁺ and NO₃ ^? are the observed products, they provide an experimental procedure for distinguishing the McClure and Weiss models. It is also shown that the same type of agreement between McClure-type theory and experiment occurs for oxycoboglobin + NO, cytochrome P450 monooxygenases, and related hydrogen atom transfer reactions. In the appendices, the results of density functional theory and multireference molecular orbital calculations for oxyhemes are related to one formulation of the increased-valence wavefunction for the McClure model, and theory is presented for the calculation of approximate weights for the Lewis structures that are components of the McClure increased-valence structure.     

Magnetic circular dichroism analysis of the ihp effect on spin equilibria in human ferric hemoglobins1

Magnetic circular dichroism (MCD) spectroscopy has been used to explore the connection between optical spectra and the high spin population of several hemoglobins under various conditions. It is found that the effectiveness of IHP in inducing spectral changes can be markedly affected by solvent. For example, the IHP-induced spectral changes in the visible region for nitritomethemoglobin-A in mixed buffer solvent systems (glycerol or polyethylene glycol (PEG), mw 190–210) are more than double those observed in aqueous buffers. We estimate that IHP induces a mix of R/T forms in bis-tris phosphate buffers, for NO₂^?metHb that is only about 50% T form. While PEG and glycerol both lead to enhanced IHP-induced spectral differences, they behave differently in two aspects. PEG shifts the visible MCD and absorption spectra of F^?metHb-A. supposedly already biased towards the T form by ligand, in the same direction that IHP does. PEG also maximizes the spin state changes with IHP for three R form hemoglobins and N₃^?metHb-A, and so appears to stabilize the T form in all cases. Glycerol does not. In addition, the apparent binding constant for NO₂^? to H₂OmetHb-A differs between these two solvents. Comparison of the data from several hemoglobins leads to the conclusion that the changes in spin state distributions induced by IHP correlate well with quarternary structure for a given hemoglobin. An analogous correlation amongst various proteins between initial spin state distribution (IHP) absent) and quarternary structure is not found.     

Magnetic circular dichroism studies of hemoglobin: The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin

The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b₅, to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8. 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b₅ This analysis generated a K_D that depends on ionic strength and hemoglobin tetramer conformation, a V_max that was independent of these factors, and an inhibition constant that was equal to K_d. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b₅ and hemoglobin and the electron transfer.     

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.     

Method for the complete separation of radioactively labeled human γ-globin chains from pre-βA chains on CM-cellulose chromatography: Use of cold carrier globin chains

Carboxymethyl (CM)-cellulose chromatography of globins is the technique used most frequently in analysis of hemoglobin synthesis. However, if this method is to be reliable in cases where only small amounts of fetal hemoglobin (α₂γ₂) compared to adult hemoglobin (α₂β₂^A) are synthesized, it is important to obtain a clean separation of γ chains from pre-β^A chains. In the past, it has been found that small amounts of pre-β^A chains tend to elute with the γ chains. Radioactively labeled γ chains can be completely and reproducibly separated from small amounts of labeled pre-β^A chains by the addition of unlabeled β^J chains (Hb J Baltimore = β16 Gly → Asp) which elute at the same position as the pre β^A chains, thus increasing the quantity of this peak and allowing a clean separation from the γ chains.     

Synthesis, structure and Mössbauer characterization of polymeric iron(II) complexes with bidentate thiourea ligands

Complexes of FeCl₂ with the known bis(3-methyl-2-thione-imidazolyl)methane (L¹) and the new bis(3-tert-butyl-2-thione-imidazolyl)methane (L²) are reported. For both [L¹FeCl₂]_n (3) and [L²FeCl₂]_n (4) X-ray crystallography reveals that 1D-polymeric chain structures are present in the solid state, with the two mercaptoimidazolyl units of L¹ and L² coordinating to different metal ions. Complexes 3 and 4 are further characterized by Mössbauer spectroscopy and SQUID magnetometry. NMR spectroscopy suggests that the complexes largely dissociate in polar solvents. X-ray structures of L² and its precursor bis(imidazolium) salt are also reported.     

Structures and oxygen affinities of crystalline human hemoglobin C (β6 Glu->Lys) in the R and R2 quaternary structures

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K₄) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2–3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.     

Synthesis,spectroscopic and redox properties of some ruthenium(II) thiosemicarbazone complexes: Structural description of four of these complexes

Sixteen neutral mixed ligand thiosemicarbazone complexes of ruthenium having general formula [Ru(PPh₃)₂L₂], where LH = 1-(arylidine)4-aryl thiosemicarbazones, have been synthesized and characterized. All complexes are diamagnetic and hence ruthenium is in the +2 oxidation state (low-spin d⁶, S = 0). The complexes show several intense peaks in the visible region due to allowed metal to ligand charge transfer transitions. The structures of four of the complexes have been determined by single-crystal X-ray diffraction and they show that thiosemicarbazone ligands coordinate to the ruthenium center through the hydrazinic nitrogen and sulfur forming four-membered chelate rings with ruthenium in N₂S₂P₂ coordination environment. In dichloromethane solution, the complexes show two quasi-reversible oxidative responses corresponding to loss of electron from HOMO and HOMO − 1. The E⁰ values of the above two oxidations shows good linear relationship with Hammett substituents constant (σ) as well as with the HOMO energy of the molecules calculated by the EHMO method. A DFT calculation on one representative complex suggests that there is appreciable contribution of the sulfur p-orbitals to the HOMO and HOMO − 1. Thus, assignment of the oxidation state of the metal in such complexes must be made with caution.     

Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria: A STUDY BY LOW TEMPERATURE FLASH PHOTOLYSIS AND RAPID WAVELENGTH SCANNING OPTICAL SPECTROMETRY

The reaction of mixed valence state cytochrome oxidase (Cu_A²⁺a³⁺ · Cu_B⁺a₃²⁺) with O₂ at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The “pure” difference spectra of the two species, I_M and II_M, relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I_M is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II_M by an α band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O₂⁻ → Cu_B²⁺ and O₂⁻ → a₃²⁺ charge transfer transitions which would imply that O₂⁻ forms a bridging ligand between Cu_B and the iron atom of cytochrome a₃ in species II_M. The kinetics of the reaction and the spectral characteristics of species I_M and II_M obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.     

Characterization of hemoglobin from Phoronis architecta (phoronida)

• 1.1. Phoronis architecta hemoglobin is composed of four distinct hemoglobin subunits with minimum MW's of 16–17,000 or 17–19,000 daltons. All four hemoglobins are monomeric when oxygenated. Two of the monomers combine to form dimers when bound with carbon monoxide.
• 2.2. In cellulo, Phoronis architecta hemoglobin has a half-saturation (P₅₀) value of 1.3 ± 0.1 mm Hg, shows cooperative oxygen binding (Hill coefficient = 2.7 ± 0.3), and no Bohr effect from pH 6.6 to 7.9. In vitro, the hemoglobin has a P₅₀ of 0.76 ± 0.21 mm Hg but shows no cooperativity (0.90 ± 0.15 (SD)).
• 3.3. The oxygen dissociation constant (K_off) from hemoglobin is 2.7 ± 0.2 sec⁻¹, and the computed oxygen association constant (K_on) is 2.5 × 10⁶ M⁻¹ · sec⁻¹ (1.9–3.6 × 10⁶ M⁻¹ · sec⁻¹).

     

Cadmium tri-tert-butoxysilanethiolates: Structural and spectroscopic models of metal sites in proteins

Syntheses and crystal structures of two molecular, heteroleptic cadmium complexes with CdS₂NO₂ and CdS₂N₂ kernels are described. Bis(tri-tert-butoxysilanethiolate)(1-methylimidazole)cadmium(II) and bis(tri-tert-butoxysilanethiolate)bis(1-methylimidazole)cadmium(II) coexist at equilibrium in chloroform solutions with varying concentrations of bis[bis(tri-tert-butoxysilanethiolate)cadmium(II)] and 1-methylimidazole. The equilibrium is characterized by solution ¹¹³Cd NMR spectra. Solid state CP MAS ¹³C, ²⁹Si, ¹¹³Cd NMR data for the complexes are also reported, analyzed and compared with the results obtained for cadmium-substituted proteins. The similarities and differences between the structures of cadmium complexes and their zinc analogues are discussed.     

Disc electrophoretic studies of hemoglobin dissociation by p-hydroxymercury benzoate

The reaction of excess p-mercurybenzoate with human hemoglobin produces five electrophoretic species on polyacrylamide gel. Only two of these bands have been previously observed when starch gel was employed. The chemical and electrophoretic studies presented in this paper illustrate that the appearance of an “extra” band in the β zone is due to the ability of PAGE to separate the βb₂^PMB ? β^PMB equilibrium to its discrete components. The remaining bands are accounted for by the superior resolution of PAGE over starch gel electrophoresis which allowed the detection of two (αβ)^PMB dimer species.     

Gelation of sickle cell hemoglobin IV. Phase transitions in hemoglobin S gels: Separate measures of aggregation and solution-gel equilibrium

Two assays of equilibrium properties in the gelation of deoxyhemoglobin S were carried out by analytical ultracentrifugation on the same sample: C_sat, the monomer concentration in equilibrium with the fully formed gel, was obtained as the supernatant concentration after sedimentation of a preformed gel. The presence of a plateau region during sedimentation of the supernatant and the rate of sedimentation of the boundary from which C_sat was measured indicate that centrifugation did not alter the pre-existing equilibrium and that the supernatant consisted of monomers. The centrifugation was then continued to equilibrium to obtain a distribution showing a sharp increase in molecular weight at C_agg, the monomer concentration at which a small amount of polymerization to large aggregates begins.The primary result is that C_sat > C_agg under all conditions. The different values of the two parameters indicate that they reflect two separate transitions and that the overall monomer to gel process has a limited co-operativity. Within the limits of the method C_sat is independent of total hemoglobin concentration. The two transitions divide the overall range of total hemoglobin concentration into an essentially monomeric region at concentrations below C_agg, a region in which isotropically oriented polymers exist, occurring when monomer concentration lies between C_agg and C_sat, and a two-phase region of conjugate isotropic and anisotropic phases when monomer concentration equals C_sat. These regions correspond to zones in the ultracentrifuge equilibrium distribution. In this scheme C_agg depends only on the interaction energy of polymerization. C_sat depends on entropic factors which induce tactoid formation as well. C_sat, while a monomer concentration, reflects a saturation not of monomers in relation to a polymeric phase, but of polymers in the isotropic phase in relation to the anisotropic or tactoidal polymerized phase. As such, C_sat represents a supersaturated state of isolated monomers.The ratio $\text{C}{}_{\mathrm{<mtext>sat</mtext>}}\text{C}{}_{\mathrm{<mtext>agg</mtext>}}\text{= 1.23}$ in stripped hemoglobin³ and equilibrium distributions in the zone of isotropically oriented polymers were both used to obtain an order of magnitude estimate of polymer size, found to be much smaller than that of hemoglobin S fibers. This further confirms that gelation does not consist of a single transition and phase change with near infinite co-operativity of polymerization.C_sat as well as C_agg are lowered by 2,3,diphosphoglycerate and inositol hexa-phosphate. Decreasing pH near 7 also favors gelation; in stripped hemoglobin a pH optimum for gelation occurs near pH 6.8. The apparent van't Hoff ΔH for stripped hemoglobin is about 3 kcal/mol for C_sat and 2 kcal/mol for C_agg.