Absence Of Organic Phosphates Research Articles

Role of β/δ101Gln in Regulating the Effect of Temperature and Allosteric Effectors on Oxygen Affinity in Woolly Mammoth Hemoglobin

The oxygen affinity of woolly mammoth hemoglobin (rHb WM) is less affected by temperature change than that of Asian elephant hemoglobin (rHb AE) or human normal adult hemoglobin (Hb A). We report here a biochemical-biophysical study of Hb A, rHb AE, rHb WM, and three rHb WM mutants with amino acid substitutions at β/δ101 (β/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the β/δ101 residue. The β/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride. The rHb WM mutants studied generally have higher affinity for oxygen under various conditions of pH, temperature, and salt concentration, and in the presence or absence of organic phosphate, than do rHb WM, rHb AE, and Hb A. Titrations for the O2 affinity of these mutant rHbs as a function of chloride concentration indicate a lower heterotopic effect of this anion due to the replacement of β/δ101Gln in rHb WM. The alkaline Bohr effect of rHb WM and its mutants is reduced by 20-50% compared to that of Hb A and is independent of changes in temperature, in contrast to what has been observed in the hemoglobins of most mammalian species, including human. The results of our study on the temperature dependence of the O2 affinity of rHb WM and its mutant rHbs illustrate the important role of β/δ101Gln in regulating the functional properties of these hemoglobins.

Mutations of the βN102 Residue of HbA Not Only Inhibit the Ligand-Linked T to ReState Transition, but Also Profoundly Affect the Properties of the T State Itself

The properties of three HbA variants with different mutations at the beta102 position, betaN102Q, betaN102T, and betaN102A, have been examined. All three are inhibited in their ligand-linked transition from the low affinity T quaternary state to the high affinity Re quaternary state. In the presence of inositol hexaphosphate, IHP, none of them exhibits cooperativity in the binding of oxygen. This is consistent with the destabilization of the Re state as a result of the disruption of the hydrogen bond that normally forms between the beta102 asparagine residue and the alpha94 aspartate residue in the Re state. However, these three substitutions also alter the properties of the T state of the hemoglobin tetramer. In the presence of IHP, the first two substitutions result in large increases in the ligand affinities of the beta-subunits within the T state structure. The betaN102A variant, however, greatly reduces the pH dependencies of the affinities of the alpha and beta subunits, K1(alpha) and K1(beta), respectively, for the binding of the first oxygen molecule in the absence of IHP. In the presence of IHP, the T state of this variant is strikingly similar to that of HbA under the same conditions. For both hemoglobins, K1(alpha) and K1(beta) exhibit only small Bohr effects. In the absence of IHP, the affinities of the alpha and beta subunits of HbA for the first oxygen are increased, and both exhibit greatly increased Bohr effects. However, in contrast to the behavior of HbA, the ligand-binding properties of the T state tetramer of the betaN102A variant are little affected by the addition or removal of IHP. It appears that along with its effect on the stability of the liganded Re state, this mutation has an effect on the T state that mimics the effect of adding IHP to HbA. It inhibits the set of conformational changes, which are coupled to the K1 Bohr effects and normally accompany the binding of the first ligand to the HbA tetramer in the absence of organic phosphates.

Alkaline Bohr effect of bird hemoglobins: the case of the flamingo

The hemoglobin (Hb) substitution His-->Gln at position alpha89, very common in avian Hbs, is considered to be responsible for the weak Bohr effect of avian Hbs. Phoenicopterus ruber ruber is one of the few avian Hbs that possesses His at alpha89, but it has not been functionally characterized yet. In the present study the Hb system of the greater flamingo (P. ruber roseus), a bird that lives in Mediterranean areas, has been investigated to obtain further insight into the role played by the alpha89 residue in determining the strong reduction of the Bohr effect. Functional analysis of the two purified Hb components (HbA and HbD) of P. ruber roseus showed that both are characterized by high oxygen affinity in the absence of organic phosphates, a strong modulating effect of inositol hexaphosphate, and a reduced Bohr effect. Indeed, in spite of the close phylogenetic relationship between the two flamingo species, structural analysis based on tandem mass spectrometry of the alpha(A) chain of P. ruber roseus Hb showed that a Gln residue is present at position alpha89.

Oxygen-binding properties of total hemoglobin and isolated components of the terrestrial tortoise Geochelone carbonaria

The hemoglobin of the terrestrial tortoise Geochelone carbonaria was separated into two major components by ion exchange chromatography. The oxygen binding properties of both the unfractionated hemolysate and the separated components were investigated. The stripped hemoglobin of G. carbonaria displayed an oxygen affinity of approximately 6.3 mm Hg at pH 7.4 with a Bohr effect of about −0.55. The Hb-O 2 binding properties were also studied in the presence of the organic polyphosphates ATP and IHP. Inositol hexaphosphate (IHP) was more effective in lowering oxygen affinity, yielding a P 50 of 20.1 mm Hg at pH 7.4. The n 50 value were quite low in the stripped form of the protein (1.0–1.9) but increased in the presence of both organic phosphates, attaining a value of approximately 2.9 at pH 7.9. The oxygen binding properties of the major hemoglobin components were also investigated in the presence and absence of organic phosphates, and the values were compared with those obtained for the total hemolysate.

Physiological Relevance of the Overall ΔH of Oxygen Binding to Fetal Human Hemoglobin

Human fetal hemoglobin is known to display, at 20°C, a lower affinity than human adult hemoglobin for oxygen when both proteins are in the absence of organic phosphates. The physiologically important reverse situation is achieved at 37°C upon addition of 2,3-bisphosphoglycerate (DPG), whose lower effect on fetal hemoglobin is related to some amino acid substitutions present in γ-chains. However, the difference in oxygen affinity observed at 37°C is not solely due to the different modulation power of DPG with respect to adult and fetal hemoglobins. In fact, the results presented here reveal new aspects linked to the interplay of temperature and organic phosphates.In particular, the lower effect of DPG on fetal hemoglobin renders almost identical the oxygen affinity of the two hemoglobins at 20°C, abolishing the difference observed in the absence of the effector. Successively on going from 20°C to 37°C, by virtue of the lower overall heat of oxygenation (ΔH) displayed by fetal hemoglobin when in the presence of DPG, adult hemoglobin shows a lower oxygen affinity, as it should if oxygen has to be transferred from maternal to fetal blood.

Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82.beta.1 and 82.beta.2 lysyl residues by bis(3,5-dibromosalicyl) fumarate

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.

Equilibrium Oxygen Binding to Human Hemoglobin Cross-linked Between the α Chains by Bis(3,5-dibromosalicyl) Fumarate

Oxygen equilibrium curves of human hemoglobin Ao (HbAo) and human hemoglobin cross-linked between the α chains (α α Hb) by bis(3,5-dibromosalicyl) fumarate were measured as a function of pH and chloride or organic phosphate concentration. Compared to HbAo, the oxygen affinity of α α Hb was lower, cooperativity was maintained, although slightly reduced, and all heterotropic effects were diminished. The major effect of α α-cross-linking appears to be a reduction of the oxygen affinity of R-state hemoglobin under all conditions. However, while the oxygen affinity of T-state α α Hb was slightly reduced at physiologic chloride concentration and in the absence of organic phosphates, KT was the same for both hemoglobins in the presence of 2,3-diphosphoglycerate (or high salt) and higher for α α Hb in the presence of inositol hexaphosphate. The reduced O2 affinity arises from smaller binding constants for both T- and R-state α α Hb rather than through stabilization of the low affinity conformation. All four Adair constants could be determined for α α Hb under most conditions, but a3 could not be resolved for HbAo without constraining a4, suggesting that the cross-link stabilizes triply ligated intermediates of hemoglobin.

Spin equilibrium in human methemoglobin: effects of inositol hexaphosphate and bezafibrate as measured by resonance Raman spectroscopy

The spin equilibria of several derivatives of human methemoglobin were probed by resonance Raman scattering. The intensity of lines in the Raman spectrum gives a measure of the high-spin (S = 5/2) to low-spin (S = 1/2) ratio which agrees well with the spin equilibria determined from direct magnetic susceptibility measurements. The addition of bezafibrate (BZF) to methemoglobin in the absence of organic phosphate, IHP, has very little effect on the spin equilibrium, whereas in the presence of IHP it augments the change in spin significantly. When both IHP and BZF are added to the mixed-spin derivatives (H2O, SCN-, OCN-, and NO2-) of human methemoglobin, the spin equilibrium is shifted toward higher spin by about 700 cal/mol, similar to the spin change detected in derivatives of carp methemoglobin upon addition of IHP alone. These data support a general mechanism for the allosteric transition in which a constant fraction of the cooperative energy (approximately 20%) is detected at the heme of the ferric ligand-bound forms.

Arctic life and adaptation—III. the function of whale ( Balaenoptera acutorostrata) hemoglobin

1. 1. The oxygen binding properties of the hemoglobin from the Lesser Rorqual, Balaenoptera acutorostrata, has been investigated with respect to the possible effects of organic phosphates on gas transport in arctic environments. 2. 2. The intrinsic oxygen affinity of the hemoglobin is high and strongly modulated by the effects of organic phoshates. 3. 3. In the absence of organic phosphates, the temperature sensitivity of oxygen binding expressed by the heat of oxygenation, ΔH is − 16.2 kcal/mol when corrected for the heat of oxygen in solution. 4. 4. In the presence of organic phosphates there is a marked decreased in the temperature sensitivity ΔH ∼ − 5 kcal/mol) . 5. 5. This feature is of great importance for oxygen unloading in the flippers and the tail, where the temperature is lower than the trunk of the whale. 6. 6. Furthermore the organic phoshates strongly increase the Bohr coefficient, Δlog P 50/Δ pH , from less than −0.3 in stripped hemoglobin to about −1.5 when the hemoglobin is saturated with P 6-inositol. 7. 7. This feature may be of great physiological importance by reducing the CO 2 tension and acidosis after a prolonged dive.

Blood oxygen equilibria and theoretical models. I. effect of protons in trout ( Salmo gairdneri) and human red cells, in absence of organic phosphates

1. 1. Oxygen equilibrium curves were measured on red cells that had been depleted of organic phosphates, for rainbow trout red cells between pH 7–9, at 15 and 20°C, and for human red cells between pH 6.8–8.0, at 37°C. 2. 2. The data were fitted to the models of Adair and of Monod et al. (MWC model). Parameters were estimated by the non-linear least-squares method, from which the number of Bohr protons released during oxygenation was calculated. 3. 3. For trout as for human red cells, the pH affects the first step of oxygenation and the overall oxygenation in nearly equal proportion. For human red cells, the association constant for binding of the last oxygen, expressed by k 4 or k R , is not affected by pH, indicating that the R structure is free of constraints. For trout red cells, there is a pronounced pH dependence of this constant at low pH, which is about 10-fold increased between pH 7–9. This corresponds to the Root effect that impairs the T → R transition, and confirms previously published data for normal trout and human red cells. 4. 4. For trout red cells, small functional heterogeneity is evidenced, despite Hb multiplicity, and effect seems linked to the level of temperature. 5. 5. The specific effect of organic phosphates, evident for human red cells, contrasts with a lack of effect for trout red cells, when taking into account the intracellular pH values.

Relationship between tetramer-dimer assembly and the stability of Hb Malmö ( α2β297Gln)

The effects of the mutation at the α 1 β 2 contact in Hb Malmö α 2β 2 97( FG4) His a ̊ Gln ) on oxygen-binding properties, ease of dissociation into dimeric hemoglobin and stability were studied. The P 50 value of Hb Malmö in the absence of organic phosphates was 1.9. mmHg, in contrast to 8.8 mmHg of Hb A. The n-value of Hb Malmö was 1.6. The overall free energy of interaction of oxygen with Hb Malmö was about 25% that of Hb A. The Adair constant, K 1, of Hb Malmö was about 10-times larger than that of Hb A, but the K 4 of Hb Malmö was similar to that of Hb A. The liganded form of Hb Malmö was found to dissociate into dimers more readily than Hb A by gel filtration on Sephadex G-100. Dissociation into dimeric hemoglobin was enhanced in dilute solutions. Increased instability during mechanical agitation of diluted samples was greater for Hb Malmö than for Hb A. The denaturation rate constants of tetramers of the oxyform of Hb A and Hb Malmö were about 20-times greater than those of dimers of these hemoglobins. The instability of Hb Malmö depends on a greater α 1 β 2 dissociation constant compared with that of Hb A. These findings allow an examination of the role of the intersubinit contact in determining the functional properties and the stability of the hemoglobin molecule.

Temperature independence of the alkaline Bohr effect in pig red cells and pig haemoglobin solutions

The influence of temperature on the oxygen affinity and the alkaline Bohr effect of pig red cells and pig heamoglobin solutions has been compared to that of human adult red cells and human adult hemoglobin. Pig red cells and pig Hb evidence a lower affinity for oxygen in various conditions of pH, temperature and salt concentration, in the presence as well as in the absence of organic phosphates. It has been observed that the alkaline Bohr effect of pig Hb was reduced by 20–25% compared to Hb A 0 and independent of changes in temperature, contrary to human Hb A 0. Titrations of pig Hb with Cl − indicate a lower heterotropic effect of this anion at low concentration of the salt. It is concluded that this may be the origin of the temperature independence of the alkaline Bohr effect in pig Hb. Conversely, the temperature dependence of the alkaline Bohr effect of Hb A 0 should be related to the oxygen-linked binding of Cl − at the α 1-α 2 interface.

Evidence of the incorporation of normally nonaggregating hemoglobins into crystalline aggregates of deoxy hemoglobin S.

Changes in the solubility of both components of binary mixtures of sickle-cell deoxyhemoglobin (Hb degrees S) and some normally nonaggregating hemoglobins were determined by direct compositional analysis of the solution phage following sedimentation of crystalline aggregates generated from them. Increases in turbidity associated with the suspension of the crystals in solution were also monitored for each mixture. Correlations between changes in species solubility and "excess' turbidity levels were observed which define two distinct forms of interaction between hemoglobin additives and Hb degrees S. The R-state quaternary structures, COHbS and metHbS in the absence of organic phosphate, are excluded from the aggregate phase and act to lower the solubility of Hb degrees S through increased excluded volume contributions to the activity of the solution phase. Increases in turbidity which parallel this solubility change reflect the increased population of highly asymmetric Hb degrees C crystals in solution. Deoxyhemoglobin A and metHbS fixed in the deoxy conformation lower the solubility of Hb degrees S in a more complex manner, which may include the establishment of a minimum solubility. The solubility of these additives also decreases, as does the turbidity due to the resultant crystals, when the proportion of additive exceeds 40% of the total. Analysis of the composition of pellets isolated from both hybrid and nonhybrid mixtures with Hb degrees A confirms its incorporation into the aggregates. Such "mixed" crystals appear to have solubility and morphological properties different from those of Hb degrees S alone. Comparison of phase composition data from mixtures prepared with varying initial concentrations of Hb degrees S suggests that the degree of HbA incorporation into HbS aggregates is a function of the amount of Hb degrees S itself available for self-aggregation and the stage of crystal development at which HbA participates.

The influence of β-93 sulfhydryl groups, organic phosphate and heme concentration on methemoglobin reduction

Native and modified methemoglobin (β-93-SH groups blocked) were reduced by NADH-dependent methemoglobin reductase in the absence and the presence of organic phosphate (inositol hexaphosphate). These experiments were performed with dilute as well as concentrated methemoglobin solutions (physiological heme concentration). It is shown that: (a) in dilute solutions the blockage of β-93-SH groups lowers the rate of methemoglobin reduction in the absence of organic phosphate but the rates of native and modified methemoglobin reduction are similar in the presence of organic phosphate; (b) at physiological heme concentration the blockage of β-93-SH groups does not affect the rate of reduction but the organic phosphate stimulates the reduction of both native and modified methemoglobins in a similar fashion, as it does in dilute solutions. It is concluded that, although in dilute solutions the blockage of β-93-SH groups alters the reduction rate, at physiological heme concentration the presence of free β-93-SH groups does not have any significant effect on methemoglobin reduction. On the contrary, the organic phosphates do accelerate the rate of reduction at all ranges of heme concentration.

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27 degrees C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H(2)O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H(2)O) as a function of oxygenation, the amount of oxygen bound to the alpha and beta chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential O(2) binding to the alpha or beta chains; (ii) in the presence of organic phosphates, the alpha hemes have a higher affinity for O(2) as compared to the beta hemes; (iii) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (iv) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.

Sequence of oxygen binding by hemoglobin

A nitroxide spin-label probe was attached directly to a propionic acid group of heme in either the alpha or the beta chain of hemoglobin. The electron paramagnetic resonance (EPR) spectrum of the spin label is altered by the spin-state change of the heme iron to which the spin label is attached. These hybrid hemoglobins showed normal optical and functional properties, indicating that the attachment of the spin label did not perturb the function of hemoglobin. Upon deoxygenation of alpha-heme-spin-labeled hemoglobin, EPR signals changed proportionally with oxygen saturation (determined by measuring absorption spectra). This result indicates that there is no binding preference between the alpha and beta chains of hemoglobin. However, the cross plot for the fraction of the EPR changes vs. the fraction of oxygen saturation deviated significantly from the diagonal straight line in response to the addition of 2,3-diphosphoglycerate and inositol hexaphosphate. The deviation indicated that the EPR change precedes the optical change at low oxygen tension. This result implies that, in the presence of organic phosphate, oxygen binds preferentially to the alpha subunit of deoxyhemoglobin. This conclusion was supported by the result obtained with beta-heme-spin-labeled hemoglobin: the direction of the deviation for beta-heme-spin-labeled hemoglobin in the presence of diphosphoglycerate and inositol hexaphosphate was opposite to that obtained for alpha-heme-spin-labeled hemoglobin. However, the curve deviated even in the absence of organic phosphate. This deviation for beta-heme-spin-labeled hemoglobin can be explained by the intersubunit interaction of hemoglobin. From these results, it was concluded that, in the absence of organic phosphate, oxygen combines with the alpha and beta chains with equal probability whereas, in the presence of organic phosphate, oxygen binds preferentially to the alpha chains of hemoglobin.

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 C sat C agg = 1.23 in stripped hemoglobin ‡ ‡ Stripped indicates hemoglobin in the absence of organic phosphates. 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.