Jacinto Steinhardt

A temperature-dependent latent-period in the aggregation of sickle-cell deoxyhemoglobin

Summary No viscosity differences have been found between the oxy and deoxy forms of hemoglobins A and S, or between like forms of the mutant and normal proteins prior to gelation of the HbS. However, measurements at concentrations up to 22 g/100 ml, at temperatures between 13.6° and 25.0°, show that no increase in viscosity occurs for periods between a few minutes at 25° to more than 14 hours at 13.6° after raising the temperature from 2°. When the viscosity finally rises, gelling occurs in 15 minutes or less. The length of the slow latent period depends strongly on temperature (indicating an energy of activation of over 60,000 calories), but the velocity of the faster process of viscosity-increase does not appear to depend on temperature. The dependence on concentration of the rates of the latent period reaction suggest a highly concerted process of aggregation without loss of compactness or radical change in molecular shape. An alternative mechanism, based on branched chain formation is also described.

Crystallization of sickle hemoglobin from gently agitated solutions--an alternative to gelation.

A new, crystalline state of deoxygenated sickle hemoglobin is described which is formed spontaneously in agitated concentrated hemoglobin S solutions under conditions otherwise favoring the well-known transition to the gel state. The birefringent, needle-like sickle hemoglobin crystals, which may subsequently grow to exceed 1 mm in length, are completely dissolved upon dropping the temperature to 0°C. Gelation can thereafter be induced by raising the temperature again without agitating the solution. It is shown by means of solubility and crystal-seeding experiments that hemoglobin S gels are thermodynamically unstable with respect to the crystalline form near the physiological values of pH and ionic strength. The rigid Hb S‡ gel is slowly transformed into a free-flowing crystal suspension after being “seeded” with a minute amount of crystalline sickle hemoglobin. The kinetics of crystallization, as a function of concentration and temperature, together with the enthalpy of crystallization, are compared with the corresponding quantities for gelation.

Formation of needle-like aggregates in stirred solutions of hemoglobin S1

Summary When concentrated solutions of hemoglobin S are gently stirred under conditions such that a gel would be formed in the absence of stirring, gellation does not occur. Instead, as light scattering measurements have shown, after an initial latent period, which can range from minutes to several hours — depending upon the experimental conditions — a highly turbid but fluid suspension of particles of aggregated hemoglobin is rapidly produced. The tubidity is fully reversed upon cooling to 0° C, nad subsequent gellation can be induced by refraining from stirring upon raising the temperature. The turbid solutions are stable for weeks under the conditions of their formation, even when in contact with gels of hemoglobin S. The particles giving rise to the turbidity have been viewed and photographed by light and electron microscopy, and are seen as highly asymmetrical rigid filaments with lengths ranging to about 15 microns.

Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation☆

Abstract The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation. The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS ‡ concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol. Gelation is not accompanied by a detectable volume change (limits 5 × 10−5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm3/mol when the aggregates represent one half of the HbS available for polymerization.

Binding of Organic Ions by Proteins

Publisher Summary nThis chapter describes the binding of organic ions by proteins. Although proteins differ with respect to whether they show measurable tendencies to bind small anions, all proteins that have been examined bind large organic anions (the longer-chain fatty acids, ionic detergents, and such aromatic compounds as dyes) to some extent. In general, within a homologous ligand series, the binding affinity increases with the size of the ion. Under some conditions, the binding of these large ions results in precipitation of the complex formed. However, binding and precipitation do not go together in any simple way. The binding of anions by those proteins that have been investigated in detail cannot be expressed as the result of simple multiple equilibria that involve sets of identical binding groups, modified only by the electrostatic effects resulting from changes in charge as the protein interacts with different numbers of ions. These difficulties may be partly because of the fact that detailed ion-binding measurements on soluble proteins have been made on a very small number of proteins.

Thermodynamic study of the crystallization of sickle-cell deoxyhemoglobin (Hemoglobin S solubility/saturation concentration/enthalpy of crystallization/entropy of crystallization)☆☆☆

Abstract The solubility of crystalline deoxygenated sickle-cell hemoglobin has been determined by a new turbidometric technique. In the absence of the allosteric effector, inositol hexaphosphate, the solubility of sickle deoxyhemoglobin crystals is about 30% less than that of gels. The dependence of the solubility of crystals on temperature at pH 7.1 is very much the same as that of gels at pH 6.48. The change in Vant Hoff enthalpy with crystallization is positive but small (3.11 kcal mol −1 ) and essentially independent of temperature below 30 °C. The presence of inositol hexaphosphate cuts the solubility almost in half. The entropy change when crystals form, just under 40 cal K −1 mol −1 , is larger than the values of ΔS ° found for gels. None of the measurements reported required the estimation of pellet volumes and compositions.

Metal-Ion Binding

Publisher Summary nThis chapter describes metal–ion binding. Two general classes of proteins are considered for describing metal–ion binding. These are (1) systems in which the metal ion occupies a small number of very high energy sites and is essential for the biological function of the macromolecule (e.g., alkaline phosphatase, carboxypeptidase) and (2) systems in which metal binds reversibly to specific amino acid residues in the polypeptide chain but is not required for biological activity and indeed may even impair protein function or disrupt protein structure. Metal ions, like protons, share electron pairs from the donor atoms of a ligand molecule and, thus, form partially covalent bonds with characteristic heats of formation. This type of binding is distinguished from binding to proteins of neutral molecules or large organic ions such as detergents, where the large binding forces are primarily entropie in origin. All metal ions have sets of characteristic coordination numbers that represent the number of hybrid bonds available for ligands. The binding of metal ions to proteins can be measured by equilibrium dialysis.

The effects of diverse proteins on the solubilization of various hydrophobic probes by protein · detergent complexes

The solubilization behavior of various protein.detergent complexes with respect to a particular water-insoluble organic substance (hydrophobic probe) dimethylaminoazobenzene, was reported in earlier studies. The present report describes further the solubilization of other hydrophobic probes (e.g. Sudan II, naphthalene, anthracene and azobenzene) in various protein.sodium dodecyl sulfate complexes, in order to enlarge the scope of our understanding of these phenomena, which undoubtedly play a part in the transport of different water-insoluble organic substances in the living organisms. Solubilization by the various protein.SDS complexes is found to be specific for each probe. The amount of a particular probe solubilized is nearly always equal to the amounts which are solubilized by pure SDS micelles equivalent in amount to the SDS bound. Serious exceptions are found with two heme proteins (e.g. myoglobin and hemoglobin) and a few others. The hemeprotein.SDS complexes also exhibit regions of flat plateaus in the solubilization curves, whereas the binding equilibria show progressively larger amounts of SDS bound. The solubilization of probes by cationic detergent (cetylpyridinium chloride and cetyltrimethylammonium bromde).protein complexes indicate that the solubilization phenomena are related to the environment of the binding sites (the cationic detergents are known to bind at different sites on the protein than the anionic detergents, i.e. SDS in the present case). With anionic detergents the effective chain length of the pseudo-micellar protein.detergent clusters is sufficient to cause an increase in solubilizing effectiveness of about 1.5 between the complexes and pure micelles. When small probes such as naphthalene are used such ratios are found. With larger probes the effectiveness ratio is reduced to 1.0 or even less as a result of steric interference with the formation of the protein.detergent.probe clusters. The solubilization energy exhibited by each protein.detergent complex is largely determined by the individual protein, and by the charge on the detergent.

Comparison of the resistance of human and horse ferrihemoglobin ligand derivatives to acid denaturation

Abstract The velocity of denaturation of human ferrihemoglobin by dilute acid is greatly reduced by complexing with ligands (CN−, N3−) that form low-spin complexes with the heme iron; it is similarly, but much less strongly, affected by the formation of high-spin complexes with other ligands (formate, F−, OCN−, SCN−). The effects are similar to those previously found with horse hemoglobin, but partial stabilization by high-spin ligands is more marked in human hemoglobin. The mechanism of stabilization appears to be strengthening of the stabilizing heme protein interaction so that lower pH values are required to initiate heme separation, which seems to be required for unfolding; neutralization of the positive charge on the ferriheme also seems to be involved. Use has been made of the more stable CN− derivatives to confirm conclusions based on earlier measurements of the numbers of basic groups unmasked incident to unfolding by acid, by extending measurements to lower pH. The results confirm earlier findings that more histidines per heme are masked in human than in horse hemoglobin, although the difference may be smaller than reported earlier. As in carbonylhemoglobin, one or two other more basic groups per heme are also masked in the native low-spin cyanoferriheme protein. It is shown that reversible changes in absorbance (Soret band) incident to unfolding are paralleled by changes in optical rotation (both Soret region and far ultraviolet).

Comparison of the acid denaturation of several hemoglobins which differ in amino acid sequence

Abstract There are pronounced differences in kinetic and thermodynamic stability between human and horse hemoglobins. Since the amino acid sequences of the α, β dimers of horse and human hemoglobins differ in 61 locations, it is difficult to account for them in terms of specific direct or indirect effects of the sequence differences. Rhesus hemoglobin differs from human in only 12 locations and its stability resembles that of human more closely than does horse, although pronounced differences remain. The stabilities of rhesus ferrihemoglobin and deoxyhemoglobin (Hb+ and Hb °) are intermediate between those of the corresponding high-spin forms of horse and human hemoglobin; but there are only small or negligible differences between the low-spin forms (carbonylhemoglobin and oxyhemoglobin) of the two species. The equilibrium isotherm between native and acid unfolded forms of rhesus Hb+ resembles that of horse more than that of human, but it is slightly more stable and slightly less cooperative. The effects of octanol on the rates of unfolding of rhesus ferrihemoglobin are only slightly smaller than with human. There is no effect of octanol on the unfolding rate of any of the CO hemoglobins. Unlike the equilibria of horse and human, octanol is also without effect on the unfolding equilibrium of rhesus ferrihemoglobin, and thus qualifies as a true catalyst of the initial stage of the acid unfolding reaction of the monkey ferriprotein. Differences in stability are tentatively attributed to a limited number of the 12 differences between the two proteins.