The inhibition of hemoglobin C crystallization by hemoglobin F

We have reported that circulating CC erythrocytes containing HbO2 C crystals exhibit little or no Hb F suggesting that Hb F may inhibit the crystallization of Hb C. We report now that Hb F inhibits in vitro crystallization of HbO2 and HbCO C when compared to the effect of Hb A in a wide range of mixture proportions. For example, while HbCO C solutions form tetragonal C crystals within 25 min, no crystals form within 2 h with 30% Hb F, whereas 550 crystals/mm3 form with 30% Hb A. Furthermore, an increase in the percent of Hb A is correlated with a greater number of orthorhombic crystal formation rather than the tetragonal morphology observed with 100% Hb C. We also report that Hb A2 (containing delta chains that exhibit 10 sequence differences with beta chains) and Hb Lepore Boston-Washington (a fusion mutant of delta and beta chains that contains only six of these differences) both inhibit Hb C crystallization. By comparing the sequences of the three inhibitory hemoglobins, we conclude that position Gln-87 in the gamma chains is, at least partially, the cause of the inhibitory effect of Hb F on the crystallization of Hb C.     

Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.     

Phase separation and crystallization of hemoglobin C in transgenic mouse and human erythrocytes

Individuals expressing hemoglobin C (β6 Glu→Lys) present red blood cells (RBC) with intraerythrocytic crystals that form when hemoglobin (Hb) is oxygenated. Our earlier in vitro liquid-liquid (L-L) phase separation studies demonstrated that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS or HbA, and that L-L phase separation preceded and enhanced crystallization. We now present evidence for the role of phase separation in HbC crystallization in the RBC, and the role of the RBC membrane as a nucleation center. RBC obtained from both human homozygous HbC patients and transgenic mice expressing only human HbC were studied by bright-field and differential interference contrast video-enhanced microscopy. RBC were exposed to hypertonic NaCl solution (1.5-3%) to induce crystallization within an appropriate experimental time frame. L-L phase separation occurred inside the RBC, which in turn enhanced the formation of intraerythrocytic crystals. RBC L-L phase separation and crystallization comply with the thermodynamic and kinetics laws established through in vitro studies of phase transformations. This is the first report, to the best of our knowledge, to capture a temporal view of intraerythrocytic HbC phase separation, crystal formation, and dissolution.     

Ice nucleation and crystallization in water-glycerol mixtures

Ice nucleation and crystallization processes play a very important role in research and, particularly, in crybiology. In this domain, water-glycerol mixtures are often used. The cryoprotective action of such solutions is not completely known but crystallization of water seems to play a dominant role. The aim of this work is to precisely define the devitrification conditions of such mixtures. Three different techniques are used in this study. Three temperature ranges have been distinguished in which nucleation, growth, and then Ostwald ripening are successively detected.     

Intermolecular effects in the polymerization of hemoglobin S

Monolayer cultures of astrocytes from newborn rat brain hemispheres have been analysed for the glial-specific protein S-100, during their growth cycle. In primary cultures S-100 protein level increases with a pattern close to that observed with rat brain hemispheres $\text{in vivo}$. This finding suggests that some biochemical maturation of the astrocytes occurs $\text{in vitro}$. In secondary cultures the level of S-100 protein decreases and then increases at the end of the proliferation phase. This modulation, similar to that observed in a clonal culture of tumor cells from rat brain (C6) provides a model to study the relationship between gene expression and the phase of growth of the cells and will allow parallel investigations in normal and tumor cells.     

Lipid bilayers: thermodynamics, structure, fluctuations, and interactions

This article, adapted from our acceptance speech of the Avanti Award in Lipids at the 47th Biophysical Society meeting in San Antonio, 2003, summarizes over 30 years of research in the area of lipid bilayers. Beginning with a theoretical model of the phase transition (J.F.N.), we have proceeded experimentally using dilatometry and density centrifugation to study volume, differential scanning calorimetry to study heat capacity, and X-ray scattering techniques to study structure of lipid bilayers as a function of temperature. Electron density profiles of the gel and ripple phases have been obtained as well as profiles from several fluid phase lipids, which lead to many structural results that compliment molecular dynamics simulations from other groups. Using the theory of liquid crystallography plus oriented lipid samples, we are the first group to obtain both material parameters (KC and B) associated with the fluctuations in fluid phase lipids. This allows us to use fully hydrated lipid samples, as in vivo, to obtain the structure.     

The thermodynamics of water-protein interactions

Information about the effects of water on protein structure and function can be obtained from studies on freeze dried protein powders of varying water content. Sorption isotherms of water on proteins can be used to obtain thermodynamic quantities for water-protein interactions. Since such isotherms show hysteresis, there is doubt in regard to their interpretation.General expressions for the thermodynamic quantities of sorption are derived. If isotherms represent data at equilibrium, it is possible to calculate these thermodynamic quantities.There are two types of hysteresis, non-equilibrium hysteresis and equilibrium hysteresis. Absorption and desorption isotherms can show equilibrium hysteresis if different protein conformations, which are only slowly interconvertible, can be present. In this case valid thermodynamic quantities can be obtained. Experimental tests for equilibrium hysteresis are presented. More experiments are needed before definite conclusions can be drawn in regard to isotherms in the literature.If the protein conformation in a protein powder is similar to the protein conformation in aqueous solution, equilibrium data obtained from sorption isotherms can be used to approximate thermodynamic quantities for the interaction of water with proteins in aqueous solution. Examination of what experimental evidence is available indicates that the protein in powders prepared by desorption of water should have a conformation similar to that in solution. Further study of such samples will help to clarify the thermodynamics of water-protein interactions in aqueous solution.     

Statistical thermodynamics of oligomer-polymer interactions

A new method for formulating partition functions for a system of particles interacting on a one-dimensional lattice has recently been developed.^3,4 In this paper the method is applied to oligomer–polymer systems. The details of the connection between this method and the matrix formulation are first established. The method is then used to find expressions for the partition function of an oligomer–polymer system when the oligomers have a distribution of lengths and an alternating sequence.     

Intermolecular interactions during ultrafiltration of pegylated proteins

Recent studies have demonstrated the feasibility of using membrane ultrafiltration for the purification of pegylated proteins; however, the separations have all been performed at relatively low protein concentrations where intermolecular interactions are unimportant. The objective of this study was to examine the behavior at higher PEG concentrations and to develop an appropriate theoretical framework to describe the effects of intermolecular interactions. Ultrafiltration experiments were performed using pegylated α‐lactalbumin as a model protein with both neutral and charged composite regenerated cellulose membranes. The transmission of the pegylated α‐lactalbumin, PEG, and α‐lactalbumin all increase with increasing PEG concentration due to the increase in the solute partition coefficient arising from unfavorable intermolecular interactions in the bulk solution. The experimental results were in good agreement with a simple model that accounts for the change in Gibbs free energy associated with these intermolecular interactions, including the effects of concentration polarization on the local solute concentrations upstream of the membrane. These intermolecular interactions are shown to cause a greater than expected loss of pegylated product in a batch ultrafiltration system, and they alter the yield and purification factor that can be achieved during a diafiltration process to remove unreacted PEG. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:655–663, 2013     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

Intermolecular interactions between DNA and methamphetamine,amphetamine, ecstasy and their major metabolites

In this work, we carried out a theoretical investigation regarding amphetamine-type stimulants, which can cause central nervous system degeneration, interacting with human DNA. These include amphetamine, methamphetamine, 3,4-Methylenedioxymethamphetamine (also known as ecstasy), as well as their main metabolites. The studies were performed through molecular docking and molecular dynamics simulations, where molecular interactions of the receptor–ligand systems, along with their physical–chemical energies, were reported. Our results show that 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine (ecstasy) present considerable reactivity with the receptor (DNA), suggesting that these molecules may cause damage due to human-DNA. These results were indicated by free Gibbs change of bind (ΔG_bind) values referring to intermolecular interactions between the drugs and the minor grooves of DNA, which were predominant for all simulations. In addition, it was observed that 3,4-Dihydroxymethamphetamine (ΔG_bind = ?13.15 kcal/mol) presented greater spontaneity in establishing interactions with DNA in comparison to 3,4-Methylenedioxymethamphetamine (ΔG_bind = ?8.61 kcal/mol). Thus, according with the calculations performed our results suggest that the 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine have greater probability to provide damage to human DNA fragments.     

Dissecting the thermodynamics of GAP-RhoA interactions

We describe a detailed study of the RhoA-binding epitope of the GAP domain of Graf, including the determination of the thermodynamic and kinetic parameters of the interaction of wild-type domain, and of its 15 single-site mutants, with cognate GTPases. We show that residues important for the structural integrity of the Arg-finger loop are critical for binding Rho and for the catalytic activity of GAP, but GTPase selectivity appears to be modulated by a much more subtle interplay of electrostatic and hydrophobic interactions involving residues on the periphery of the main interface. The eight residues targeted in this study are involved in three distinct patches on the surface, two of which appear to interact with highly conserved regions of the GTPase, while the third plays a role in GTPase selectivity.     

Control of nucleation in the crystallization of lysozyme.

This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample.     

Protein nucleation and crystallization by homologous protein thin film template

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.     

The kinetics of nucleation and growth of sickle cell hemoglobin fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times theta of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within <1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of <5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E(A)=50 kJ mol(-1), a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong theta(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.     

Purification, crystallization and preliminary analysis of hemoglobin from rabbit (Oryctolagus cuniculus)

Hemoglobin (Hb) is a tetrameric protein, which contains four heme prosthetic groups, and each one is associated with a polypeptide chain. Herein, we report the rabbit hemoglobin which has intrinsically high oxygen affinity and possess highest sequence identity with human hemoglobin. The purified hemoglobin has been tried to crystallize in different crystallization conditions owing to its formation of various crystal systems. The rabbit Hb crystals were grown using PEG 3,350 as the precipitant at 18 degrees C. The crystals of rabbit Hb belongs to triclinic space group P1 with one molecule (alpha2beta2) in the asymmetric unit.     

Two-step mechanism of homogeneous nucleation of sickle cell hemoglobin polymers

Sickle cell anemia is a debilitating genetic disease that affects hundreds of thousands of babies born each year worldwide. Its primary pathogenic event is the polymerization of a mutant, sickle cell, hemoglobin (HbS); and this is one of a line of diseases (Alzheimer's, Huntington's, prion, etc.) in which nucleation initiates pathophysiology. We show that the homogeneous nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The evidence comes from data on the rates of fiber nucleation and growth and nucleation delay times, the interaction of fibers with polarized light, and mesoscopic metastable HbS clusters in solution. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. The variations of these concentrations in patients might account for the high variability of the disease in genetically identical patients. In addition, these components can potentially be utilized for control of HbS polymerization and treatment of the disease.