Dielectric properties and oxygenation of hemoglobin

Dielectric properties of human and horse hemoglobin were studied at frequencies ranging from 20 kc./sec. to 7 Mc./sec. The relative errors in the measurements were usually less than 10^?3 even for mildly conducting solutions (10^?3M KCl). The experimental setup allowed us variation and measurement of the degree of oxygenation of the protein and to determine its dielectric parameters. Our main conclusion is that it was not possible to find any variation of the dielectric increment for hemoglobin oxygenation levels of 25, 50, 75, and 100%, approximately. This result is at variance with some previous reports. We cannot give the reason for this discrepancy but discuss some possible explanations. The specific dielectric increment, Δε_s/c, of human hemoglobin was shown to be significantly smaller than that of horse hemoglobin (0.28 against 0.32). This physical property is lowered with increasing ionic strength I: Δε_s/c = 0.28 and 0.20 for I = 10^?4 and 10^?3, respectively (human protein).     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Dielectric study of the urea denaturation of delipidated and relipidated bovine serum albumin

Dielectric relaxation and viscosity measurements were performed on delipidated and relipidated samples of bovine serum albumin (BSA) at urea concentrations between O and 6M. By the combined interpretation of these two hydrodynamic methods the characterization of conformational changes of the molecule during urea denaturation is possible. The denaturation of delipidated BSA results from two mechanisms. The first one is a slow, time-dependent elongation of the molecule; the second one is a rapid swelling which becomes most pronounced at urea concentrations higher than 4M. For relipidated albumin, the slow elongation mechanism occurs but the presence of fatty acids protects the protein aganist molecular swelling. In both cases these conformational changes are accompanied by an increased disymmetry of charge repartition and a concomitant increase of the dipole moment. From these results it follows that lipidated albumin (as occurs under physiological conditions) is less sensitive to denaturation than delipidated albumin.     

Dielectric properties of polyelectrolytes. V. Dielectric dispersion of heavy meromyosin

Dielectric constant and dielectric loss of heavy meromyosin (HMM) were measured with varying pH. HMM showed a broader dispersion pattern than that with a single relaxation time especially on the high-frequencey side. The dielectric increment increased sharply with pH, above p^{H 6}, whereas the mean relaxation time and whole dispersion pattern were unchanged in the same region. The values of the increment and the mean relaxation time were much larger than those of usual globular proteins. The dispersion profile, pH dependence, and values of the increment are well explained by Oosawa's counterion fluctuation theory. Other mechanisms are more or less inadequate to our results. In the low pH region below the isoelectric precipitation region, both the increment and the mean relaxation time decreased; this is probably due to partial denaturation and suppression of the dissociation of carboxyl groups. An experiment on a urea-denatured sample supports this assumption. The biological significance of the pH dependence is discussed.     

pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.     

The nature of alkylurea and urea denaturation of alpha-chymotrypsinogen.

The optical rotatory dispersion of alpha-chymotrypsinogen in aqueous solution became less levorotatory upon the addition of ethyl-, propyl-, or butylurea; less negative values for the Moffitt-Yang parameter, alphao, were also obatined. This change in optical rotation of alpha-chymotrypsinogen induced by the alkylureas was similar in direction and magnitude to that observed for alcohols but was opposite to that caused by unsubstituted urea. It appears, therefore, that the alkylureas share with the alcohols an ability to rearrange alpha-chymotrypsinogen into a non-native yet regularly ordered conformation. The effectiveness of the alkylureas and alcohols as denaturants for this protein increased in the order ethyl less than propyl less than butyl derivatives. An identical rank-order was observed for the ability of the alkylureas and alcohols to diminish attractive forces between aliphatic groups, as measured by a model system based upon the extent of aggregation of glass beads coated with methyl groups. These findings indicate that the denaturing action of alkylureas for alpha-chymotrypsinogen is a function of the substituted aliphatic group and is predominantly hydrophobic in character. Non-hydrophobic interactions of unsubstituted urea with alpha-chymotrypsinogen appear to be critical for unfolding of the protein to a random-coil configuration.     

Dielectric properties of hemoglobin and myoglobin. II. Dipole moment of sperm whale myoglobin

This paper is concerned with the molecular origin of the dipole moment of sperm whale myoglobin as it can be calculated from the dielectric dispersion at 1 Mcps on the basis of a mechanism of orientational polarization. It was possible to compare the dielectric increment of native myoglobin and its change during the reaction with bromo acetate with dipole moments calculated according to the known coordinates of the charged groups of the molecule. The agreement between the two shows that in myoglobin only the permanent dipole moment due to these charged groups is important, and that contributions from other possible sources remain within the limits of experimental error.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Temperature dependence of macromolecular interactions in dilute and concentrated hemoglobin solutions

We studied the temperature-dependent effects of intramolecular interactions on the mutual diffusion coefficient of normal human oxygenated hemoglobin in salt solution. We used photon correlation spectroscopy to observe this temperature dependence of the mutual diffusion coefficient of two protein concentrations (1.25 and 17.0 g %) between 13 and 37°C. This coefficient was our probe for monitoring temperature-dependent structural changes of hydrated hemoglobin in solution. Comparison of our measured diffusion coefficient with that predicted by the Stokes-Einstein relationship in terms of solvent or solution viscosity showed a clear transition in the conformation of hemoglobin at approximately 22°C, independent of the hemoglobin concentration. We postulated that at this physiological temperature, a considerable quaternary rearrangement of the hemoglobin chains takes place. We believe this rearrangement changes the effective volume and the hydration sphere of the hemoglobin macromolecule.     

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N₂ or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.     

Dielectric properties of hemoglobin and myoglobin. I. Influence of solvent and particle size on the dielectric dispersion

Dielectric dispersion measurements with aqueous solutions of hemoglobin and myoglobin have been performed in the frequency range from 100 kcps to 15 Mcps. The influence of preparation, particle size, and solvent conditions was studied. The results are analyzed in terms of an orientational polarization mechanism.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer.

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20 degrees C to 35 degrees C. Polymer domain size at 20 degrees C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S-CO reaction rate decreased with increasing temperature from 20 degrees C to 35 degrees C, and increased with decreasing domain size. In comparison, Hb A-CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S-CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.