Aqueous Hemoglobin Solutions Research Articles

Studies of Light Scattering by Hemoglobin Molecules under the Effect of Iron (III) Chloride and Various pH Levels

A lot of literature sources discuss diseases associated with the hemoglobin protein [1, 2]. In the modern world, more than 800 million people suffer from anemia [3]. Assessing the level of hemoglobin in the circulatory system is one of the ways to diagnose anemia. Hemoglobin protein is also a promising source of bioactive peptides [4]. The aim of the work was to identify the reasons leading to conformation changes of this protein. It still remains unclear what exactly causes disruption of its functionality. Human hemoglobin from Sigma H7379 was studied in the work; all experiments were conducted on a dynamic light scattering spectrometer — Photocor Complex. Experimental data were obtained using optical methods of static and dynamic light scattering. The work included an analysis of the behaviour of hemoglobin protein molecules in aqueous and aqueous-saline solutions with changes in solution parameters (pH, addition of iron chloride III). At values of pH< (3.56±0.15) and pH> (10.4±0.2), the hemoglobin molecule underwent conformational changes, resulting in the disintegration of the quaternary structure into αβ−dimers and individual α− and β−globules. As a result of the study, it was found that the addition of FeCl3 to aqueous solutions of hemoglobin increases the size and mass of scattering particles, which can be explained by the adsorption of Fe3+ ions on the protein surface. However, upon reaching a certain concentration of FeCl3, the pH of the solution was lowered to such an extent that it caused conformational changes in hemoglobin, leading to the disintegration of its quaternary structure. These results can be taken into account when creating medicinal drugs for the treatment of anemia and other diseases associated with the hemoglobin protein.

ВПЛИВ ЕЛЕКТРОМАГНІТНОГО ВИПРОМІНЮВАННЯ МІЛІМЕТРОВОГО ДІАПАЗОНУ НА ОПТИЧНІ ВЛАСТИВОСТІ ГЕМОГЛОБІНУ

The biological activity of millimeter electromagnetic radiation (MEMR) has long been known that is the basis for its use in medicine. But the primary biophysical mechanisms of this factor at the molecular level remain poorly understood and debated. Because of it the features of the influence of microwave radiation on the optical properties of water solutions of hemoglobin with the aim to characterize the certain structural state of hemoglobin were investigated in this research. The object of the study was the optical properties of aqueous solutions of human hemoglobin in the concentration range of 7 and 15 μM and in the temperature range 10–40°C. The optical spectra were recorded by using a BiospecMini (Shimadzu) spectrophotometer in the wavelength range of 190–1100 nm. In order to increase the accuracy of the analysis of spectral data the spectra were normalized relative to the baseline which was taken as a line that ran parallel to the horizontal axis due to the minimum value of the optical density in the absorption spectrum. The values of the absorption maxima and the optical density were used as the main parameters of the absorption spectra. The B-spline function was used to more accurately determine these parameters. Microwave exposure of hemoglobin solutions was performed at wavelength of 7,1 mm with power that calculated to be approximately 6mW. This power of electromagnetic radiation corresponds to the conditional limit range between thermal and non-thermal action of this factor. The dependence of the optical properties of hemoglobin in the spectral range of heme absorption on temperature was analyzed and effects of microwave radiation on the optical properties of hemoglobin solutions were considered in frame of hypochromic effects induced by temperature. It was revealed that the parameters of the absorption spectrum of an aqueous solution of hemoglobin depend on temperature. The optical density in the Sore band and the spectral bands of the oxygenated form of hemoglobin decreases upon temperature increase. The observed temperature hypochromic effect is associated with increased protein aggregation. The effect of MEMR leads to a small but statistically significant hypochromism in the spectra of hemoglobin absorption that testifies an increase in protein aggregation under the action of this physical factor which cause the changes in the hydration of protein molecules. This effect depends on the protein concentration and is not detected at low concentrations.

Surface imprinted bacterial cellulose nanofibers for hemoglobin purification

There is a significant need for the development of the novel adsorbents in the field of protein purification. In this study, thin hemoglobin imprinted film (MIP) was fabricated onto the bacterial cellulose nanofibers’ (BCNFs) by surface imprinting method using metal ion coordination interactions with N-methacryloyl–(L)-histidinemethylester (MAH) and copper ions. The hemoglobin surface imprinted bacterial cellulose nanofibers (MIP-BCNFs) was applied to selective recognition of hemoglobin and purification from hemolysate. The characterization of the MIP-BCNFs was carried out by the Fourier Transformed Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), micro-Computerized Tomography (μCT), atomic force microscopy (AFM) and surface area measurements. The adsorption experiments of hemoglobin onto the MIP-BCNFs and NIP-BCNFs from aqueous hemoglobin solutions were investigated in a batch system. The results showed that MIP-BCNFs are promising materials for purification of hemoglobin with high adsorption capacity, significant selectivity and reusability.

A simple quantitative method to study protein-lipopolysaccharide interactions by using liquid crystals.

The interaction of proteins with endotoxins has divergent effects on lipopolysaccharide (LPS)-induced responses, which serve as a basis for many clinical and therapeutic applications. It is, therefore, important to understand these interactions from both theoretical and practical points of view. This paper advances the design of liquid crystal (LC)-based stimuli-responsive soft materials for quantitative measurements of LPS-protein binding events through interfacial ordering transition. Micrometer-thick films of LCs undergo easily visualized ordering transitions in response to proteins at LPS-aqueous interfaces of the LCs. The optical response of the LC changes from dark to bright after aqueous solutions of hemoglobin (Hb), bovine serum albumin (BSA), and lysozyme proteins (LZM) are in contact with a LPS-laden aqueous-LC interface. The effects of interactions of different proteins with LPS are also observed to cause the response of the LC to vary significantly from one to another; this indicates that manipulation of the protein-LPS binding affinity can provide the basis for a general, facile method to tune the LPS-induced responses of the LCs to interfacial phenomena. By measuring the optical retardation of the 4'-pentyl-4-cyanobiphenyl (5CB) LC, the binding affinity of the proteins (Hb, BSA, and LZM) towards LPS that leads to different orientational behavior at the aqueous interfaces of the LCs can be determined. The interaction of proteins with the LPS-laden monolayer is highest for LPS-Hb, followed by LPS-BSA, and least for LPS-LZM; this is in correlation with their increasing order of binding constants (LPS-Hb>LPS-BSA>LPS-LZM). The results presented herein pave the way for quantitative and multiplexed measurements of LPS-protein binding events and reveal the potential of the LC system to be used as quantitative LC-based, stimuli-responsive soft materials.

Red blood cell as a universal optoacoustic sensor for non-invasive temperature monitoring.

Optoacoustic (photoacoustic) temperature imaging could provide improved spatial resolution and temperature sensitivity as compared to other techniques of non-invasive thermometry used during thermal therapies for safe and efficient treatment of lesions. However, accuracy of the reported optoacoustic methods is compromised by biological variability and heterogeneous composition of tissues. We report our findings on the universal character of the normalized temperature dependent optoacoustic response (ThOR) in blood, which is invariant with respect to hematocrit at the isosbestic point of hemoglobin. The phenomenon is caused by the unique homeostatic compartmentalization of blood hemoglobin exclusively inside erythrocytes. On the contrary, the normalized ThOR in aqueous solutions of hemoglobin showed linear variation with respect to its concentration and was identical to that of blood when extrapolated to the hemoglobin concentration inside erythrocytes. To substantiate the conclusions, we analyzed optoacoustic images acquired from the samples of whole and diluted blood as well as hemoglobin solutions during gradual cooling from +37 to -15 °C. Our experimental methodology allowed direct observation and accurate measurement of the temperature of zero optoacoustic response, manifested as the sample's image faded into background and then reappeared in the reversed (negative) contrast. These findings provide a framework necessary for accurate correlation of measured normalized optoacoustic image intensity and local temperature in vascularized tissues independent of tissue composition.

High Frequency Dynamics in Hemoglobin Measured by Magnetic Relaxation Dispersion

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at β-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two β-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to ∼1 ps. Both formate and acetate ions are found to bind specifically within 5 Å of the β-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to β-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between β-93 positions on different chains.

Solid-phase extraction of phospholipids from hemoglobin solutions using Empore styrene–divinylbenzene disks

Styrene–divinylbenzene Empore disks were investigated for the extraction of phospholipids from red blood cells or aqueous solutions of hemoglobin as a means to reduce the time and solvent use required in sample preparation. Red blood cells are the source for hemoglobin used in the preparation of a hemoglobin-based oxygen carrier which is being developed to replace blood in transfusion therapy. Phospholipids are a major component of the membrane of red blood cells, and are toxic when administered directly into the vasculature. Sensitive analytical methods are required to detect phospholipids to ensure that concentrations in purified hemoglobin are well below toxic levels. This requires isolation from large volumes of purified hemoglobin solutions. The method described utilizes Empore disks to extract phospholipids from 30 ml of stroma free Hb preparations. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and sphingomyelin were recovered with an average of 92% yield. The recovery of phosphatidylserine was 65%. The use of solvent and time required for sample preparation were reduced by an average of 80% relative to liquid–liquid extraction. The capacity of the 47-mm disk for the total of five phospholipids exceeds 0.3 mg. The method has been used for quantitation of phospholipids in red blood cells and stroma free hemoglobin solutions.

Large-scale blood substitute production using a microfluidizer.

Microfluidization has been tested as a way to disperse phospholipids in aqueous hemoglobin solutions. Spherical and stable liposomes of 2 to 3 microns were obtained. Lipid incorporation (up to 85%) and hemoglobin encapsulation (up to 15%) in liposomes have been improved with respect to previous investigations. However, results show that a more efficient dispersion system using lower concentrations of lipid is required to obtain a high liposome hemoglobin concentration (limited actually to 150 g/l) and an economically and biologically suitable process for artificial blood production at large scale.

Low-frequency ultrasound absorption in aqueous solutions of hemoglobin, myoglobin, and bovine serum albumin: the role of structure and pH.

New measurements on ultrasound absorption of aqueous solutions of the three globular proteins, bovine hemoglobin, horse heart myoglobin, and bovine serum albumin, at 20 degrees C are reported for the frequency range below 1.2 MHz and, in the case of hemoglobin and bovine serum albumin, to a limited extent for the range 2-30 MHz. The effect of protein conformation has been investigated by use of a range of denaturants and by change of pH. A much more detailed description of protein interactions emerges from the low-frequency studies than was possible hitherto. Specific absorption processes for myoglobin and bovine serum albumin peak in the neighborhood of pH 4.2. The process for myoglobin has a relaxation frequency between 1 and 2.6 MHz, while for bovine serum albumin the frequency is as low as 70 kHz. Both processes can be related to conformational changes in the molecules. In the case of hemoglobin, an absorption process with a relaxation frequency of 600 kHz is found that can be attributed to the dissociation equilibrium of the quaternary structure of the molecule and can be considerably enhanced by denaturants or pH change from neutral to acid conditions. Measurements at frequencies down to 200 kHz have also permitted a more thorough investigation of proton-transfer reactions at carboxyl and amino groups in these proteins and of the effect of pH on these reactions.

Isolation of intermediate valence hybrids between ferrous and methemoglobin at subzero temperatures.

Quenching a hemoglobin solution partially saturated with carbon monoxide into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound hemoglobin molecules. Since each Fe3+ heme carries one extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between hemoglobin and carbon monoxide, which was originally present in solution. In this study we report: 1) the development of a simple and rapid method to quench aqueous hemoglobin solutions into a hydro-organic solvent at subzero temperatures; 2) the determination of suitable experimental conditions to isolate valence hybrids between carbonmonoxy- and methemoglobin by isoelectric focusing at temperatures as low as -25 degrees C; and 3) the identification and isolation of all valence hybrids of different charge between carbonmonoxy- and methemoglobin.

Effects of Gamma Irradiation on the Structure and Function of Human Hemoglobin

SZWEDA-LEWANDOWSKA, Z., PUCHALA, M., LEYKO, W. Effects of Gamma Irradiation on the Structure and Function of Human Hemoglobin. Radiat. Res. 65, 50-59 (1976). In this paper physicochemical and functional properties of irradiated aqueous hemoglobin solutions are presented. HbO2 solutions (5%) were irradiated in air with doses ranging from 0.5 to 5 Mrad at the efficiency of 1 Mrad/hr. At doses exceeding 1 Mrad, coagulation of small amounts of protein was observed. Hemoglobin which remained in the solution consisted of a mixture of HbO2 and MetHb. At doses of 4 and 5 Mrad the presence of hemochromogen was established. The appropriate hydrodynamic parameters-molecular weight, sedimentation constant, and limiting viscosity valuedo not differ from the control values up to a dose of 1 Mrad. At greater doses sedimentation constants and the limiting viscosity number increase. The decrease in the values of the second virial coefficient and the electrophoretic behavior in polyacrylamide gel suggest some changes in the electric charge of the molecules. Simultaneously, a considerable part of the molecules exhibits an increased affinity for oxygen and a decreased

Ultrasonic examination of the hemoglobin dissociation process in aqueous solutions of guanidine hydrochloride.

The ultrasonic absorption was determined, in aqueous solutions of hemoglobin undergoing dissociation as a function of the strong electrolyte guanidine hydrochloride (GuHCl), up to 5.4M (molar), over the frequency range 1–50 MHz. With increasing GuHCl concentration to 0.7M, the Hb molecule dissociates into half-molecules without loss of tertiary structure, and at GuHCl concentrations greater than 2.5M, the Hb molecule dissociates further into its four individual polypeptide chains with complete loss of tertiary structure. The ultrasonic absorption coefficient appears to be insensitive to the dissociation process, though a small contribution appears at the lower frequencies, owing to the expansion of the polypeptide molecule. A maximum in the absorption occurs near 2M GuHCl concentration, which does not correspond to dissociation or expansion of the Hb molecule, and it is thought to be associated with the proton transfer reaction.

Ultrasonic Examination of the Hemoglobin Dissociation Process

The ultrasonic absorption and velocity were determined in aqueous solutions of hemoglobin (Hb) undergoing dissociation. The dissociation of Hb in aqueous solutions of the strong electrolyte guanidine hydrochloride (GuHCl) has been studied extensively [K. Kawahara, A. G. Kirschner, and C. Tanford, Biochemistry 4, 1203 (1965)]. With increasing concentration of GuHCl to 0.7M (molar), the Hb molecule dissociates into half-molecules without loss of tertiary structure. At GuHCl concentrations greater than 2.5M, the Hb molecule dissociates further into its four individual polypeptide chains with complete loss of tertiary structure. The acoustical absorption coefficient within the frequency range 1–50 MHz appears to be insensitive to these conformational changes. Within the neighborhood of 2-M GuHCl concentration, a maximum in the excess ultrasonic absorption coefficient occurs which does not correspond to dissociation or expansion of the Hb molecule, and it is speculated that this maximum can be attributed to the proton transfer reaction.

X‐Ray and Neutron Small‐Angle Scattering from Hemoglobin in Aqueous Solution and in Crystal

The small‐angle scattering function of hemoglobin for X‐rays and neutrons has been calculated from the atomic coordinates of hemoglobin, established by X‐ray diffraction analysis. These theoretical scattering functions were compared with the experimental scattering data on aqueous hemoglobin solutions reported previously. The good agreement of the two sets of data in and beyond the region of the Guinier approximation gives confidence to the statement that the three‐dimensional structures of hemoglobin in single crystal and in aqueous solution are the same.

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

AbstractDielectric 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.

Molecular Mechanisms Contributing to Ultrasonic Absorption by Tissues

The interpretation of data presented by ultrasonic visualization techniques may be expected to benefit from an understanding of the mechanisms of the predominant contributions to the total ultrasonic absorption coefficients of tissues. Since aqueous solutions of hemoglobin have been most extensively investigated, analyses of specific molecular mechanisms have been undertaken with relation to the structure of this macromolecule and its environment. The contributions that could arise from reactions occurring at the charged side chains of residues of arginine, lysine, aspartic acid, and glutamic acid were considered since these residues occupy positions with highest frequency of occurrence at the hydrophilic exterior of the hemoglobin molecule. On the assumption of a structural mechanism, a family of possible single relaxation curves was calculated from which one member could be selected with knowledge of an experimental value of relaxation time. We conclude that basic side chains make significant contributions, which are larger than those of acidic side chains. [Work supported by a grant from the National Institutes of General Medical Sciences.]

The Use of Atomic Absorption Spectroscopy in Hemoglobinometry: I. The Determination of Iron in Hemoglobin

Atomic absorption is finding wide acceptance as a convenient method of metal analysis in biologic materials. The specificity and accuracy of the method, and the convenience of direct analysis of samples, requiring no preparation other than simple dilution, make the use of this technic highly desirable for iron analysis of hemoglobin. In order to take full advantage of the potentials of the technic, we were interested in investigating whether the analysis of aqueous hemoglobin solutions by direct aspiration into the flame gave accurate values for iron. Interfering effects might be observed from the incorporation of the iron in the hemoglobin molecule itself, or from the presence of other components of the sample matrixderiving from whole blood constituents. We were able to show that very accurate quantitative iron determinations can be obtained when simple aqueous dilutions of toluene hemolysates of hemolyzed washed cells or of hemolyzed whole blood were analyzed. No effect on the iron values was seen from the concomitant constituents of hemolysates or of whole blood when dilutions of 100-fold or greater were used. Instrumentation and technics. A PerkinElmer atomic absorption spectrophotometer model 303 (Perkin-EImer Corporation, Norwalk, Connecticut) was used. The standard instrument settings were: neon-filled iron hollow cathode tube, operating current, 30 ma.; analytical resonance line, 24S3 A; monochromator slit, 3; flame, acetylene-air, stoichiometric. The baffle system of the burner was modified as described previously. The aspirator capillary was replaced by a 10-in. length of polyethylene tubing of 0.030 in. i.d. (Clay-Adams Intramedic Pol}'ethylene tubing, PE 60). A wide-slot

Methemoglobinemia Induced by X-Irradiation

Methemoglobin forms in aqueous solutions of hemoglobin and in intact rats after exposure to ionizing radiation. For moderate doses, the degree of methemoglobinemia is a linear function of dose. In the sub-lethal dose range, the methemoglobinemia is comparable to the random variation in normal values. A secondary methemoglobinemia was observed in rats 3 to 8 hours postirradiation.