Change in Osmotic Pressure Influences the Absorption Spectrum of Hemoglobin inside Red Blood Cells.

Absorption spectra of red blood cell (RBC) suspensions are investigated in an osmolarity range in the medium from 200 mOsm to 900 mOsm. Three spectral parameters are used to characterize the process of swelling or shrinkage of RBC-the absorbance at 700 nm, the Soret peak height relative to the spectrum background, and the Soret peak wavelength. We show that with an increase in the osmolarity, the absorbance at 700 nm increases and the Soret peak relative height decreases. These changes are related to the changes in the RBC volume and the resulting increase in the hemoglobin intracellular concentration and index of refraction. Confocal microscopy and flow cytometry measurements supported these conclusions. The maximum wavelength of the Soret peak increases with increasing osmolarity due to changes in the oxygenation state of hemoglobin. Using these spectrum parameters, the process of osmosis in RBCs can be followed in real time, but it can also be applied to various processes, leading to changes in the volume and shape of RBCs. Therefore, we conclude that UV-Vis absorption spectrophotometry offers a convenient, easily accessible, and cost-effective method to monitor changes in RBC, which can find applications in the field of drug discovery and diagnostics of RBC and hemoglobin disorders.

Radiofrequency dielectric spectroscopy study: Effects of pH, hydrogen bond donors and acceptors on the attachment of spectrin skeleton to the lipid membrane of erythrocytes.

Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, ßsp (1.4 MHz) and γ1sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the ßsp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the ßsp and γ1sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the ßsp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1sp relaxation.

Hemolysis by Saponin Is Accelerated at Hypertonic Conditions.

Saponins are a large group of organic amphiphilic substances (surfactants) mainly extracted from herbs with biological activity, considered as one of the main ingredients in numerous remedies used in traditional medicine since ancient times. Anti-inflammatory, antifungal, antibacterial, antiviral, antiparasitic, antitumor, antioxidant and many other properties have been confirmed for some. There is increasing interest in the elucidation of the mechanisms behind the effects of saponins on different cell types at the molecular level. In this regard, erythrocytes are a very welcome model, having very simple structures with no organelles. They react to changing external conditions and substances by changing shape or volume, with damage to their membrane ultimately leading to hemolysis. Hemolysis can be followed spectrophotometrically and provides valuable information about the type and extent of membrane damage. We investigated hemolysis of erythrocytes induced by various saponin concentrations in hypotonic, isotonic and hypertonic media using measurements of real time and end-point hemolysis. The osmotic pressure was adjusted by different concentrations of NaCl, manitol or a NaCl/manitol mixture. Unexpectedly, at a fixed saponin concentration, hemolysis was accelerated at hypertonic conditions, but was much faster in NaCl compared to mannitol solutions at the same osmotic pressure. These findings confirm the colloid-osmotic mechanism behind saponin hemolysis with pore formation with increasing size in the membrane.

Role of Plasma Membrane at Dielectric Relaxations and Intermembrane Interaction in Human Erythrocytes.

Dielectric relaxations at 1.4 MHz (ßsp) and 9 MHz (γ1sp) on the erythrocyte spectrin network were studied by dielectric spectroscopy using dense suspensions of erythrocytes and erythrocyte ghost membranes, subjected to extraction with up to 0.2% volume Triton-X-100. The step-wise extraction of up to 60% of membrane lipids preserved γ1sp and gradually removed ßsp-relaxation. On increasing the concentration up to 100 mM of NaCl at either side of erythrocyte plasma membranes, the ßsp-relaxation was linearly enhanced, while the strength of γ1sp-relaxation remained unchanged. In media with NaCl between 100 and 150 mM ßsp-relaxation became slightly inhibited, while γ1sp-relaxation almost disappeared, possibly due to the decreased electrostatic repulsion allowing erythrocytes to come into closer contact. When these media contained, at concentrations 10-30 mg/mL dextran (MW 7 kDa), polyethylene glycol or polyvinylpyrrolidone (40 kDa), or albumin or homologous plasma with equivalent concentration of albumin, the γ1sp-relaxation was about tenfold enhanced, while ßsp-relaxation was strengthened or preserved. The results suggest the Maxwell-Vagner accumulation of ions on the lipid bilayer as an energy source for ßsp-relaxation. While ßsp-relaxation appears sensitive to erythrocyte membrane deformability, γ1sp-relaxation could be a sensitive marker for the inter-membrane interactions between erythrocytes.

Segmental flexibility of spectrin reflects erythrocyte membrane deformability.

The frequency-dependent contribution of spectrin, the main cytoskeletal protein of red blood cell (RBC) membrane, to the complex admittance and capacitance of suspended RBCs have revealed two dielectric relaxations, ßsp (1.4 MHz) and γ1sp (7 MHz). The strength of these relaxations was related to the ability of RBC membrane to deform. In this study the two relaxations were inhibited by N-ethylmaleimide (up to 5 mM), known to impair the RBC deformability, and the degree of inhibition, i.e., the number of accessible SH-groups on spectrin, depended on the deformation of RBC membrane. Dithiothreitol (up to 5 mM), which does not affect RBC deformability, did not affect the above dielectric relaxations in line with the absence of S-S groups on spectrin. Phenylhydrazine (up to 3 mM) and hydrogen hydroperoxide (up to 450 µM) are known to denature the haemoglobin of RBCs producing nanoparticles (globins) that bind to spectrin turning the RBC membrane rigid. At the same concentrations they were shown to inhibit progressively the two relaxations on spectrin. The results are in line with the involvement of some globin-sized segments of spectrin in the dielectric activity of spectrin and in the ability of RBC plasma membrane to deform.

Differential dielectroscopic data on the relation of erythrocyte membrane skeleton to erythrocyte deformability and flicker.

Two dielectric relaxations, ßsp (1.5 MHz) and γ1sp (7 MHz), have been detected on spectrin-based membrane skeleton (MS) of red blood cells (RBCs) using the plot of admittance changes at the spectrin denaturation temperature (Ivanov and Paarvanova in Bioelectrochemistry 110: 59-68, 2016, Electrochim Acta 317: 289-300, 2019a). In this study, we treated RBCs and RBC ghost membranes with agents that make membranes rigid and suppress membrane flicker, and studied the effect on ßsp and γ1sp relaxations. Diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) (up to 0.85 mM), taurine mustard (tris(2-chloroethyl)amine) (up to 2 mM), known to specifically cross-link and stiffen spectrin, and glutaraldehyde (up to 0.044%) all inhibited the relaxations in RBC ghost membranes. Similar inhibition was obtained resealing RBC ghost membranes with 2,3-diphosphoglicerate (up to 15 mM), binding WGA (wheat germ agglutinin) (up to 0.025 mg/ml) to exofacial aspect of RBCs, incubating RBCs in hypotonic (200 mOsm) and hypertonic (600-900 mOsm) media and depleting RBCs of ATP. By contrast, concanavalin A (1 mg/ml) and DIDS (4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid) (75 µM, pH 8.2), both known to bind specifically band 3 integral protein of RBCs without effect on RBC membrane rigidity, did not affect the relaxations. We conclude there might be a relation between the strength of dielectric relaxations on MS spectrin and the deformability and flicker of RBC membrane.

Species-dependent variations in the dielectric activity of membrane skeleton of erythrocytes.

Previously detected ßsp and γ1sp dielectric relaxations on the spectrin-based membrane skeleton (MS) of human red blood cells (RBCs) have been shown sensitive to the attachment of MS to the lipid-protein membrane. Such relaxations were now detected on the MS of mammal (rat, horse, bovine, sheep and goat) and "unstrained" chicken RBCs. To become "unstrained" chicken RBCs were subjected consecutively to cold (4°C, >20 h) and either colchicine (15 mM) or vinblastine (30 µM) (4°C, 1 h) that led to irreversible disassembly of their marginal band and an additional portion of their cytoskeleton. With the exception of bovine RBCs, the critical frequency (fc) of either relaxation increased, although at different rates, with the decrease in the volume of RBC species. The strong increase in fc of γ1sp relaxation from 2.5 MHz ("unstrained" chicken RBCs) to 13 MHz (goat RBCs) could indicate denser state of MS in smaller RBC species. The low values of fc of γ1sp relaxation in "unstrained" chicken RBCs (2.5 MHz) and bovine RBCs (4.5 instead of 9 MHz) could be related to their extraordinary thermal stability at the temperature of spectrin denaturation.

Drug Exchange between Albumin Nanoparticles and Erythrocyte Membranes.

The effects of thioridazine (TDZ) and chlorpromazine (CPZ) and bovine serum albumin nanoparticles (BSA-NPs) on erythrocyte membranes have been investigated. Two kinds of hemolytic assays were used; hemolysis under hypotonic conditions and hemolysis in physiological conditions. Under hypotonic conditions for 50% hemolysis, both TDZ and CPZ have a biphasic effect on membranes; namely, stabilization at low concentrations and destabilization after reaching a critical concentration. In physiological conditions, there are other critical concentrations above which both drugs hemolyse the erythrocites. In each case, the critical concentrations of TDZ are lower than those of CPZ, which is consistent with the ratio of their partition coefficients. When BSA-NPs are added to the erythrocyte suspension simultaneously with the drugs, the critical concentrations increase for both drugs. The effect is due to the incorporation of a portion of drug substances into the BSA-nanoparticles, which consequently leads to the decrease of the active drug concentrations in the erythrocyte suspension medium. Similar values of the critical concentrations are found when the BSA-NPs are loaded with the drugs before their addition to the erythrocyte suspension in which case the events of the partition are: desorption of the drug from BSA-NPs, diffusion through the medium, and adsorption on erythrocyte membranes. This result suggests that the drugs are not influenced by the processes of adsorption and desorption onto and out of the BSA-NPs, and that the use of BSA-NPs as drug transporters would allow intravenous administration of higher doses of the drug without the risk of erythrocyte hemolysis.

Effects of heat and freeze on isolated erythrocyte submembrane skeletons.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.