The kinetics of malonamide-induced haemolysis of mammalian erythrocytes II. The Eyring activation parameters

• 1.1. The temperature dependence of the malonamide-induced haemolysis of erythrocytes of rabbit, guinea pig, rat, cattle, sheep, dog, cat and pig has been further evaluated by an empirical application of the Eyring equation.
• 2.2. Numerical values are presented for the Eyring parameters, ΔH^≠, enthalphy of activation, ΔS^≠, entropy of activation, ΔG^≠, free energy of activation and K^≠ the activation equilibrium constant.
• 3.3. From a consideration of the dependence of these parameters on the concentration of the haemolysing solution, and on their variation with animal species, it is concluded that these data fully substantiate the view that the hydration structure of the erythrocyte membrane is the predominant factor in the mechanism of hemolysis.

     

Radioprotection of bovine erythrocytes to haemolysis

The effect of several free-radical scavengers on the radiation-induced haemolysis of bovine erythrocytes (50 krad, 60Co, cell concentration: 0.05 v/v) was studied. A two-phase effect of the additives was found with a maximum of the protective action in the millimolar concentration range. A correlation was established between the degree of protection at a fixed concentration (5mM) and the rate constants of reactions of respective compounds with OH. A radiosensitizing effect of diethyldithiocarbamate, an inhibitor of superoxide dismutase (SOD) was not confirmed and no specific protective effect of SOD was seen.     

Water relations in the malonamide-induced haemolysis of mammalian erythrocytes

A revised procedure is described for deriving enthalpy-entropy relations in the haemolysis kinetics of mammalian erythrocytes and its application demonstrates that previously reported linear enthalpy-entropy correlations are statistical artefacts with no real physical basis.Physically valid linear enthalpy-entropy relations do exist between species at constant osmotic concentration, but these are the result of the mutual dependence of the activation parameters on erythrocyte solvent volume. Non-linear enthalpy-entropy dependence on osmotic concentration, which is also physically valid and occurs within species, is attributed to erythrocyte solvent volume variation due to the osmotic properties of haemoglobin.Further development of the data indicates that malonamide-induced haemolysis is essentially an osmotic phenomenon and that the water permeability of all those cells is probably the same.From a consideration of the process in relation to the molecular dynamics of water it appears that the activation enthalpy, entropy and internal energy of haemolysis may refer to the molecular mobility of water during osmosis.     

The kinetics of drug-membrane interactions in human erythrocytes

The time course of the shape transformations and vesicle release in human erythrocytes caused by lysophosphatidylcholine and chlorpromazine was monitored using a light microscope-video recording technique. The time required for the erythrocytes to reach a stage I echinocytic shape decreased from 4.0 to 2.0 s, when the concentration of the lysophosphatidylcholine solution injected was increased from 1 to 25 microM. The time required to reach stage II decreased from 8.3 to 3.5 s and that required for vesicle release and the formation of stage IV spherocytes decreased from 78.0 to 11.6 s. Correspondingly, the time needed for the formation of stage I stomatocytes decreased from 2.3 to 1.0 s and that for stage II stomatocytes from 3.1 to 2.0 s, when the ambient chlorpromazine concentration was increased from 50 to 200 microM. The kinetics of the shape transformations of the erythrocytes were dependent on the ambient drug concentration. The rate of shape transformations could be predicted from a formula derived for the kinetics of the incorporation of the detergent into the cell membrane, providing that the affinity coefficient and mass transfer coefficient for drugs changed as a function of the free drug concentration. The results give a time scale for the drug-membrane interactions, i.e., the formation of stages I and II for drug-lipid bilayer interactions and the release of vesicles for drug-cytoskeleton interactions.     

Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes

The lateral diffusion coefficients (D) and the mobile fractions of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) and of membrane proteins labelled with fluorescein isothiocyanate, were measured by fluorescence photobleaching recovery on erythrocytes from healthy persons and from a hereditary spherocytosis patient. Measurements of lipid probe mobility were performed on ghosts labelled by NBD-PE exclusively at the external monolayer, or at both sides of the membrane. Our results indicate the following: (1) The mean values and the temperature dependence of D are different at the external and internal membrane leaflets. (2) In both normal and HS ghosts the mobile fraction of NBD-PE in the external monolayer does not depend significantly on temperature. On the other hand, the mobile fraction in the internal monolayer is reduced as the temperature is decreased. (3) At low temperatures, the mobile fraction of NBD-PE in the internal monolayer of spherocytic ghosts is significantly lower than the mobile fraction in the internal monolayer of normal ghosts. (4) No differences were observed between the mobilities of membrane proteins in normal and in spherocytic ghosts. However, differences were observed between the two cell populations in the temperature-dependence of the intrinsic fluorescence of unlabelled membrane proteins. The implications of these results for membrane phospholipid asymmetry and for cytoskeletal interactions with the internal lipid monolayer are discussed.     

The release of membrane components prior to haemolysis during extraction of intact erythrocytes with bile salts

Glycocholate removed significant amounts of acetylcholinesterase and membrane phospholipid from human erythrocytes prior to cell lysis. The phospholipids were relatively enriched in phosphatidylcholine. These results may represent selective attack on the outer leaflet of the plasma membrane and also may provide a model for some aspects of hepatic bile formation.     

The kinetics of drug-membrane interactions in human erythrocytes from neonates

The kinetics of drug-membrane interactions of erythrocytes from neonates were compared with those from adults by monitoring the time course of the shape transformations and vesicle release caused by drugs, using a light microscope--video recording technique. Both crenation caused by lysophosphatidylcholine (LPC) and cupping caused by chlorpromazine (CPZ) took place more slowly in the neonatal cells than in the cells from adults. The equilibrium concentrations of LPC and CPZ in erythrocytes did not differ significantly between the neonates and adults, however. The slower responses of the neonatal erythrocytes can be explained by the presence of negatively charged phosphatidylethanolamine and phosphatidylserine in the outer layer of the erythrocyte membrane, which may reduce the rate of incorporation of amphipathic LPC and attract cationic CPZ to remain in the outer membrane layer, lowering the rate of inward bending of the membrane normally caused by CPZ.     

Cadmium uptake kinetics in human erythrocytes

Cross-membrane transport of cadmium in human erythrocytes was studied using 109Cd+(+) and liquid scintillation counting. Uptake rates were determined by depletion of radioactivity in the incubation medium and the amount of hemolyzate radioactivity taken up by the erythrocytes. Both saturable and nonsaturable components for cadmium transport were observed. The mean maximum uptake rate (Jmax) of the saturable component was 4.9 X 10(-6) mol/L/h. The transport constant (Kt) was estimated at 6.9 X 10(-5) mol/L. The diffusion constant (Kd) of the non-saturable component was 1.4 X 10(-2)/h. Both Jmax and Kt of cadmium generally decreased when Zn+(+) was present, with a biphasic response in the presence of Cu+(+). Kd of cadmium increased as Zn+(+) or Cu+(+) levels were increased. It is suggested that cadmium may penetrate human red cells via cation transport sites owing to its behavior as an analog of one or more nutrient species.     

Effect of an interaction between haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine erythrocytes.

The effect of the haematocrit and cryoprotectant concentration on freeze-thaw haemolysis of bovine red cells was studied. Two-milliliter samples of bovine blood with an haematocrit of either 20 or 60% were diluted with 2 ml of either 5, 4, 3, 2, or 1 m glycerol or DMSO in PBS or with PBS alone. The degree of haemolysis after freezing to ?79 °C and thawing was least in blood diluted with 4m cryoprotectant. At the lower concentrations of cryoprotectant, haemolysis was greater in blood with the higher haematocrit, but this effect decreased as the cryoprotectant concentration was increased and was negligible at the optimal concentration.     

Glutaraldehyde effect on the osmotic fragility and rate of haemolysis of human erythrocytes: a kinetic study

The effect of glutaraldehyde treatment on the osmotic fragility and rate of haemolysis of human erythrocytes was studied in the temperature domain 22-42 degrees C at different aldehyde concentrations from 0.010 to 0.040% (w/v). The osmotic fragility linearly decreases at increasing temperatures for all glutaraldehyde concentrations with approximately the same slope as for the untreated erythrocytes. Rather similar effects are produced on the rate of haemolysis. It was not possible to define unique activation energies. The maximal haemolysis becomes smaller after the treatment with increasing concentrations of glutaraldehyde but more erythrocytes are no longer able to tolerate normally non-haemolysing osmotic stretching, this suggesting an artificial "ageing" of erythrocytes. The formation of a membrane protein skeleton as a result of cross-linking is inferred, while the changes in the cytoskeletal spectrin network seem to be less important.