HEMOLYSIS BY SAPONIN AND SODIUM TAUROCHOLATE,WITH SPECIAL REFERENCE TO THE SERIES OF RYVOSH

1. The series of Ryvosh is obtained when hemolysis of the red cells of the animals concerned occurs with saponin as the lytic agent. 2. The series of Ryvosh is not obtained when R _∞ is taken as the resistance constant and sodium taurocholate is used to hemolyse the cells of the same animals. 3. The hemolysin sodium taurocholate has been found to differ from saponin in that the time-dilution curves are found to approach their respective asymptotes with different values of κ.     

THE EFFECT OF HEMOLYTIC SUBSTANCES ON WHITE CELL RESPIRATION

Substances such as saponin, the bile salts, etc., which produce lysis of red cells also produce cytolysis of white cells from rabbit peritoneal exudates, the arbitrary criterion of their cytolytic effect being their ability to depress the O₂ consumption of the leucocytes. The amount of cytolysis increases regularly as the amount of the added lysin is increased, and sufficiently large quantities of saponin, sodium taurocholate, sodium glycocholate, or sodium oleate are capable of virtually abolishing the O₂ consumption altogether. At the same time, it can be shown that a lysin such as saponin is used up in combining with the white cells in much the same way as it is used up in combining with red cells, and the reduction in oxygen consumption appears to be roughly proportional to the amount so combined. The action of these lytic substances on white cells, in fact, is very similar to their action on red cells, due allowance being made for the fact that the cytolysis of the white cell is probably not an all-or-none process like hemolysis. White cell respiration is also depressed in hypotonic solutions, the respiration being virtually linear with the tonicity.     

COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S'' was delayed, and in this property, streptolysin S'' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S'' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S'', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase.     

THE ACCELERATION OF SAPONIN HEMOLYSIS BY PROFLAVINE

Proflavine is a very powerful accelerator of saponin hemolysis of rabbit, human, and dog erythrocytes. Lysolecithin hemolysis, on the other hand, is inhibited. Dog erythrocytes in the presence of proflavine undergo marked changes in shape, finally becoming rods of about 13 µ in length. Rabbit and human erythrocytes are not altered in form under these conditions.     

Enzyme loading of nucleated chicken erythrocytes

Exogenous enzymes can be loaded into nucleated chicken erythrocytes by hypotonic hemolysis. The hemolysed and resealed cells retain the ability to activate transport ions and to survive while circulating in the blood of chickens. It is proposed that these cells may be useful for the investigation of several aspects of cellular biochemistry.     

A mixing chamber to enucleate avian and fish erythrocytes: preparation of their plasma membrane

Biochemical studies of the plasma membrane and the cytoskeleton of nucleated erythrocytes are strongly limited by the difficulties encountered in enucleating large quantities of cells. We describe an easily built hydrodynamic system which allows rapid preparation of large amounts of avian and fish erythrocyte plasma membranes. The contents of two 25-ml syringes containing hemolyzed nucleated erythrocytes are forced through four capillaries to a specially designed mixing chamber which fills a collecting syringe. The 50-ml erythrocyte suspension can be processed in 2 s. The high speed flow is achieved with a hand-activated piston. The turbulences in the mixing chamber are carried to an optimal efficiency by the vis-à-vis disposition of the four mixing jets. The enucleated membranes are separated from the nuclei and residual nucleated cells by differential centrifugations. They do not show contamination with nuclear material. Erythrocytes from chicken and trout have been used. They present striking differences in their stability toward hydrodynamic disruption, erythrocytes from chicken being far more stable. Ninety-five percent of trout erythrocytes are enucleated after only one run through the mixing chamber. Two runs performed at the maximal flow rate are necessary to enucleate chicken erythrocytes with a yield of 80%. In the former case most of the purified enucleated plasma membranes are fragmented in small vesicles while they retain a large size in the case of chicken erythrocytes. The proteins of the membranes thus prepared are characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: we found that erythrocyte membranes from trout are remarkable for their small spectrin content compared to those from chicken.     

Experimental variations in sonic fragility of red cells

A magnetostriction oscillator has been used for determining the relative fragility of human red cells to 9000 cycle vibrations under some different environmental conditions. The destruction of the cells is a logarithmic function of time according to the equation See PDF for Equation. Hypertonic saline solution, saponin, preheating, alcohol, and ether in subhemolytic concentrations decrease the fragility of human red cells subjected to sonic oscillation. Hypotonic saline, preheating to 50°C. for 3 minutes, and hemolytic concentrations of ether increase the fragility of red cells to 9 kc. vibrations. Following preheating of the cells, and in the presence of saponin the destruction deviates slightly from a true logarithmic rate of hemolysis.     

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.     

Response of erythrocytes to heat in the presence of D2O, glycerol, and anisotonic saline

Mammalian erythrocytes that lack cytoplasmic organelles and a nucleus are a useful model for studying the effect of heat on the cell membrane and cytoskeleton. The effect of heat on the membrane bilayer and cytoskeleton of erythrocytes is remarkably similar to that observed in nucleated cells. Some concentrations of D2O and glycerol can effectively protect erythrocytes from heat-induced damage to the membrane and cytoskeleton. These results are similar to observations in nucleated cells. Heating erythrocytes in some concentrations of anisotonic NaCl solutions reduced damage, an observation that does not apply to enhanced killing of nucleated cells. This difference implies that some components of the cytoplasm or nucleus, or both, may contribute to the enhancement of cytotoxicity of nucleated cells when they are heated in the anisotonic NaCl solution. Incremental heating, dividing a heat treatment into two fractions, and preheating of erythrocytes all modify the effect of heat on erythrocytes slightly, but the results suggest little, if any, development of thermotolerance. The response of chicken erythrocytes is similar to that of mammalian erythrocytes, although higher temperatures are required to produce a heat effect in chicken erythrocytes. These observations suggest that the characteristic differences in heat sensitivity in nucleated and enucleated cells involve components other than the cell membrane.     

The kinetics of progressive reactions in systems containing saponin,digitonin, and sodium taurocholate

The relations between lysin concentration, percentage hemolysis at the moment at which the lysin concentration is reduced by dilution, and the amount of hemolysis which follows the dilution as a result of the reaction being "progressive" point to there being an "internal" phase at the red cell surfaces, in which the lysin is less affected by the dilution than in the system as a whole. A second possibility, i.e. that the combination of lysin molecules with certain components of the cell surface has an ultimate effect on neighboring components which depend on the former for their stability cannot, however, be ruled out. In systems containing digitonin or sodium taurocholate, this internal phase, once formed, seems to be almost unaffected by the dilution of the system; i.e., these lysins are very firmly held at the cell surfaces. In systems containing saponin the lysin is less firmly attached, so that dilution of the system affects its concentration appreciably.     

Histone H5 in nucleated erythrocytes of fish as determined by radioimmunoassay

Tissue-specific histone H5 in the nucleated erythrocytes of dogfish, scup, skate, tautog, sea robin and toad fish was studied. The presence of this histone was inferred by its electrophoretic mocility on polyacrylamide gels containing either acid-urea or sodium dodecyl sulfate. By radioimmunoprecipitation assays, cross reaction was observed between fish histones and an anti-H5chicken antibody. The antibody was specific to chicken histone H5; purified chicken histone H1 and calf thymus total histones did not cross react. It is concluded that fish histone H5 shares common antigenic determinants with the chicken H5 histone.     

Hyperosmotic hemolysis and antihemolytic activity of the saponin fraction and triterpene glycosides from Panax ginseng C. A. Meyer

A saponin fraction and triterpene glycosides Rb1, Rb2 and Rg1 from Panax ginseng C. A. Meyer (saponins) were shown to inhibit the hyperosmotic hemolysis of erythrocytes. Using scanning electron microscopy and light scattering, saponins were found to restore the shape of the cells by inhibiting their contraction and the formation of echinocytes and microspherocytes. Scanning microcalorimetry demonstrated that an increase in osmomolarity and ionic strength of the medium eliminated structural transitions of erythrocyte ghost membranes. The hyperosmotic conditions were shown to cause the destruction of the erythrocyte membrane skeleton, except for the cytoplasmic and membrane domains of band 3 proteins. Saponins changed the parameters of all membrane structural transitions, induced new reversible transitions in the membranes and eliminated the ripple phase of dipalmitoylphosphatidylcholine. The mechanism of the antihemolytic action is discussed based on the bilayer couple model hypothesis.     

The role of ecto-ATPases of erythrocyte plasma membrane in hemodynamics of fishes

The review addresses varied aspects of physiological and biochemical mechanisms aimed at creating special rheological conditions for blood flow termed non-Newtonian blood properties. We conducted a comparative analysis of structural features and phospholipid repertoire of the erythrocyte plasma membranes and cytoskeleton, extracellular ATP pool, and ecto-ATPase enzymatic activity in nucleated and non-nucleated erythrocytes in vertebrates, as well as a study of thermal effects in nucleated red blood cells. Based on data from the literature and our own research, we hypothesize that the phenomenon of non-Newtonian blood properties is underlain by a decrease in the relative blood viscosity due to thermal hydrolysis of extracellular ATP that erythrocytes release onto their surface most actively under capillary deformation stress. We believe that in fishes an important role in this process may belong to erythrocyte plasma membrane ecto-ATPases. Due to a heat released during hydrolysis of extracellular ATP, the marginal blood plasma layer, adjoining the capillary wall, appears to warm up. This may modify the structure of the membrane bilayer and deform the cytoskeleton, thus providing special rheological conditions for blood flow. The heat-producing ability, that we found in fish nucleated erythrocytes, may serve an additional evidence for the existence of this mechanism.     

Damage to erythrocytes caused by the interaction of nitrite-ions with hemoglobin

The formation of two hemoglobin forms (methemoglobin and nitrite methemoglobin) in native human erythrocytes in the presence of sodium nitrite in suspension was shown. In normal erythrocytes, the interaction of intracellular oxyhemoglobin with nitrite ions results in the formation of methemoglobin, whereas in metabolically exhausted erythrocytes, this leads predominantly to the formation of nitrite methemoglobin. The nitrite methemoglobin reacts with hydrogen peroxide to form reactive intermediates (e.g. peroxynitrous acid) and the products of hemoglobin destruction. During the storage of erythrocyte suspensions containing methemoglobin and modified nitrite methemoglobin, differences in the forms of erythrocytes and the degree of their hemolysis were revealed. It is assumed that the formation of methemoglobin leads to the destruction of erythrocytes.     

Regulatory differences between Ca(2+)-ATPase in plasma membranes from chicken (nucleated) and pig (anucleated) erythrocytes

Kinetic and regulatory properties of the plasma membrane Ca(2+)-ATPase activity from chicken (nucleated) erythrocytes were studied and compared to those from pig (anucleated) erythrocytes. In the absence of known activators: (1) Ca(2+) affinity for the Ca(2+)-ATPase activity from nucleated erythrocytes was 12-fold higher than that from pig erythrocytes, and thus the enzyme is sensitive to physiological Ca(2+) concentrations; (2) the enzyme from chicken erythrocytes showed two apparent Km values for ATP, as compared to one apparent Km value displayed by pig erythrocytes; (3) Ca(2+)-ATPase inserted in chicken erythrocyte membranes showed a low sensitivity to activation by phosphatidylinositol-4-phosphate; (4) when p-NPP was used as substrate, the activity of chicken erythrocytes was high, similar to that attained by pig erythrocytes, but barely sensitive to activation by dimethylsulfoxide and calmodulin. ATP hydrolysis was 10-fold lower than that displayed by pig erythrocytes and the maximal velocity was activated three-fold by calmodulin. The enzyme was insensitive to alkaline phosphatase treatment and showed a single phosphorylation band in electrophoresis, ruling out the possibility of previous modulation by endogenous kinases and/or by partial proteolysis. The differences may be attributed to some endogenous modulator, to distinct isoforms, or to a difference in the E(1)/E(2) states of the enzyme.     

Reevaluation, using marker enzymes, of the ability of saponin and ammonium chloride to free Plasmodium from infected erythrocytes

Saponin and ammonium chloride lysis have been applied for some time to the separation of erythrocyte membranes from malarial-infected erythrocytes, allowing easy isolation of the parasites. We present a reevaluation of the use of saponin and ammonium chloride as tools for isolating Plasmodium (knowlesi or falciparum) parasites. Acetylcholine esterase (EC 3.1.1.7) was used as an erythrocyte membrane marker and CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) as a parasite membrane marker to monitor fractionation by these agents. Both saponin and ammonium chloride produced hemolysis of uninfected and infected erythrocytes, but failed to separate host erythrocyte membrane from the parasite, regardless of its stage. Thus, saponin and ammonium chloride can be used to isolate whole infected erythrocytes, depleted of hemoglobin, by selective disruption of uninfected cells.     

Rat embryonic and foetal erythrocytes. High 2,3-bisphosphoglycerate and ATP and low oxygen affinity in vitro for nucleated embryonic cells.

Embryonic nucleated red cells of the rat have high ATP and 2,3-bisphosphoglycerate and relatively low oxygen affinity. During foetal life they are replaced by large non-nucleated red cells with high ATP, low bisphosphoglycerate and high oxygen affinity. After birth, small non-nucleated red cells with high bisphosphoglycerate and low oxygen affinity rapidly predominate.     

Effects and mechanisms of action of ionophorous antibiotics valinomycin and salinomycin-Na on Babesia gibsoni in vitro

Valinomycin and salinomycin-Na, 2 ionophorous antibiotics, exhibited in vitro antibabesial activities against Babesia gibsoni that infected normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes, which completely lack Na,K-ATPase activity. The level of parasitemia of B. gibsoni was significantly decreased when the parasites were incubated in culture medium containing either 10(-1) ng/ml valinomycin or 10(2) ng/ml salinomycin-Na for 24 hr. Four-hour incubation in the culture medium containing 5 μg/ml salinomycin-Na led to the destruction of most parasites. In contrast, when the parasites infected canine erythrocytes containing high potassium (HK) and low sodium concentrations, i.e., HK erythrocytes, the in vitro antibabesial activities of both ionophorous antibiotics seemed to be weakened, apparently due to the protection by the host cells. Therefore, differential influences of ionophorous antibiotics on LK and HK erythrocytes were observed. In LK erythrocytes, the intracellular concentrations of potassium, sodium, and adenosine triphosphate (ATP) were not modified, and hemolysis was not observed after incubation in the medium containing each ionophorous antibiotic. These results suggested that these ionophorous antibiotics did not affect cells without Na,K-ATPase, and directly affected B. gibsoni. In HK erythrocytes, the ionophorous antibiotics increased the intracellular sodium concentration, and decreased the intracellular potassium and ATP concentrations, causing obvious hemolysis. Additionally, the decrease of the intracellular ATP concentration and the hemolysis in HK erythrocytes caused by valinomycin disappeared when the activity of Na,K-ATPase was inhibited by ouabain. These results indicate that modification of the intracellular cation concentrations by the ionophorous antibiotics led to the activation of Na,K-ATPase and increased consumption of intracellular ATP, and that the depletion of intracellular ATP resulted in hemolysis in HK erythrocytes. Moreover, the antibabesial activity of valinomycin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations. This showed that the intracellular cation concentration in the parasites was not modified in those media and would remain the same.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.