Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Characteristics of the interaction of a treponemal hemolysin with rabbit erythrocytes

The mechanism of action on rabbit red cells of Treponema hyodysenteriae hemolysin was studied using volume analysis and release of hemoglobin. While fixation of the hemolysin on the erythrocytes is temperature independent, it appears that hemolysis is temperature dependent. The kinetics of hemolysis proceed according to a sigmoid curve characterized by a prelytic lag. The duration of the prelytic lag varies inversely with the quantity of hemolysin but the rate and the maximum value of hemolysis are directly proportional to the quantity of hemolysin. The effect of sucrose and trypan blue on the hemolysin and the red cells suggest that erythrocyte lysis is likely to be induced by the hemolysin in a way different from that known for other hemolytic agents.     

Some properties of the alpha-hemolysin produced by a hemolytic strain of E. coli]

It was found that alpha-hemolysin of E. coli P 678 HIy+ was maximally active against human erythrocytes at pH 6.5. The hemolytic activity is characterized in time by a distinct lag-phase and a phase of the greatest velocity of the reaction immediately following it. The duration of the lag-phase and also the rate of hemolysis depends on alpha-hemolysin concentration, whose increase is accompanied by a decrease of the lag-phase and acceleration of hemolysis. There is a definite limit below which the duration of the lag-phase remains unchanged with further increase of hemolysin concentration. There was noted a linear relationship between the amount of erythrocytes taken for the test and the rate of hemoglobin release and also a temperature activation of the hemolytic reaction.     

Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.     

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.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Bacteriocin (hemolysin) of Streptococcus zymogenes

The sensitivity of Streptococcus faecalis (ATTC 8043) to S. zymogenes X-14 bacteriocin depends greatly on its physiological age. Sensitivity decreases from the mid-log phase on and is completely lost in the stationary phase. The sensitivity of erythrocytes to the hemolytic capacity of the bacteriocin showed considerable species variation. The order of increasing sensitivity was goose < sheep < dog < horse < human < rabbit. However, when red cell stromata were used as inhibitors of hemolysis in a standard system employing rabbit erythrocytes the order of increasing effectiveness was sheep < rabbit < human < horse < goose. When rabbit cells were used in varying concentrations with a constant hemolysin concentration, there was a lag of about 30 min, which for a given hemolysin preparation was constant for all red cell concentrations. Furthermore, the rate of hemolysis increased with increasing red cell concentration. If red cells are held constant and lysin varied, the time to reach half-maximal lysis varies directly with lysin but is not strictly proportional. Bacterial membranes were one to three orders of magnitude more effective than red cell stromata as inhibitors. The order of increasing effectiveness seems to be Escherichia coli < Bacillus megaterium < S. faecalis < Micrococcus lysodeikticus. In addition to membranes, a d-alanine containing glycerol teichoic acid, trypsin in high concentration, and deoxyribonuclease also inhibited hemolysis. Ribonuclease, d-alanine, l-alanine, dl-alanyl-dl-alanine, N-acetyl-d-alanine, N-acetyl-l-alanine did not inhibit hemolysis.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.     

Effect of dose-rate and dose fractionation on radiation-induced hemolysis of human erythrocytes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit 2%) were exposed under air to gamma radiation at a dose rates of 2.2 kGy.h-1 and 4.2 kGy.h-1. The dose-response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose-rate. At both dose-rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy + 2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Similarities between complement-mediated and streptolysin S-mediated hemolysis

The oxygen-stable hemolysin streptolysin S (SLS) of Streptococcus pyogenes is encoded in part by the pel/sagA gene product. Antibodies to a synthetic peptide from the C terminus of the Pel/SagA open reading frame inhibited hemolysis mediated by both culture supernatants from multiple M serotypes of S. pyogenes isolates or a commercially available SLS preparation. Analysis of the SLS-mediated hemolytic reaction demonstrated that it was temperature- and concentration-dependent. Like complement-mediated hemolysis it conforms to the prediction of a one-hit mechanism of hemolysis. A number of intermediates in the SLS-mediated hemolysis of sheep erythrocytes could be distinguished. SLS could bind to erythrocytes below 17 degrees C; however, lysis could only occur at temperatures >23 degrees C. Following binding of SLS and washing, a papain-sensitive intermediate could be distinguished prior to insertion of the SLS complex into the erythrocyte membrane, which resulted in formation of a transmembrane pore and led to irreversible osmotic lysis of the cell. These intermediates were similar to those described previously during complement-mediated hemolysis.     

Erythrocyte membrane structural features that are critical for the lytic reaction of Spirographis spallanzani coelomic fluid hemolysin

1. Hemolytic activity of Spirographis spallanzani coelomic fluid depends on factor(s) strongly influenced by calcium but not by sulfhydril or disulfide reagents.2. The lytic reaction was suppressed by low zinc ion concentrations but it was not influenced by the presence of proteinase inhibitors.3. These data indicate that S. spallanzani hemolysin is a non-enzymatic, calcium-dependent, zincinhibitable factor that occurs naturally in the coelomic fluid.4. In the absence of calcium, enzymatic desialization converted sheep erythrocytes into susceptible targets, suggesting the involvement of erythrocyte surface sialic acid.5. However, the inhibitory effect of the sugar on anti-rabbit lysis was partially removed by addition of calcium.6. Attempts to characterize membrane components that are critical for hemolysis were performed by inhibition experiments.7. We found that saccharides, glycoproteins, mucosubstances as well as rabbit erythrocyte soluble tryptic fragments were ineffective in inhibiting hemolysis.8. Sonicated dispersion of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanol, sphingomyelin and cholesterol did not influence the hemolytic reaction.9. Rabbit erythrocyte extracted from membrane lipids (chloroform phase) did not modify the lytic activity against rabbit red blood cells.10. Conversely, the methanol phase consistently reduced the lytic capacity of the fluid.11. The heat-stable, trypsin-resistant inhibitory factor was most probably a small molecule, since dialysis removed the inhibitory effect.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

In vivo hemolytic activity of group B streptococcus is dependent on erythrocyte-bacteria contact and independent of a carrier molecule

Experiments were performed to determine the interaction between the hemolysin of group B streptococcus (GBS) and sheep erythrocytes. Growing GBS were shown to possess a potent hemolysin at a very early stage of the growth cycle. After separation of the cells from the growth medium, all the hemolytic activity remained with the bacterial cells, and no activity could be detected in the growth medium. When fetal calf serum was added to the media, some soluble activity was detected. This activity, however was completely removed by ultracentrifugation, the hemolytic activity being found solely in the pellet. After the hemolysin had formed, no new protein synthesis was needed to cause hemolysis because the addition of chloramphenicol to cells caused no difference in their hemolytic potential. For proof that no short-lived, soluble factors are produced by the bacteria, bacteria and sheep erythrocytes were incubated in contiguous media, separated by a 0.22-m membrane. No hemolytic activity was detected on the erythrocyte side of the membrane, although high amounts of hemolysin could be extracted from the bacteria. Only when a detergent was added to the growth medium was hemolysis detected from the erythrocytes, showing that extracted hemolysin could indeed pass through the membrane. These results suggest that the hemolysin is attached to the surface of the cell and that contact is needed between the bacteria and erythrocyte to cause lysis. Where soluble activity was detected, it was connected to bacterial fragments.     

Characterization of inhibitor to leptospiral hemolysin present in bovine serum

Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform-methanol-soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin-layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective. From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.     

Liposome oxidation and erythrocyte lysis by enzymically generated superoxide and hydrogen peroxide.

Xanthine oxidase, acting on acetaldehyde under aerobic conditions, produces a flux of O2- and H2O2 which attacks artificial liposomes and washed human erythrocytes. The liposomes were peroxidized and the erythrocytes suffered oxidation of hemoglobin followed by lysis. The oxidation of hemoglobin followed by lysis. The oxidation of hemoglobin, within the exposed erythrocytes, could be largely prevented by prior conversion to carbon monoxyhemoglobin, without preventing lysis. Hemolysis thus appeared to be a consequence of direct oxidative attack on the cell stroma. The enzyme-generated flux of O2- and of H2O2 also inactivated the xanthine oxidase. Superoxide dismutase or catalase, present in the suspending medium, protected the liposomes against peroxidation, the erythrocytes against lysis, and the xanthine oxidase against inactivation. Scavengers of O2('deltag), such as histidine or 2,5-dimethylfuran, which do not react with O2- or H2O2, also prevented peroxidation of liposomes and lysis of erythrocytes when present at low concentrations. In contrast a scavenger of OH-, such as mannitol was ineffective at low concentrations and provided significant protection only at much higher concentrations. It is proposed that O2- and H2O2 cooperated in producing OH- and O2('deltag), which were the proximate causes of lipid peroxidation and of hemolysis.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphospatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

Effect of retinol on the hemolysis and lipid peroxidation in vitamin E deficiency

A study on the effect of retinolin vitro on the hemolysis of vitamin E deficient rat red blood cells showed that retinol enhanced the lysis of the E deficient cells as compared to the lysis of normal cells. The lipid peroxidation present during hydrogen peroxide induced lysis of E deficient cells was however markedly inhibited in the presence of retinol without affecting the rate of lysis. In an actively peroxidising system of non-enzymatic lipid peroxidation of rat liver or brain homogenates and of brain lysosomes incubated with human erythrocytes, no lysis was obtained; incorporation of retinol in such systems resulted in lysis but no peroxidation. Hydrogen peroxide generating substances almost completely inhibited the lysis of normal human erythrocytes by retinol, but linoleic acid hydroperoxide and auto-oxidised liver or brain homogenates and ox-brain liposomes increased the lysis. It is concluded that vitamin E deficient erythrocyte hemolysis may be augmented by retinol, an anti-oxidant, having a lytic function without the peroxidation of stromal lipids