The combination between hemolysins and red cells or ghosts,as studied with a radioactive lysin and with new color reactions

The quantity of a radioactive hemolysin, sodium dodecyl sulfonate-S³⁵, taken up by red cells from concentrations too small to produce hemolysis varies with the lysin concentration, and does so in a way which can be described by an adsorption isotherm. Attempts to use color reactions or surface tension measurements to determine the quantity of digitonin, saponin, and the bile salts taken up by red cells from hypolytic concentrations have failed, principally because chromogenic, and also surface-active, substances are liberated from the cells when the lysin is added. Color reactions with the anthrone reagent show that digitonin and saponin are both taken up by or fixed to red cell ghosts; the extent of the uptake, however, is uncertain, again because of the liberation of chromogenic substances. Comparison of the results of the various methods which measure the apparent amount of lysin fixed, or utilized in reactions between lysins and red cells or ghosts show discrepancies between results given by direct methods (measurement of radioactivity or of color) and indirect methods (addition of a second population after lysis of a first, and dependence of the position of the asymptote of the time-dilution curve on the number of red cells). The discrepancies are traceable to the inhibitory effects of substances liberated from the red cells or ghosts. The ease with which a lysin, once taken up by red cells, can be detached by diluting the system determines the extent to which the hemolytic reaction is "progressive," but has no observed connection with the quantity taken up in the first place. There is now ample evidence that lysis in systems containing simple hemolysins is a process involving two stages in time and two phases, and that it is usually complicated by reactions between the hemolysin and liberated inhibitory material.     

Studies on the mechanism of action of oxygen-labile haemolysins.

The sensitivities of the binding step and the lytic step of haemolysis by pneumolysin to the action of various inhibitors and to variations in the assay conditions were studied. Binding was inhibited by HgCl2 and N-ethylmaleimide. Lysis by previously fixed lysin was insensitive to HgCl2 and only slightly sensitive to N-ethylmaleimide. Binding of pneumolysin was independent of ionic strength. Binding of pneumolysin and streptolysin O decreased above pH 8-0 and 8-4 respectively. These results suggest that binding requires a non-ionized unsubstituted sulphydryl group. Incubation of erythrocytes with NaF caused inhibition of pneumolysin, indicating that some metabolic function of the cell may be involved in lysis. The action of streptolysin O was not affected by NaF.     

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.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

The inhibition of hemolysis,as studied by the technique used for investigating progressive reactions,and by a technique using radioactive hemolysins

Inhibition of hemolysis by plasma has been studied in systems containing saponin, digitonin, and sodium lauryl sulfate, using the methods developed for the study of the kinetics of progressive reactions. The results are that the progressive nature of the hemolytic reaction in saponin systems becomes less when the inhibitor is added, that the addition of inhibitor to digitonin systems has no effect on the final result although the velocity of the progressive reaction is reduced, and that the effect of plasma in lauryl sulfate systems is intermediate between the effects in saponin systems and digitonin systems. A simple explanation is that the lysin is very strongly fixed, to form an internal phase, to the cell surfaces in digitonin systems, less strongly in laurate systems, and still less strongly in saponin systems. To answer the question as to whether, in a system in which some of the lysin forms as internal phase, the addition of an inhibitor results in a redistribution of the lysin between the internal phase and the bulk phase, sodium lauryl sulfate-S³⁵ and sodium cetyl sulfate-S³⁵ were prepared, and their distribution between the internal phase and the bulk phase was measured before and after the addition of plasma, the lysins being added to the cells either before or after the addition of the inhibitor. The results show that there is a large uptake of these lysins at the red cell surfaces when they are added first, and that the subsequent addition of plasma greatly reduces the quantity of lysin held in the internal phase. Further, if the inhibitor is added first and the lysin subsequently, the internal lysin phase is very incompletely formed. Serum albumin, used in place of plasma, gives essentially similar results.     

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.     

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.     

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.     

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.     

THE MECHANISM OF THE INHIBITION OF HEMOLYSIS

The principal conclusion of this investigation is that the inhibitory effect of plasma or serum on hemolysis by saponin and lysins of the same type is similar in nature to the inhibitory effects of certain sugars and electrolytes, which again are similar to the acceleratory effects produced by indol, benzene, and other substances already studied. All these effects, both inhibitory and acceleratory, are the result of reactions between the inhibitors or accelerators and those components of the red cell membrane which are broken down by lysins. The inhibitory effect of plasma on saponin hemolysis has a number of properties in common with the inhibition produced by sugars and electrolytes and with accelerations in general. (a) The temperature coefficient is small and negative. (b) The extent of the inhibition depends on the type of red cell used in the hemolytic system. (c) The most satisfactory measure of the extent of the inhibition, the constant R, is a function of the concentration of lysin in the system, and (d) R is a linear function of the quantity of inhibitor present. It is also shown that the inhibitory effect of plasma, and serum is not entirely dependent on its protein content. The process underlying the phenomenon of lysis and its acceleration or inhibition seems to be one in which the lysin reacts with a component or components of the cell membrane in such a way as to break down its semipermeability to hemoglobin, and in which the accelerator or inhibitor also reacts with the same component in such a way as to increase or decrease the effectiveness of the lysin in producing breakdown. The membrane is considered as being an ultrastructure made up of small areas or spots of varying degrees of resistance to breakdown, the resistances being distributed according to a negatively skew type of frequency curve, and the process of lysis seems to begin with the least resistant spots breaking down first. These spots may be arranged in some regular spatial pattern, and the membrane has also to be regarded as possessing spots of varying rigidity of form. The accelerator or inhibitor changes the resistance of every reactive spot in the ultrastructure by a factor R, which suggests that acceleration and inhibition are results of some over-all effect, such as that of changing the extent to which lysin is concentrated at the surface or partitioned between the material of the membrane and the surrounding fluid. Some kind of combination between the accelerator or inhibitor and the material of the ultrastructure is presumably involved; at first the combination seems to be a loose one and partly reversible, but later some of the loose links are replaced by more permanent combinations involving the same types of bond as are broken down by the lysins themselves.     

Factors affecting the hemolytic action of "lysolecithin" upon rabbit erythrocytes

1. Lysolipid was prepared by the action of snake venom on egg yolk, and a study was made of the factors affecting its hemolytic action upon rabbit erythrocytes. 2. Lysis proceeded very rapidly at first, then ceased within a few minutes at room temperature. A given amount of lysin appeared to hemolyze a fixed number of cells, under specified conditions. 3. The more dilute erythrocyte suspensions required relatively more lysin per cell, for 50 per cent hemolysis of the suspension. There may be an equilibrium between the lysin dissolved in the medium and that adsorbed on the cells. 4. The degree of hemolysis for varying lysin concentrations was measured, and the cells showed a typical distribution of resistance to hemolysis. 5. As the temperature was lowered lysis was more extensive. Adsorption of the lysin on the cell surface was apparently increased. 6. The resistance of the erythrocytes to lysis increased slightly as the pH was raised from 5.5 to 7.8. 7. Resistance to lysis was independent of the tonicity of the medium and of initial cell volume. The magnitude of the cell surface was probably the determining factor. 8. A marked shrinkage of the erythrocytes was observed in the presence of calcium ions and lysin, but not in the absence of the lysin. 9. Hemolytic resistance curves obtained by the Wilbrandt technique were of the "colloid-osmotic" type. However, there was no evidence of prolytic loss of potassium ions. 10. Hypotonic fragility of the cells was slightly increased in the presence of the lysin. The rate of penetration of thiourea was greatly increased.     

Comparison of bacterial cardiotoxins: thermostable direct hemolysin from Vibrio parahaemolyticus, streptolysin O and hemolysin from Listeria monocytogenes.

Some properties of the bacterial cardiotoxins, thermostable direct hemolysin from Vibrio parahaemolyticus (vibriolysin), and streptolysin O and hemolysin from Listeria monocytogenes (listeriolysin), were compared. These toxins had rapid lethal effects on mice when injected intravenously. The electrocardiographic changes of rats after intravenous injections of these toxins were very similar, showing bradycardia and inhibition of atrio-ventricular conduction. These toxins also caused cessation of the spontaneous beating and degeneration of cultured foetal mouse heart cells. When equal hemolytic units of these three toxins were administered, vibriolysin had the most potent effects on mice and cultured mouse heart cells. Differences in the kinetics of the hemolysis by each toxin and in the effects of cholesterol of their hemolytic actions suggest that the mode of action of vibriolysin is different from those of streptolysin O and listeriolysin.     

猪源链球菌溶血性的检测

用平板法和上清法检测了19株猪源链球菌的溶血性,并通过加入还原剂或氧化剂分别作活化和抑制试验,对各菌株溶血素的性质做了初步鉴定,其中8株猪链球菌2型菌株在血平板上的溶血性较弱,仅为α溶血或弱β溶血,但在THB和5%血清THB中都可产生很强的溶血性,其溶血性可被还原剂活化,被氧化剂和胆固醇所抑制,卵磷脂对其活性则没有影响,属于巯基活化类（类SLO）溶血素,9株马链球菌兽疫亚种菌株在血平板上呈显的β溶血,在不含血清THB中不能产生溶血性,在含血清的THB中可产生较强的溶血性,其活性不被还原剂活化,不被氧化剂抑制,胆固醇能抑制大部分活性,卵磷脂则可全部抑制,属于类SLS溶血素,两株非典型兰氏C群链球菌菌株在血平板上呈显的β溶血,在THB中显示了非常强的溶血性,其活性不被还原剂活化,不被氧化剂抑制或卵磷脂抑制,但被胆固醇强烈抑制,属于非SLO,非SLS溶血素。     

Immunogenic properties of Clostridium perfringens type A anatheta-hemolysin

Guinea-pigs were immunized with anatheta-hemolysin preparations adsorbed on aluminum hydroxide, as well as with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated. Anatheta hemolysin preparations were obtained with the use of homogeneous theta hemolysin, as well theta hemolysin of various purification degrees. As a result, antatheta hemolytic guinea-pig sera capable of neutralizing 2,000-8,000 HU of theta-hemolysin were obtained. Tests made to establish the degree of protection in the immunized guinea-pigs did not show that the animals immunized with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated, had any advantages in the degree of protection over the animals immunized with the toxoid alone. But there is no doubt that this component plays a positive role under the conditions of natural gas gangrene when the hemolytic action of Cl. perfringens toxin becomes considerably pronounced.     

In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.     

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.