Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract ofP. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37 ℃, and at pH 7.0; Hg2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Biological activity of sea anemone proteins: II. Cytolysis and cell line toxicity

Potent cytolytic activity was exhibited by proteins extracted from three sea anemones viz. Heteractis magnifica, Stichodactyla haddoni and Paracodylactis sinensis by affecting the red blood corpuscles (RBC) and the mouse fibroblast cell line (L929) and leukemia cell line (P388). Crude toxin of all the three anemone species induced spontaneous hemolysis of chicken, goat and human erythrocytes. The crude toxin of H. magnifica (0.98 mg/ml) elicited hemolysis at levels of 4096, 512 and 4096 HU (hemolytic unit) in chicken, goat and human erythrocytes respectively. Subsequently, the crude toxin of S. haddoni (0.82 mg/ml) exhibited a hemolytic activity of 256, 128 and 512 HU and that of P. sinensis (0.60 mg/ml) had a hemolytic activity of 128, 4096 and 512 HU. Most of the partially purified proteins of these anemones also exhibited the activity against the three different erythrocytes. The viability of L929 and P388 was adversely affected on adding the crude toxins. The symptoms of toxicity shown by the cells were rounding, lysis and detachment from the substratum. These effects were the least in S. haddoni, as compared to those the crude toxins of the other two species. Inhibition of growth of L929 exhibited by the toxin of the three species ranged between 61.08 and 93.38%. Similarly, inhibition of the growth of P388 ranged between 51.32 and 86.16%. The present investigation reveal the cytotoxic nature of anemone toxins.     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract of P. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37℃, and at pH 7.0; Hg^2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg^2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.     

Kinetic aspects of the aggregation of Clostridium perfringens theta-toxin on erythrocyte membranes. A fluorescence energy transfer study

Fluorescence resonance energy transfer was used to monitor aggregation kinetics of the "thiol-activated" cytolysin (perfringolysin O (PFO) or theta-toxin) of Clostridium perfringens on erythrocyte membranes. PFO was labeled with the isothiocyanate derivatives of either fluorescein or tetramethylrhodamine. No detectable change in the hemolytic activity of PFO was detected after modification with either fluorophore at a ratio of 1:2 fluorophore molecules/cytolysin molecule. Fluorescence energy transfer (FET) between the donor (fluorescein.PFO or PFOD) and the acceptor (tetramethylrhodamine.PFO or PFOA) was detected by both quenching of donor fluorescence (520 nm) and by enhancement of acceptor fluorescence (575 nm) upon aggregation of labeled cytolysin molecules. FET was only observed when PFOD and PFOA were incubated in the presence of membranes. FET was not observed when PFOD and PFOA were incubated in a membrane-free solution or when unlabeled toxin was substituted for PFOA. FET was also found to be temperature-dependent. The temperature-dependent rates of change in FET upon mixing labeled toxin with erythrocyte membranes proceeded without a lag phase and displayed an activation energy of 18.7 kcal/mol. At all temperatures aggregation of PFO was virtually complete before the onset of hemolysis, the latter exhibiting a distinct lag phase. The lag period before onset of hemolysis was temperature-dependent and exhibited an activation energy of 23.2 kcal/mol. These results suggest that the aggregation of membrane-associated PFO is necessary to initiate the hemolytic process, and the lag phase which occurs before onset of hemolysis reflects the kinetics of PFO monomer to polymer conversion.     

The relationship between calcium and the metabolism of plasma membrane phospholipids in hemolysis induced by brown spider venom phospholipase-D toxin

Brown spider venom phospholipase-D belongs to a family of toxins characterized as potent bioactive agents. These toxins have been involved in numerous aspects of cell pathophysiology including inflammatory response, platelet aggregation, endothelial cell hyperactivation, renal disorders, and hemolysis. The molecular mechanism by which these toxins cause hemolysis is under investigation; literature data have suggested that enzyme catalysis is necessary for the biological activities triggered by the toxin. However, the way by which phospholipase-D activity is directly related with human hemolysis has not been determined. To evaluate how brown spider venom phospholipase-D activity causes hemolysis, we examined the impact of recombinant phospholipase-D on human red blood cells. Using six different purified recombinant phospholipase-D molecules obtained from a cDNA venom gland library, we demonstrated that there is a correlation of hemolytic effect and phospholipase-D activity. Studying recombinant phospholipase-D, a potent hemolytic and phospholipase-D recombinant toxin (LiRecDT1), we determined that the toxin degrades synthetic sphingomyelin (SM), lysophosphatidylcholine (LPC), and lyso-platelet-activating factor. Additionally, we determined that the toxin degrades phospholipids in a detergent extract of human erythrocytes, as well as phospholipids from ghosts of human red blood cells. The products of the degradation of synthetic SM and LPC following recombinant phospholipase-D treatments caused hemolysis of human erythrocytes. This hemolysis, dependent on products of metabolism of phospholipids, is also dependent on calcium ion concentration because the percentage of hemolysis increased with an increase in the dose of calcium in the medium. Recombinant phospholipase-D treatment of human erythrocytes stimulated an influx of calcium into the cells that was detected by a calcium-sensitive fluorescent probe (Fluo-4). This calcium influx was shown to be channel-mediated rather than leak-promoted because the influx was inhibited by L-type calcium channel inhibitors but not by a T-type calcium channel blocker, sodium channel inhibitor or a specific inhibitor of calcium activated potassium channels. Finally, this inhibition of hemolysis following recombinant phospholipase-D treatment occurred in a concentration-dependent manner in the presence of L-type calcium channel blockers such as nifedipine and verapamil. The data provided herein, suggest that the brown spider venom phospholipase-D-induced hemolysis of human erythrocytes is dependent on the metabolism of membrane phospholipids, such as SM and LPC, generating bioactive products that stimulate a calcium influx into red blood cells mediated by the L-type channel.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

A hemolysin of Vibrio mimicus (VMH) stimulates cells to produce ATP and cyclic AMP which appear to be secretory mediators

The hemolysin of Vibrio mimicus(VMH) is a pore-forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP-dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.     

On the interaction of cobra venom protein cardiotoxins with erythrocytes

The principally active hemolytic toxin (cardiotoxin) previously purified from the venom of the Thailand cobra, Naja naja siamensis, was shown to produce spontaneous twitching, contractures and membrane depolarization in sartorius muscles from the frog, Rana pipiens. Spontaneous twitching, observed at concentrations greater than 0.1 uM was completely abolished by addition of tetrodotoxin and not affected by d-tubocurarine. Dose and time dependent membrane depolarization of muscle fibers was observed to occur within 10-30 min at 0.2 to 1.0 uM concentrations of the toxin. These observations, taken together with an amino acid analysis characteristic of previously described cobra venom cardiotoxins, characterized this hemolytic toxin as a cardiotoxin. In the absence of EDTA the initial velocities of erythrocyte hemolysis for this toxin showed a sigmoidal concentration dependence which became hyperbolic in the presence of EDTA. The largest increases in hemolysis rates on addition of 1 mM EDTA were observed at low toxin concentrations. In the presence of EDTA extracellular and membrane associated divalent cations are complexed, thus alleviating their competition with toxin for binding to the membrane, a key and apparently rate-determining initial step which leads to hemolysis. In the presence of EDTA hemolysis rates increased linearly at low toxin concentration and reached an extrapolated maximum value at toxin concentrations at which, given its molecular dimensions, there are just sufficient toxin molecules to cover the entire membrane surface area provided by the erythrocytes.     

Role of the essential thiol group in the thiol-activated cytolysin from Clostridium perfringens

A hemolysin, 0-toxin, produced by Clostridium perfringens has one cysteinyl residue in the free thiol form which is essential for its hemolytic activity. The cysteinyl residue was shown to be located at a position about 5 kDa from the C terminus of the molecule by the method of cysteine-specific chemical cleavage. Modification of the residue with a thiol-blocking agent, 5,5'-dithiobis(2-nitrobenzoic acid), reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding at low cell concentrations (0.5%). Thus the failure of hemolysis at low cell concentrations is primarily ascribed to a decreased affinity of the toxin for erythrocytes. Effects of the modification on the lytic processes were examined using high cell concentrations where considerable amounts of modified toxin bound to the cells. The modified toxin hemolyzes erythrocytes once it binds to them; however, the efficiency of hemolysis is reduced by the modification. These, and additional results indicating that modification alters the sensitivity of toxin molecules to protease digestion, show that thiol-modification inactivates the toxin by affecting both binding and the subsequent lytic processes, probably through a conformational change introduced in the toxin molecules.     

米氏凯伦藻溶血毒素的溶血反应特征

探讨了温度、pH值、二价阳离子等对米氏凯伦藻(Karenia mikimotoi Hasen)溶血毒素溶血活性的影响,分析了米氏凯伦藻溶血毒素的溶血反应特征.结果表明,实验室培养米氏凯伦藻的溶血活性约为64.69±6.43 HU L-1,单个藻细胞的溶血活性为6.17±0.61×10-6 HU;在实验温度(0～37℃)下,溶血活性随温度的增加而增加;pH6.0时的溶血活性最高;Cu2+、Mg2+、Mn2+、Ca2+、Co2+、Zn2+和Hg2+等对米氏凯伦藻的溶血活性的影响不同.离子浓度为5 mmol/L时,Hg2+的抑制作用最强.高浓度Hg2+对红细胞的集合效应不但阻止了Hg2+进入血细胞诱导的溶血作用,而且阻止了毒素对细胞膜的破坏,但这种抑制作用可被EDTA消除.     

Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens

A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin. The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity. C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C. At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin. It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis. The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

Trypsin Action on the Growth of Sendai Virus in Tissue Culture Cells II. Restoration of the Hemolytic Activity of L Cell-Borne Sendai Virus by Trypsin

Sendai virus grown in L cells (L Sendai) caused little hemolysis, whereas the one grown in fertile eggs (egg Sendai) induced distinct hemolysis. Enzymatic treatment with trypsin at low concentrations markedly enhanced the hemolytic activity of L Sendai but not that of egg Sendai. Both sonic treatment and freezing and thawing greatly enhanced the hemolytic activity of egg Sendai, but they gave little enhancing effect on that of L Sendai which could, however, be greatly increased by successive treatment with trypsin. Dose response and kinetic experiments on the trypsin effect have suggested that a similarity exists in the inhibitory mechanism of infectivity for L cells and hemolytic activity of L Sendai. Treatment of L cells with trypsin at later stages of infection released a highly hemolytic L Sendai from those cells. The present study, by reference to the density centrifugation studies in a previous report (4), has shown that a variation in infectivity for L cells and in the hemolytic activity of L Sendai is a type of host-controlled modification distinguishable from the density variation.     

Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.     

Role in hemolysis of the interaction of tellurium compounds with glutathione

We have shown that tellurite and tellurate require the interaction with reduced glutathione (GSH) to hemolyze human erythrocytes. The study of the nature of this interaction is the main object of this paper. The degree of hemolysis was determined by the method of Angelone. The addition of extracellular 1 mM GSH or cysteine increased the rate of hemolysis. Concanavalin A (0.3 mg/mL) and/or 4 mg/mL adenosine did not affect the hemolysis by 0.1 mM tellurite. One tenth to 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS) inhibited this hemolysis by 60–100%. Millimolar GSH released this inhibition. Incubation of 0.1 mM tellurite with 1 mM GSH for 90 min at 37°C, produced a hemolytic agent when prepared and tested under nitrogen, but one that was not active when prepared in air. The hemolysis byp-hydroxymercuribenzoate orp-hydroxymercuriphenylsulfonate did not involve GSH. Scanning electron micrographs showed a sphero-echinocyte transformation, in the pre-hemolytic stage, with all the agents tested. The rate of penetration of tellurite plays a role in determining the rate of hemolysis, as shown by the effect of SITS. The release by GSH of the inhibition by SITS poses questions concerning the site of action and cell membrane penetration of the hemolytic agent. Telluride or some intermediate in the interaction of GSH with tellurite is the actual hemolytic agent.     

Mutational and comparative analysis of streptolysin O, an oxygen-labile streptococcal hemolysin

The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.     

The mixture of aldehydes and hydrogen peroxide produced in the ozonation of dioleoyl phosphatidylcholine causes hemolysis of human red blood cells

Dioleoyl phosphatidylcholine (PC) liposomes were ozonized and the ozonized liposomes were tested for their lytic potency on human red blood cells (RBC). Ozonation of PC liposomes generated approximately 1 mole equivalent of hydrogen peroxide (H2O2) and 2 mole equivalents of aldehydes, based on the moles of ozone consumed. The time necessary for 50% hemolysis induced by ozonized liposomes (a convenient measure of hemolytic activity) was found to depend on the extent of ozonation of the PC liposomes, indicating the formation and accumulation of hemolytic agents during ozonation. Hemolysis was also observed when RBC were incubated with nonanal, the expected product of the ozonation of oleic acid, the principle unsaturated fatty acid in the liposomes. Hydrogen peroxide, another product of PC ozonation, did not induce hemolysis; however, a combination of H2O2 and nonanal was significantly more hemolytic than nonanal alone. A ratio of 1:2 H2O2/nonanal (the ratio observed in the ozonized liposomes) provided hemolytic activity comparable to that observed with ozonized dioleoyl PC. Among different antioxidants tested, ascorbate, catalase, and glutathione peroxidase partially inhibited hemolysis induced by ozonized liposomes and by H2O2/nonanal mixtures, but they were not protective against the nonanal-induced hemolysis. Identification of H2O2 and aldehydes as cytotoxic chemical species generated from the ozonation of unsaturated fatty acids may have an important bearing on the in vivo toxicity of ozone on the lung as well as on extrapulmonary tissues.