sodium-dodecyl-sulfate and Hemolysis

It has been argued that the mol/cell metric is more universal than concentration of the toxic agent since in many cases the effect of dose expressed as mol/cell is independent of ex-perimental setup. We confirmed it for hemolysis of erythrocytes in phosphate-buffered saline induced by hypochlorite where the amount of femtomoles/cell of hypochlorite needed for 50% hemolysis was independent of erythrocyte concentration. However, in the presence of blood plasma this metric became dependent on cell concentration. Similarly, the effect of 3-bromopyruvic acid (3-BP) on PEO1 cells as a function of mol/cell ratio depended on the volume of the 3-BP containing medium, due to the reaction of 3-BP with components of the medium. Hemolytic amounts of sodium dodecyl sulfate and Triton X-100 expressed as mol/cell decreased with increasing cell concentration while the effect of DMSO on the viability of a constant number of fibroblasts was independent of the volume of DMSO-containing medium. These results demonstrate that the mol/cell metric is still dependent on experimental conditions when the toxic agent interacts with components of the medium or when its physical state is modified by the target cells, and the effect is independent of the mol/per cell ratio for high excess of a cell damaging agent.

Topics: Cell Line; Cell Line, Tumor; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Erythrocytes; Fibroblasts; Hemolysis; Humans; Hypochlorous Acid; Octoxynol; Pyruvates; Sodium Dodecyl Sulfate

For miltefosine (MIL), a zwitterionic alkylphospholipid approved for leishmaniasis treatment, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy has indicated that the interaction of MIL with membrane proteins has similarities to that of ionic surfactants. A general concern about leishmanicides is their high hemolytic potential, so we decided to compare the interactions of MIL and three ionic surfactants with the erythrocyte membrane. Measurements with two different spin labels indicated that the surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) as well as MIL increase the dynamics of erythrocyte membrane proteins in a concentration-dependent manner. SDS produced the smallest increases in protein dynamics and was also the least hemolytic for measurements in PBS and in whole blood. Spin label EPR measurements performed directly in the blood plasma detected increased albumin stiffness caused by 2.5 mM SDS due to electrostatic/hydrophobic interactions. For 10 mM concentrations of the compounds, the EPR spectra showed a fraction of albumin with greater mobility and another with the same as that of the untreated plasma. The zwitterionic compounds MIL and HPS did not present significant differences in this study.

Topics: Animals; Antiprotozoal Agents; Bis-Trimethylammonium Compounds; Cattle; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Membrane Proteins; Micelles; Phosphorylcholine; Quaternary Ammonium Compounds; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Spin Labels; Static Electricity

Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.

Topics: Antimicrobial Cationic Peptides; Cell Membrane; Circular Dichroism; Deuterium Oxide; Hemolysis; Lipid Bilayers; Liposomes; Micelles; Microbial Sensitivity Tests; Protein Conformation; Sodium Dodecyl Sulfate; Solutions

Toxicity is one of the main concerns limiting the use of surfactants. Many efforts have been devoted to the development of new amphiphilic molecules characterized by a lower toxicological profile and environmental impact. N-acyl amino acids are a class of anionic surfactants that can find applications in different technological fields as an alternative to sulphate-based surfactants (e.g., sodium dodecyl sulphate). The understanding of the relationship between chemical structure and toxicological profile is fundamental for the disclosure of the full potential of these amphiphiles. With this aim, two series of N-acyl surfactants, with different length of the hydrophobic tails and serine or alanine as polar head, were synthesized and fully characterized. The correlation between the surface and toxicological parameters allowed highlighting the role exerted by the length of the hydrocarbon chain and the polar head on cytotoxicity. The length of the hydrocarbon chain mainly influences surface properties and toxicological parameters, while the amino acid polar head may play a key role only on cellular toxicity. Overall, our data suggest that minor differences in the polar head, not significantly affecting CMC values, may have an impact on cytotoxicity.

Topics: Alanine; Amino Acids; Caco-2 Cells; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Design; Electron Spin Resonance Spectroscopy; Gases; Hemolysis; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; L-Lactate Dehydrogenase; Light; Magnetic Resonance Spectroscopy; Micelles; Scattering, Radiation; Serine; Sodium Dodecyl Sulfate; Solvents; Surface Properties; Surface-Active Agents; Temperature

To evaluate the influence of age on the relationships between biochemical and hematological variables and stability of erythrocyte membrane in relation to the sodium dodecyl sulfate (SDS) in population of 105 female volunteers between 20 and 90 years.. The stability of RBC membrane was determined by non-linear regression of the dependency of the absorbance of hemoglobin released as a function of SDS concentration, represented by the half-transition point of the curve (D50) and the variation in the concentration of the detergent to promote lysis (dD).. There was an age-dependent increase in the membrane stability in relation to SDS. Analyses by multiple linear regression showed that this stability increase is significantly related to the hematological variable red cell distribution width (RDW) and the biochemical variables blood albumin and cholesterol.. The positive association between erythrocyte stability and RDW may reflect one possible mechanism involved in the clinical meaning of this hematological index.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Aging; Blood Glucose; Dose-Response Relationship, Drug; Erythrocyte Indices; Erythrocyte Membrane; Erythrocytes; Female; Hematocrit; Hemoglobins; Hemolysis; Humans; Linear Models; Lipids; Middle Aged; Multivariate Analysis; Serum Albumin; Sodium Dodecyl Sulfate; Spectrophotometry; Surface-Active Agents; Young Adult

Shuttle-drug: Self-assembled vesicles of a pharmaceutically active ionic liquid are shown to be an efficient drug delivery system, which realizes the controlled release of its pharmaceutically active component directly.

Topics: Administration, Oral; Amitriptyline; Animals; Drug Carriers; Erythrocytes; Half-Life; Hemolysis; Ionic Liquids; Rabbits; Sodium Dodecyl Sulfate; Surface Tension

Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amino Acid Sequence; Amino Acid Substitution; Animals; Anti-Bacterial Agents; Calorimetry, Differential Scanning; Cattle; Erythrocytes; Escherichia coli; Hemolysis; Humans; Lactoferrin; Micelles; Molecular Sequence Data; Peptide Fragments; Phosphatidylglycerols; Protein Binding; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Streptococcus sanguis; Thermodynamics; Unilamellar Liposomes

Attenuated total reflectance mid-infrared spectra of serum and blood samples were obtained from 4,000 to 600 cm(-1). Models for the determination of albumin, immunoglobulin, total globulin, and albumin/globulin coefficients were established for serum samples, using reference data obtained by capillary electrophoresis. Based on the use of the amide bands I and II regions, the relative root mean square error of prediction (RRMSEP) was 4.9, 14.9, 4.5, and 7.1% for albumin, immunoglobulin, total globulin, and albumin/globulin coefficients, respectively, determined in an independent validation set of 120 samples using 200 samples for calibration. Additionally, the use of Kennard-Stone method for the selection of a representative calibration subset of samples provided comparable results using only 60 samples. For whole blood analysis, hemoglobin was determined in 40 validation samples using models built from 40 calibration independent samples with RRMSEP of 8.3, 5.5, and 4.9% with models built from direct spectra in the first case and from sample spectra recorded after lysis by sodium dodecyl sulfate and freezing, respectively, for the last two ones. The developed methodologies offer green alternatives for patient diagnosis in a few minutes, minimizing the use of reagents and residues and being adaptable for its use as a point-of-care method.

Topics: Calibration; Electrophoresis, Capillary; Erythrocytes; Green Chemistry Technology; Hemoglobins; Hemolysis; Humans; Immunoglobulins; Point-of-Care Systems; Serum Albumin; Sodium Dodecyl Sulfate; Spectroscopy, Fourier Transform Infrared

LL37 and histatin 5 are antimicrobial peptides. LL37 exhibits killing activity against a broad spectrum of pathogens, whereas histatin 5 is primarily an antifungal agent. Head-to-tail cyclization of histatin 5 did not affect its antimicrobial and haemolytic activity. The cyclic LL37 exhibits identical antifungal and haemolytic activity as does LL37. Its antimicrobial activity varied in one dilution depending on the kind of bacteria. The structure of cyclic peptides was studied by circular dichroism spectroscopy. Both peptides undergo a conformational change leading to stabilisation of their α-helical structure in the presence of negatively charged sodium dodecyl sulfate micelles. However, with cyclic histatin 5, the presence of Zn(2+) ions is also necessary to fuse the peptide to the micelle. The specific action of the Zn(2+) ions is attributed to the presence of a zinc-binding motif, His-Glu-X-X-His. It has been speculated that this zinc complexing may be related to the well-established anticandidal activity. In the case of cyclic LL37, also the presence of a zwitterionic dodecylphosphocholine micelle induces formation of the helical structure. A microwave-assisted procedure for the cleavage of a peptide from the 2-chlorotrityl chloride resin was, for the first time, successfully used to obtain protected peptide fragments that can be applied to the preparation of head-to-tail cyclopeptides or to condensation of peptidic fragments.

Topics: Antifungal Agents; Antimicrobial Cationic Peptides; Candida; Cathelicidins; Circular Dichroism; Hemolysis; Histatins; Humans; Micelles; Microwaves; Peptides, Cyclic; Sodium Dodecyl Sulfate

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH(2)), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria. The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu(2) and Val(21). Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys(3) and Val(21). Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala(19) do not interact with the micelles.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Chlorides; Cyclic N-Oxides; Hemolysis; Humans; Lysine; Manganese Compounds; Membrane Lipids; Membranes, Artificial; Micelles; Microbial Sensitivity Tests; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phospholipid Ethers; Phosphorylcholine; Protein Conformation; Sodium Dodecyl Sulfate; Solvents; Spin Labels; Structure-Activity Relationship; Trifluoroethanol; Valine; Water

Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.

Topics: Antifungal Agents; Circular Dichroism; Hemolysis; Hydrogen Bonding; Ion Channels; Lipids; Magnetic Resonance Spectroscopy; Micelles; Molecular Dynamics Simulation; Peptides, Cyclic; Phospholipids; Protein Conformation; Rifampin; Sodium Dodecyl Sulfate; Water

The stability of human erythrocytes to sodium dodecyl sulfate (SDS) was assessed spectrophotometrically in the presence of different concentrations of bovine serum albumin (BSA) and at different temperatures (27-45 °C). The absorbance at 540 nm (A₅₄₀) was correlated with the SDS concentration by sigmoidal regression based on the Boltzmann equation. Erythrocyte stability was characterized on the basis of the SDS concentration that induces hemolysis in 50% of the cells (D₅₀). Progressive increases in the albumin concentration led to increases in the D₅₀ value. The protective effect of BSA against SDS-induced hemolysis was attributed to the binding of the surfactant to the hydrophobic binding sites of this protein. The D₅₀ values decreased sigmoidally with an increase in the temperature. This trend, which could not be explained by changes in the spectral properties of hemoglobin, maybe due to heterogeneity in the erythrocyte population.

Topics: Animals; Cattle; Erythrocytes; Hemolysis; Humans; Regression Analysis; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet; Temperature

Membrane lytic peptides are a novel class of anticancer agents that have the potential to overcome drug resistance. The limited selectivity against cancer cells, however, presents a major hurdle for the application. We aim to exploit the proteolytic activity of tumor-associated matrix metalloproteinases (MMP) to mediate the cytotoxicity of these peptides. We designed a membrane lytic peptide cyclized with a linker cleavable by membrane type 1-MMP (MT1-MMP). We showed that the cyclic peptide could be restored to the linear state on MT1-MMP digestion, and it preferentially killed MMP-overexpressing cells above a threshold concentration. Circular dichroism indicated that cyclization resulted in a more rigid structure, making it more difficult for the lytic peptide to transit from random coil to alpha-helix in a membrane-mimicking environment. Selective membrane activity of the cyclic peptide was shown by comparing cytotoxicity results on RBC and two human breast cancer cell lines of different malignancy and MT1-MMP expression: highly invasive MDA-MB-435 and noninvasive MCF-7. Above a concentration of 5 micromol/L, suppressed activity to MCF-7 and RBC was observed, whereas the toxicity against MDA-MB-435 was maintained. MMP inhibition experiments further showed that the membrane-lysing activity was enzyme dependent.

Topics: Amino Acid Sequence; Animals; Cell Death; Cell Line, Tumor; Cell Membrane; Cell Shape; Drug Screening Assays, Antitumor; Hemolysis; Humans; Matrix Metalloproteinase 14; Molecular Sequence Data; Peptides, Cyclic; Protein Structure, Secondary; Rabbits; Sodium Dodecyl Sulfate; Water

In this work, the naturally occurring antimicrobial peptides temporin A (TA) and L (TL) are studied by spectroscopic (CD and NMR) techniques and molecular dynamics simulation. We analyzed the interactions of TA and TL with sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles, which mimic bacterial and mammalian membranes, respectively. In SDS, the peptides prefer a location at the micelle-water interface; in DPC, they prefer a location perpendicular to the micelle surface, with the N-terminus imbedded in the hydrophobic core. TL shows higher propensity, with respect to TA, in forming alpha-helical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles. Hence, we have proposed a different molecular mechanism underlying the antimicrobial and hemolytic activities of the two peptides. We also designed new analogues of TA and TL and found interesting differences in their efficacy against microbial species and human erythrocytes.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Erythrocytes; Hemolysis; Humans; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Models, Molecular; Phosphorylcholine; Proteins; Sodium Dodecyl Sulfate; Spectrometry, Mass, Fast Atom Bombardment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The effect of the presence of charged amphiphiles during the incubation of human erythrocytes in a sucrose-substituted low-Cl(-) solution on the shift of the osmotic resistance profile and the net K+ efflux was investigated. Osmotic fragility was determined by fitting the complementary error function to the haemolysis resistance curve. K+ efflux was calculated from the increase in the K+ concentration in supernatant measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). The cationic amphiphile hexadecyltrimethylammonium bromide (CTAB) at 14 microM decreases, whereas the anionic amphiphile sodium dodecyl sulfate (SDS) at 50 microM increases the shift of the haemolysis resistance curve of erythrocytes incubated in isotonic sucrose by 0.069 and 0.079 %NaCl, respectively. Both the positively and the negatively charged amphiphile caused a significant change in the K+ efflux into isotonic sucrose solution: CTAB decreased and SDS increased K+ efflux by about 40%. In view of the lack of effect of the investigated compounds on the haemolysis resistance curve and K+ efflux from human erythrocytes incubated in isotonic NaCl solution, these results suggest that the insertion of charged amphiphiles into the erythrocyte membrane modulates the properties of the K+ transport pathway which is activated under low ionic strength (LIS) conditions.

Topics: Cetrimonium; Cetrimonium Compounds; Chlorides; Erythrocytes; Hemolysis; Humans; Incubators; Osmolar Concentration; Osmotic Fragility; Potassium; Sodium Dodecyl Sulfate; Solutions; Sucrose

In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.

Topics: Base Sequence; Caco-2 Cells; Calorimetry; Cations; Hemolysis; Humans; Interleukin-2; Oligonucleotides; Peptides; T-Lymphocytes

Surfactants represent one of the most common constituents in topical pharmaceutical and cosmetic applications or cleansers. Since adverse skin and ocular reactions can be caused by them, it is important to evaluate damaging effects. Amino acid-based surfactants deserve particular attention because of their low toxicity and environmental friendly properties. New lysine derivative surfactants associated with heavy and light counterions were tested. The ocular irritancy was assessed by hemolysis, and photohemolysis was employed to evaluate their phototoxicity. Cytotoxicity on HaCaT cells was determined by neutral red uptake and MTT assay to predict skin irritation. All lysine derivative surfactants were less hemolytic and thus less eye-irritating than the commercial surfactants used as model irritants. No phototoxic effects were found. All surfactants presented cytotoxic effects as demonstrated by decrease of neutral red uptake and reduction of MTT salt, with clear concentration-effect profiles. However, the rates of cytotoxicity on HaCaT for the new surfactants suggested that they were less cytotoxic and then, less skin-irritating than the reference ones; surfactants with heavy counterions were the less cytotoxic. The anionic surfactants investigated in the present work may constitute a promising class of surfactants given their low irritancy potential for pharmaceutical and cosmetic preparations.

Topics: Betaine; Cell Line; Cell Survival; Cetrimonium; Cetrimonium Compounds; Colorimetry; Erythrocytes; Hemolysis; Humans; Inhibitory Concentration 50; Irritants; Lysine; Molecular Structure; Neutral Red; Sodium Dodecyl Sulfate; Surface-Active Agents; Tetrazolium Salts; Thiazoles

The results of a study of vesicular-hemolytic action of anionic detergent sodium dodecyl sulfate on human erythrocytes are presented. Possible molecular events underlying detergent-induced pore formation in the erythrocyte membrane are discussed.

Topics: Detergents; Dose-Response Relationship, Drug; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Kinetics; Sodium Dodecyl Sulfate

The antioxidant effect of strictinin (SOH), which was extracted from green tea leaves, against the peroxidation of linoleic acid in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium (CTAB) micelles, against the peroxidation of low-density lipoprotein (LDL) and against oxidative hemolysis of human red blood cells (RBCs), has been studied. The peroxidation of linoleic acid and LDL, and oxidative hemolysis of RBCs were initiated thermally by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), and the reaction kinetics in micelles and LDL were monitored by uptake of oxygen. The synergistic antioxidant effect of SOH with alpha-tocopherol (Vitamin E) was also studied by following the decay kinetics of alpha-tocopherol. Kinetic analysis of the antioxidation process demonstrates that SOH, used either alone or in combination with alpha-tocopherol, is an effective antioxidant against lipid peroxidation, but its effects significantly depend on the reaction medium.

Topics: alpha-Tocopherol; Antioxidants; Cetrimonium Compounds; Erythrocytes; Hemolysis; Humans; Kinetics; Linoleic Acid; Lipid Peroxidation; Lipoproteins, LDL; Micelles; Oxygen Consumption; Phenols; Quaternary Ammonium Compounds; Sodium Dodecyl Sulfate; Tea; Water

The alpha-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin isolated from the marine snail Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype alpha3beta2. To improve the bioavailability of this peptide, two lipidic analogues of MII have been synthesized, the first by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII) and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII). Both lipidic linear peptides were then oxidized under standard conditions. (1)H NMR shift analysis of these peptides and comparison with the native MII peptide showed that the tertiary structure of the N-conjugated analogue, LaaMII, was consistent with that of the native conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit nAChRs of the subtype alpha3beta2 with equal potency to the parent peptide, whereas the 5LaaMII analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII, and both peptides showed negligible toxicity.

Topics: Amino Acid Sequence; Animals; Caco-2 Cells; Conotoxins; Ganglia, Parasympathetic; Hemolysis; Humans; In Vitro Techniques; Lauric Acids; Magnetic Resonance Spectroscopy; Male; Molecular Sequence Data; Neurons; Nicotinic Antagonists; Patch-Clamp Techniques; Peptides, Cyclic; Permeability; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Solubility; Structure-Activity Relationship

The effect of sodium dodecyl sulfate (SDS) upon the conformation and hemolytic activity of St I and St II strongly depends on its concentration. At relatively low surfactant concentrations (ca. 0.5-5mM range) the surfactant leads to the formation of aggregates, as suggested by the turbidity observed even at relatively low (micromolar range) protein concentrations. In this surfactant range, the proteins show an increase in intrinsic fluorescence intensity and reduced quenching by acrylamide, with an almost total loss of its hemolytic activity. At higher surfactant concentrations the protein adducts disaggregates. This produces a decrease in fluorescence intensity, increase in quenching efficiency by acrylamide, loss of the native tertiary conformation (as reported by the near UV-CD spectra), and increase in alpha-helix content (as evidenced by the far UV-CD spectra). However, and in spite of these substantial changes, the toxins partially recover their hemolytic activity. The reasons for this recovering of the activity at high surfactant concentrations is discussed.

Topics: Animals; Circular Dichroism; Cnidarian Venoms; Dose-Response Relationship, Drug; Erythrocytes; Hemolysin Proteins; Hemolysis; Humans; Organic Chemicals; Protein Conformation; Sea Anemones; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence; Surface-Active Agents

Cell surface glycans present docking sites to endogenous lectins. With growing insight into the diversity of lectin families it becomes important to answer the question on the activity profiles of individual family members. Focusing on galectins (beta-galactoside-binding proteins without Ca(2+)-requirement sharing the jelly-roll-like folding pattern), this study was performed to assess the potency of proto-type galectins (galectins-1 and -7 and CG-16) and the chimera-type galectin-3 to elicit selected cell responses by carbohydrate-dependent surface binding and compare the results. The galectins, except for galectin-1, were found to enhance detergent (SDS)-induced hemolysis of human erythrocytes to different degrees. Their ability to confer increased membrane osmofragility thus differs. Aggregation of neutrophils, thymocytes and platelets was induced by the proto-type galectin-1 but not -7, by CG-16 and also galectin-3. Cell-type-specific quantitative differences and the importance of the fine-specificity of the galectin were clearly apparent. In order to detect cellular responses based on galectin binding and bridging of cells the formation of haptenic-sugar-resistant (HSR) intercellular contacts (an indicator of post-binding signaling) was monitored. It was elicited by CG-16 and galectin-1 but not galectin-3, revealing another level at which activities of individual galectins can differ. Acting as potent elicitor of neutrophil aggregation, CG-16-dependent post-binding effects were further analyzed. Carbohydrate-dependent binding to the neutrophils' surface led to a sustained increase of cytoplasmic Ca2+ concentration in a dose-dependent manner. The ability of CG-16 to activate H2O2 generation by human peripheral blood neutrophils was primed by the Ca(2+)-ionophor ionomycin and by cytochalasin B. In a general context, these results emphasize that--besides plant lectins as laboratory tools--animal lectins can trigger cell reaction cascades, implying potential in vivo relevance for the measured activities. Within the family of galectins, the activity profiles depend on the target cell type and the individual galectin. Notably, proto-type galectins do not necessarily share a uniform capacity as elicitor.

Topics: Animals; Blood Platelets; Calcium; Carbohydrate Metabolism; Carbohydrates; Cell Membrane; Dose-Response Relationship, Drug; Erythrocytes; Galectins; Hemolysis; Humans; Hydrogen Peroxide; Lectins; Neutrophils; Protein Binding; Rats; Signal Transduction; Sodium Dodecyl Sulfate; Thymus Gland; Time Factors

Spinigerin is a linear antibacterial peptide derived from a termite insect. It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria, but has no hemolytic activities against human erythrocytes. In this study, we present a three-dimensional solution structure of spinigerin in SDS micelles. According to CD data spinigerin has an alpha-helical conformation in the presence of TFE, DPC micelles, and SDS micelles. The three-dimensional structure of spinigerin as determined by NMR spectroscopy contains a stable alpha-helix from Lys4 to Thr23. Spinigerin (4-21), an 18-residue fragment from Lys4 to Leu21, contains a similar content of alpha-helical structure compared to native spinigerin and was found to retain antibacterial activity, too. Therefore, this alpha-helical structure and the strong electrostatic attraction between four Lys and three Arg residues in spinigerin and the negatively charged polar head groups of the phospholipids on the membrane surface play important roles in disrupting membrane and subsequent cell death.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Hemolysis; Humans; Isoptera; Liposomes; Micelles; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptides; Phospholipids; Protein Conformation; Sodium Dodecyl Sulfate; Structure-Activity Relationship

Surface tension measurements were employed to monitor the erythrocyte hemolysis process induced by surfactants. Two types of surfactants were used: the cationic surfactant DTAB and the anionic surfactant SDS. During DTAB-induced hemolysis, the changes in surface tension clearly demonstrate three stages. The first stage is characterized by surface tension increase, which is explained by surfactant removal from the suspending solution, due to adsorption onto cell membranes. In the second stage, surface tension remains constant, implying that equilibrium is attained between the membrane-bound surfactant and the surfactant in solution. The third stage is characterized by surface tension decrease that begins simultaneously with measurable cell-interior release, and lasts until hemolysis is completed. With SDS-induced hemolysis, the same three stages are observed at a low concentration; however, fluctuational increase in surface tension is obtained for higher concentrations. The latter is explained by additional adsorption of surfactant to solubilized membrane fragments.

Topics: Hemolysis; Humans; Sodium Dodecyl Sulfate; Surface Tension; Surface-Active Agents

The effect of ionic strength, solute size, and osmolarity of the suspending solution on surfactant-induced erythrocyte hemolysis was studied. Two possible mechanisms of hemolysis were considered: osmotic lysis (affected by solute particle size) and solubilization (not affected by solute particle size). It was found that the ionic strength of the solution has a major effect on the hemolysis process, depending on the surfactant nature and concentration. An increase in the ionic strength lowers the rate of hemolysis induced by DTAB, and enhances SDS-induced hemolysis. Changes in ionic strength have little effect on hemolysis induced by Triton X-100. To explain these effects, it was assumed that the changes in ionic strength differently affect the adsorption of cationic and anionic surfactants to the membrane. The change in the amount of adsorbed surfactant either influences the rate of osmotic hemolysis by changing the membrane permeability or induces a transition from the osmotic mechanism to solubilization. These phenomena were observed for isotonic as well as hypertonic solutions.

Topics: Erythrocytes; Hemolysis; Membrane Fluidity; Octoxynol; Osmolar Concentration; Particle Size; Quaternary Ammonium Compounds; Sodium Dodecyl Sulfate; Solubility; Solutions; Structure-Activity Relationship; Surface-Active Agents

The influence of pH of the medium on the parameters of detergent-induced fast hemolysis and vesiculation of human erythrocytes was studied. In the range of pH 6.3-7.2 neither the extent nor the rate of the vesiculation induced by 25 microM sodium dodecyl sulfate (SDS) changed. However, a decrease of pH from 8.0 to 5.8 strongly modified both the extent and the rate of the hemolysis induced by SDS. Within the range of pH 8.0-6.4, the effect can be ascribed to the increase of the positive charge of the membrane. This could lead to the accumulation of the membrane-bound anion detergent and, hence, to the change of the hemolysis parameters. Non-charged detergent Triton X-100 did not display any pH-dependence. At pH between 6.4 and 5.8 the extent and rate of hemolysis changed in a complicated manner. The kinetic curves of hemolysis could be approximated by a single exponential within the pH range between 8.0 and 7.2. Upon further reduction of pH, a second exponential component, with a larger time constant, appeared in the kinetic curves. At 5.8 < pH < 7.2, the contribution of the "fast" hemolysis dropped virtually to zero, with pK about 6.0. This points to a structural transition of the membrane, possibly involving histidine. We suggest that the parameters of the detergent-induced hemolysis are sensitive to the changes of the charge and structural state of erythrocyte membrane.

Topics: Acetylcholinesterase; Detergents; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Mathematics; Octoxynol; Phosphorus; Sodium Dodecyl Sulfate; Spectrophotometry; Surface-Active Agents

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s).

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; Base Sequence; Detergents; DNA-Binding Proteins; DNA, Bacterial; Drug Resistance; Genes, Bacterial; Hemagglutination; Hemolysis; Lactams; Molecular Sequence Data; Mutagenesis; Sequence Homology, Amino Acid; Sodium Dodecyl Sulfate; Transcription Factors; Vibrio; Virulence

The kinetic and concentration dependences of erythrocyte vesiculation and hemolysis induced by sodium dodecyl sulfate were studied. The similarity of the slopes of the dose dependence of the SDS-induced vesiculation and slow hemolysis rates in the double logarithmic coordinates suggested a close relation between the processes of vesiculation and pore formation for slow hemolysis by the detergent. Further evidence of the competitive nature of the detergent-induced vesiculation and fast hemolysis by sodium dodecyl sulfate was obtained. The phenomenon of partial hemolysis proceeding at a rate comparable to that of cell vesiculation is explained in terms of the competition between hemolysis and vesiculation, without resorting to erythrocyte heterogeneity. New vesicular-competitive hemolysis is described. Based on it, the action of different hemolysis-inducing agents is analysed.

Topics: Detergents; Dose-Response Relationship, Drug; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Kinetics; Sodium Dodecyl Sulfate

The complement system is an essential part of the innate defense, and C3 is an integral part of this powerful system. In previously identified complement C3 deficient guinea pigs only approx. 5% of the normal serum C3 level is detectable. No differences were found between in vitro C3 protein synthesis and C3 mRNA levels of cells from C3-deficient and wild-type animals and the amino acid sequences of both C3 proteins are identical as deduced from cDNA sequencing. Previously, the principal inability to form a C3 thiolester was discussed as a possible reason for this C3-deficiency. Here we report the isolation of two functionally different C3 species from the C3-deficient animals. Only one of these C3 proteins exhibits normal hemolytic activity and contains a thiolester group. The second C3 species is exclusively present in C3-deficient animals and lacks a thiolester, explaining its failure to express hemolytic activity. The presence of a second C3 species lacking a thiolester structure only in C3-deficient animals indicates that the stability of the thiolester may play a role in C3 deficiency. However further analysis of the in vitro stability of the thiolesters of C3 from normal and C3-deficient guinea pigs revealed no differences. A decreased in vivo thiolester stability might lead to the presence of C3 with and without a thiolester or alternatively the expression of two isoforms of C3 in these animals. Considering the central role of C3 in host defense, the mechanisms of C3 thiolester formation require further analysis.

Topics: Animals; Biotin; Blotting, Western; Carbohydrates; Carbon Radioisotopes; Chemical Fractionation; Complement C3; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hemolysis; Isotope Labeling; Methylamines; Mice; Mice, Knockout; Protein Isoforms; Sodium Dodecyl Sulfate; Sulfhydryl Compounds

Polyoxyethylene alkyl ether surfactants have been shown to have excellent penetration enhancing abilities although they are associated with a high level of local toxicity. We have compared the toxicity of a range of polyoxyethylene alkyl ethers (Brij 96, Brij 76, Brij 56, 10 lauryl ether and 9 lauryl ether) to an anionic surfactant (sodium dodecyl sulphate (SDS)), an ampholytic surfactant (lysophosphatidylcholine) and a cationic surfactant (tetradecyltrimethylammonium bromide (TTAB)), in the presence and absence of egg phosphatidylcholine. The toxicity of the surfactants or phospholipid/surfactant mixtures was assessed by measuring haemolytic activity. The test samples were incubated with a suspension of red blood cells for 30 min and Drabkin's reagent was used to indicate the amount of haemoglobin released. All of the polyoxyethylene alkyl ethers, SDS, TTAB and lysophosphatidylcholine exhibited haemolytic activity at concentrations between 0.10 and 0.25 mM. The addition of egg phosphatidylcholine reduced the toxicity for all of the surfactants, with the toxicity of Brij 96 being mitigated to a greater extent than the toxicity of the other polyoxyethylene surfactants examined. The rate of haemolysis induced by Brij 96 or 10 lauryl ether was also reduced by increasing concentrations of phosphatidylcholine. As the phosphatidylcholine content of a mixed surfactant system comprising egg phosphatidylcholine: Brij 96 was replaced by lysophosphatidylcholine and fatty acid, the haemolytic action of the mixture increased markedly. The results from this study show that the toxicity of surfactants to erythrocytes can be mitigated by the addition of egg phosphatidylcholine. Synthetic surfactants combined with phosphatidylcholine may generate drug delivery systems worthy of more extensive investigation.

Topics: Drug Delivery Systems; Eggs; Erythrocytes; Female; Hemolysis; Humans; Phosphatidylcholines; Plant Oils; Polyethylene Glycols; Sodium Dodecyl Sulfate; Statistics, Nonparametric; Surface-Active Agents

The incorporation of a reduced amide bond, psi(CH(2)NH), into peptide results in an increase in the net positive charge and the perturbation of alpha-helical structure. By using this characteristic of the reduced amide bond, we designed and synthesized novel pseudopeptides containing reduced amide bonds, which had a great selectivity between bacterial and mammalian cells. A structure-activity relationship study on pseudopeptides indicated that the decrease in alpha-helicity and the increase in net positive charge in the backbone, caused by the incorporation of a reduced amide bond into the peptide, both contributed to an improvement in the selectivity between lipid membranes with various surface charges. However, activity results in vitro indicated that a perturbation of alpha-helical structure rather than an increase in net positive charge in the backbone is more important in the selectivity between bacterial and mammalian cells. The present result revealed that the backbone of membrane-active peptides were important not only in maintaining the secondary structure for the interactions with lipid membranes but also in direct interactions with lipid membranes. The present study showed the unique function of a reduced amide bond in cytolytic peptides and a direction for developing novel anti-bacterial agents from cytolytic peptides that act on the lipid membrane of micro-organisms.

Topics: Amides; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Bacteria; Candida albicans; Cell Membrane; Chromatography, High Pressure Liquid; Circular Dichroism; Coloring Agents; Erythrocytes; Fluoresceins; Hemolysis; Liposomes; Mice; Microbial Sensitivity Tests; Naphthalenes; Oxidation-Reduction; Peptides; Phospholipids; Protein Structure, Secondary; Pyridinium Compounds; Sodium Dodecyl Sulfate; Static Electricity; Structure-Activity Relationship; Substrate Specificity; Trifluoroethanol

Mammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein sigma3 and contain protein mu1/mu1C as endoprotease-generated fragments mu1delta/delta and phi. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein mu1 in both these steps. To determine whether the cleavage of mu1/mu1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked sigma3 yet retained mu1/mu1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of mu1/mu1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of mu1/mu1C to mu1delta/delta and phi during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of mu1/mu1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.

Topics: Animals; Capsid; Capsid Proteins; Cattle; Cell Line; Chymotrypsin; Detergents; DNA-Directed RNA Polymerases; Dogs; Endopeptidases; Enzyme Activation; Hemolysis; In Vitro Techniques; L Cells; Mice; Micelles; Reoviridae; Sodium Dodecyl Sulfate; Virus Replication

The size of pores arising in human erythrocytes under the action of two detergents (Triton X-100 and sodium dodecyl sulfate) and causing the slow component of hemolysis was estimated by the method of osmotic protectors. The pore diameters were found to be about 40 A. The pores responsible for the fast component of hemolysis induced by sodium dodecyl sulfate were shown to be of greater size and even molecules of polyethylene glycol 4000 could pass through them. The unusual decrease. In the rate and percentage of this hemolytic component was caused by the side action of the protectors, i.e., by the acceleration of erythrocyte vesiculation, a process that competed with pore formation. Vesiculation protected erythrocytes against fast and slow detergent-induced hemolysis. It is shown that the method of osmotic protectors can not be used for estimation of pore size in fast hemolysis by sodium dodecyl sulfate. The application of this method for hemolysis by other amphiphilic compounds is discussed.

Topics: Detergents; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Kinetics; Octoxynol; Osmolar Concentration; Sodium Chloride; Sodium Dodecyl Sulfate; Zinc Sulfate

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly- spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC- mutant was not diarrheagenic.

Topics: Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Toxins; Electrophoresis, Polyacrylamide Gel; Hemolysin Proteins; Hemolysis; Immunoblotting; Immunoglobulin Fab Fragments; Mice; Mice, Inbred BALB C; Rabbits; Sodium Dodecyl Sulfate; Type C Phospholipases; Vibrio cholerae

Rational modification of an existing cationic alpha-helical antimicrobial peptide (ESF1) for improved activity by increasing amphipathicity was undertaken. ESF1 and two variants (GR7 and SA3) were synthesized and tested for activity range, minimum inhibitory concentration, and hemolytic activity. Biological activity was related to structure as determined by circular dichroism. The substitution of arginine for glycine in position seven was found to increase antimicrobial activity without effecting hemolysis. Increased activity was related to stronger alpha-helix formation in buffer. Increased beta-sheet formation in micellar SDS was observed and speculated to be due to a stronger ability of the variants to form multimolecule complexes, a feature consistent with existing models of cationic peptide activity.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Anti-Infective Agents; Circular Dichroism; Erythrocytes; Hemolysis; Humans; Molecular Conformation; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Sodium Dodecyl Sulfate

The effect of osmotic protectors (sucrose and polyethylene glycols) and of a decrease in the detergent concentration at different points of hemolysis of human erythrocytes by sodium dodecyl sulphate on the shape of kinetic curves of hemolysis were studied. It is shown that slow detergent-induced hemolysis follows the colloid-osmotic mechanisms. Evidence is provided that rapid hemolysis by sodium dodecyl sulphate is caused by opening of large pores sufficient for the release of hemoglobin molecules rather than by the colloid-osmotic mechanism, and that the kinetics of hemolysis is mainly determined by time dependence of the opening probability of these pores.

Topics: Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Kinetics; Polyethylene Glycols; Sodium Dodecyl Sulfate; Sucrose; Surface-Active Agents

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.

Topics: Aminoglycosides; Animals; Base Sequence; Blotting, Northern; Blotting, Western; Cattle; Cells, Cultured; Coagulase; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Complementation Test; Genetic Variation; Hemin; Hemolysis; Lysostaphin; Molecular Sequence Data; Mutagenesis, Site-Directed; Sodium Dodecyl Sulfate; Staphylococcus aureus

The numbers of Triton X-100 and sodium dodecyl sulfate molecules required to form respective pores were estimated from the relationship between the detergent concentrations and the rates of fast and slow hemolysis components. It has been found that the slow hemolysis component evoked by Triton X-100 is related to the existence of two different pores. It is shown that the fast hemolysis component induced by sodium dodecyl sulfate is associated with the modification of phosphatidylcholine which determines the break in the Arrhenius plots of the hemolysis rate within the range of 20 degrees C. The shape of hemolysis kinetic curves and the dependence of hemolytic parameters on the detergent concentration and temperature are discussed based on the concept of hemolysis caused by the formation of pores in various membrane lipid regions and by releasing vesicles from erythrocytes.

Topics: Detergents; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Kinetics; Octoxynol; Sodium Dodecyl Sulfate; Temperature

Topics: Animals; Cattle; Cattle Diseases; Hemolysis; Hemolytic Plaque Technique; Microfilariae; Sodium Dodecyl Sulfate; Trypanosoma brucei gambiense

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Cell Line; Cell Membrane Permeability; Cells, Cultured; Cytotoxins; Erythrocyte Membrane; Fibroblasts; Hemolysis; Humans; Ion Channels; Keratinocytes; Molecular Sequence Data; Molecular Weight; Polymers; Rabbits; Sodium Dodecyl Sulfate; Vibrio cholerae

The study was designed to show whether band 3, anion exchange protein in human erythrocytes, is responsible for hemolysis in hypotonic solution. From ESR spectra of the spin labeled erythrocytes, it was found that the change of the osmotic condition had no effect on membrane fluidity at pH 7.4. At pH 6.0, however, the fluidity at the central part of the lipid bilayer was dependent on osmotic stress. In this case, the fluidity change as a function of KCl concentration was similar to the hemolysis curve. From circular dichroism (CD) spectra of erythrocyte ghosts, it was found that the conformational and/or associative changes of the membrane proteins correlated with the hypotonic hemolysis. In contrast to the osmotic stress, sodium dodecyl sulfate (SDS) changed the fluidity of the membranes but not the conformation of the membrane proteins regardless of the medium pH. In this case, the changes in the fluidity corresponded to the hemolysis curves in SDS solutions. Further, by using anion transport inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, eosin 5-isothiocyanate and eosin-5-maleimide, a correlation between the effect on the hypotonic hemolysis and the conformational and/or associative change of band 3 was suggested. These results demonstrate that band 3 is responsible for the hypotonic hemolysis.

Topics: Anion Exchange Protein 1, Erythrocyte; Circular Dichroism; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Hypotonic Solutions; In Vitro Techniques; Membrane Fluidity; Osmolar Concentration; Potassium Chloride; Protein Conformation; Sodium Dodecyl Sulfate; Spin Labels

A procedure based on modifications of published methods for human proteins for the isolation of rat C8 and C9 from one batch of serum is described. The procedure allows the rapid, large-scale isolation of pure and haemolytically active proteins. Rat C9 had a slightly higher molecular weight than human C9 on SDS-PAGE and similar isoelectric point. Rat C8 differed from human C8 in the molecular weight of the gamma chain (23,000 and 21,000 kD respectively), and on isoelectric focusing (pI rat C8: 6.5-6.9; pI human C8: 7.4-7.9).

Topics: Animals; Complement C1; Complement C8; Complement C9; Electrophoresis, Polyacrylamide Gel; Hemolysis; Humans; Isoelectric Focusing; Rabbits; Rats; Sodium Dodecyl Sulfate

A new bovine red blood cell (RBC) test is presented as a biological in vitro assay for rapid assessment of the membrane and protein damaging effects of tensides. The system uses the hemoglobin released during RBC damage as an indicator to determine quantitatively the concentration at which the tensioactive agents affect and disrupt the plasma membrane. The spectrophotometric assay also considers the denaturation of hemoglobin caused by high concentrations of the tensides. Thus besides the halfmaximal concentrations to cause hemolysis (H50), a denaturation index (DI in %) can be determined for each test agent. The RBC assay was validated by assessing the H50 and DI for 30 randomly selected tensioactive agents from various sources and comparing the in vitro effects with their local irritant activity as assessed by the Draize test in the conjuntiva of conscious rabbits. The relationship between H50 and DI values, expressed as the lysis/denaturation ratio, was found to give a ranking order that best characterized the membrane damaging potency of the agents tested. Tensides known to possess ocular irritancy in the Draize test were characterized by a low lysis/denaturation ratio.

Topics: Animals; Cattle; Cell Survival; Erythrocytes; Eye; Hemolysis; In Vitro Techniques; Irritants; Protein Denaturation; Rabbits; Sodium Dodecyl Sulfate; Surface-Active Agents

The nonionic detergent Triton X-100 at concentrations of about 0.003 to 0.008% causes swelling followed by the haemolysis of erythrocytes suspended in 160 mM KCl. The rate of haemolysis increases with the increase in detergent concentration. Finally all the erythrocytes are haemolysed. The resistance of erythrocytes to this detergent decreases with an increase in temperature. The effect of Triton X-100 is explained by increased membrane permeability to KCl and colloid osmotic haemolysis. The anionic detergent, sodium dodecyl sulfate (SDS), at concentrations of about 0.003 to 0.001% causes the haemolysis of a certain number of erythrocytes. This number increases with an increase in detergent concentration. The resistance of erythrocytes to SDS increases with an increase in temperature. The effect of SDS is explained by direct disruption of membranes by the detergent.

Topics: Animals; Cattle; Detergents; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Octoxynol; Polyethylene Glycols; Sodium Dodecyl Sulfate; Surface-Active Agents; Swine; Temperature

7.0 microM Sodium dodecyl sulfate induces lysis on A Rh(+) red blood cells 1.6 times faster than the cationic analog dodecyl thiouronium chloride at 37 degrees C and pH = 7.30. Hemolysis isotherms show identical values for the Hill cooperativity parameters of the organic surfactants, but different dissociation parameters. The anionic detergent destabilizes red cell membranes more efficiently than the cationic analog, probably due to the predominance of negative charges on the erythrocyte membranes at physiological pH.

Topics: Animals; Cell Membrane; Detergents; Erythrocytes; Hemolysis; Humans; Onium Compounds; Sodium Dodecyl Sulfate; Sulfonium Compounds; Surface-Active Agents

To evaluate the proteolytic activities of oncogene-transfected 3T3 cells, we have developed a copolymerized substrate electrophoretic assay that permits the detection of picogram quantities of proteases produced by cells in culture. Our assay involves a gelatin substrate copolymerized in a polyacrylamide gel. Purified cell membrane preparations were run on gels and various protease activities were detected by amido black. Ras-transfected 3T3 cells appear to have a soluble metalloprotease that may be transiently membrane bound and responsible for destruction of red blood cells (RBC). Oncogene-transfected NIH-3T3 cells have been demonstrated to have RBC cytolytic activity. We have previously shown that v-src-transfected 3T3 cells and their cell membranes cause RBC cytolysis which is inhibited by the protease inhibitor leupeptin. Here we report that both H-ras- and K-ras-transfected 3T3 cells and their cell membranes are cytolytic for RBC, but are inhibited by the metalloprotease inhibitor ethylene diamine tetraacetic acid. Using the gelatin substrate gel assay, we determined that some of the proteases were intrinsic to the oncogene expressing cells, while other proteases were secreted into the culture growth medium.

Topics: Cell Line, Transformed; Culture Media; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Genes, ras; Hemolysis; Membrane Proteins; Oncogenes; Peptide Hydrolases; Protease Inhibitors; Sodium Dodecyl Sulfate; Spectrophotometry

Decorticated Momordica charantia seeds were extracted with acidic ethanol. The pH of the extract was adjusted to 8 and the resulting precipitate was chromatographed on CM Sepharose CL-6B and then desalted on Sephadex G-10. A number of factors which exhibited antilipolytic and lipogenic activities in rat adipocytes were isolated. Two of the factors were demonstrated to be peptides with similar amino acid compositions and a molecular weight of approximately 8000. However the peptides greatly differed in their chromatographic behavior on CM-Sepharose CL-6B and in their mobilities in agarose and polyacrylamide gel electrophoresis.

Topics: Abortifacient Agents, Nonsteroidal; Amino Acids; Animals; Chromatography, Gel; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Female; Hemagglutination Tests; Hemolysis; Hypolipidemic Agents; Immunodiffusion; Lipids; Mice; Mice, Inbred ICR; Molecular Weight; N-Glycosyl Hydrolases; Peptides; Plant Proteins; Rats; Ribosome Inactivating Proteins, Type 2; Seeds; Sodium Dodecyl Sulfate

Human monocytes are known to synthesize many of the components of complement, including C1-INA. In this report we demonstrate that the human monocyte-like cell line U937 is also capable of synthesizing functional C1-INA. This was shown in several ways, including 1) incorporation of tritiated amino acids into antigenic C1-INA, immunoprecipitation, and detection by fluorography; 2) a sensitive ELISA, which allowed quantitation of antigenic C1-INA in cell lysates, and 3) a C2-dependent hemolytic assay in which the functional activity of U937 C1-INA was assayed. Data from the ELISA indicate that U937 cells contain between 2.1 to 12.8 ng of C1-INA per 1 X 10(6) cells. Furthermore, fluorescence-activated cell sorter analysis revealed that approximately 16% of U937 cells carry C1-INA as a surface bound antigen. Other proteins found to be synthesized by U937 cells include C1r, C8, and possibly alpha-2-macroglobulin. These results suggest that the U937 cell line could be a convenient and valuable model for the study of monocyte C1-INA synthesis and physiology.

Topics: Amino Acids; Antigens, Surface; Cell Line; Complement C1 Inactivator Proteins; Complement C2; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Hemolysis; Humans; Membrane Proteins; Monocytes; Proteins; Sodium Dodecyl Sulfate

Human C4 displays a structural polymorphism which is consistent with there being two closely linked genetic loci coding for this protein. These give rise to two C4 isotypes, designated C4A and C4B, which can be distinguished by charge and apparent m.w. differences in their respective alpha-chains and by the presence or absence of the Chido/Rodgers blood group antigens. Previous qualitative studies of C4 immune hemolysis activity in whole plasma had suggested that the C4B isotype was functionally more active. By using purified C4A and C4B isolated from individual donors known serologically to possess only one of the C4 isotypes, we examined the molecular basis for the differences in their respective hemolytic activities. It was found that the C4B:C4A hemolytic activity ratio was approximately 4:1. This fourfold difference could not be accounted for by a commensurate difference in the cleavage rate of the two isotypes by C1s by differences in the kinetics of assembly or intrinsic decay of the respective C3 convertase enzymes, or by differences in the rate of isotypic C4b cleavage by factor I in the presence of C4bp . However, the fourfold greater deposition efficiency of nascent C4b of the C4B isotype onto the surface of C1-bearing sheep erythrocytes quantitatively accounted for the observed difference in immune hemolysis function. It was further found that the thioester bond of nascent C4b of the C4A isotype preferentially transacylates onto amino group nucleophiles, whereas in the C4B isotype, acylation of hydroxyl groups is strongly preferred. Thus, the difference in immune hemolysis activity between the two C4 isotypes does not necessarily indicate an impairment of function in C4A; it may merely be a reflection of the relative abundance at the surface of a C1-bearing target of hydroxyl and amino groups capable of being acyl acceptors for nascent C4b. Finally, we also present evidence showing that the apparent m.w. difference between the alpha-chains of the C4A and C4B isotypes is not due to differences in protein glycosylation.

Topics: Acylation; Animals; Blood Group Antigens; Complement C3-C5 Convertases; Complement C4; Complement C4b; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hemolysis; Humans; Molecular Weight; Phenotype; Polymorphism, Genetic; Sodium Dodecyl Sulfate; Thiolester Hydrolases

proteins which were able to bind noncovalently with mouse factor B were found in cells that are nonsecretors of factor B such as mouse-established monocytic cells and L cells but not in peritoneal resident macrophages. These proteins were isolated from lysates of L cells and separated into four distinct proteins by preparative SDS-polyacrylamide gel electrophoresis, with molecular weights of 25K , 28K , 33K, and 35K . The individual proteins formed a complex with purified mouse factor B at a molecular ratio of 1: 1 and inhibited its hemolytic activity. Proteins 25K and 28K inhibited the hemolytic activity of an activated form of factor B combined with cobra venom factor as well as that of the native form. These inhibitors did not affect the hemolytic activity of the second component of complement in mouse serum. The inhibitory activity of the 25K protein was partially inhibited by antiserum raised against it in rabbits.

Topics: Animals; Cell Line; Complement Factor B; Elapid Venoms; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Hemolysis; Humans; L Cells; Macrophages; Mice; Mice, Inbred C3H; Molecular Weight; Protease Inhibitors; Rabbits; Sodium Dodecyl Sulfate

The effects of iodipamide on C3 and factor B in normal human serum and in purified form have been examined by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Temperature-dependent changes in immunoelectrophoretic profiles have been observed; however, these are not the same as those obtained after treatment of normal human serum (NHS) with cobra venom factor Naja naja. Analyses of iodipamide-treated NHS and purified C3 and factor B by reducing SDS-PAGE indicate that no macromolecular changes have occurred in C3 and factor B that can be ascribed to proteolysis (i.e., activation). The changes observed in C3 and factor B, including loss of hemolytic activity, appear to be due to direct interactions between iodipamide and C3 and factor B. In the case of factor B, iodipamide treatment at 37 degrees C induces aggregation, which is reversible upon reduction with beta-mercaptoethanol.

Topics: Complement Activation; Complement C3; Complement Factor B; Contrast Media; Depression, Chemical; Elapid Venoms; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Hemolysis; Humans; Immunoelectrophoresis; Iodipamide; Sodium Dodecyl Sulfate

Rats were given different doses (0.25-25 mg) of two forms of PVC past polymers and polystyrene by intratracheal instillation and monitored for cellular and biochemical changes in the lung lavage fluid and remaining lung tissue after 1 and 4 weeks of exposure. Changes were observed in free cell morphology, the level of pulmonary surfactant, and hydrolytic enzyme activities with all three compounds at high concentrations. Some differences in the extent of changes produced per given mass identified were noted with different formulations and the relationship between particle numbers, surface areas, and detergent coatings of samples is discussed. It is argued that PVC paste polymers and polystyrene cannot be considered totally biologically inert but that, in comparison with active cytoxic and inflammatory minerals such as chrysotile asbestos, the extent of their initial reaction in the lung is relatively weak.

Topics: Animals; Aspartic Acid Endopeptidases; Benzenesulfonates; Detergents; Disinfectants; Endopeptidases; Hemolysis; Latex; Lung; Male; Particle Size; Polyvinyl Chloride; Polyvinyls; Pulmonary Surfactants; Rats; Ribonucleases; Sodium Dodecyl Sulfate

Topics: Complement C3; Erythrocytes; Guanidines; Hemolysis; Humans; Kinetics; Macromolecular Substances; Protein Conformation; Sodium Dodecyl Sulfate

A new procedure for the isolation of the human complement component C9 is described. This procedure offers the possibility to prepare functionally pure C9 in a one-step procedure with a high recovery of 10-22% of the biological activity. The 125iodinated C9 had a molecular weight of 78,000 daltons and was only contaminated in traces with other proteins. Further purification by absorption with an "anti-impurity" column lead to a C9 preparation which behaved as a homogenous single polypeptide chain in SDS polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It formed a single bell-shaped precipitate in crossed immunoelectrophoresis with antibodies against human serum in the gel of the second dimension. The recovery of the biological activity after the second purification step was in the order of 6-10%. Both preparative steps could be performed within a few hours 450 microgram C9 protein were isolated from 135 ml human serum. A human serum completely defective in C9 was prepared by the extensive absorption of a smaller volume of human serum proteins with the anti-C9 column.

Topics: Complement C9; Glass; Glutaral; Hemolysis; Humans; Immune Sera; Immunoelectrophoresis; Methods; Molecular Weight; Sodium Dodecyl Sulfate

The conversion of the S-surfonate group in a peptide to a disulfide bond has been observed in vitro. This paper reports that the conversion of this group in human S-sulfonated gamma-globulin (S-GG) appeared to occur in vivo judging from the change in molecular weight of S-GG observed in the presence of sodium dodecyl sulfate (SDS) and the restoration of hemolytic activity.

Topics: Animals; Binding Sites; Disulfides; Electrophoresis, Disc; gamma-Globulins; Hemolysis; Humans; Macromolecular Substances; Protein Binding; Rabbits; Sodium Dodecyl Sulfate; Sulfonic Acids; Toxins, Biological

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37 degress C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0 degrees thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di- and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75 degrees C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10-second intervals. This method was more convenient than standard procedures and gave yields of di- and triphosphoinositide equivalent to that obtained by the method of Folch.

Topics: Animals; Calcium; Cell Membrane; Edetic Acid; Erythrocytes; Hemolysis; Kinetics; Magnesium; Phosphatidic Acids; Phosphatidylinositols; Polysorbates; Potassium; Rabbits; Sodium; Sodium Dodecyl Sulfate

Human leukocyte interferon, prepurified either by acid ethanol extraction or by affinity chromatography with antibodies, was further purified by gel filtration in the presence of sodium dodecyl sulfate. Interferon was eluted from gel filtration columns as an apparently homogeneous entity with a molecular weight of 26,600, resulting in an up to 50-fold additional purification during a single step. The antiviral activity could be further resolved into two components by hydroxylapatite adsorption chromatography. The isolated components (A and B) were distinguishable by isoelectric focusing and polyacrylamide gel electrophoresis. The apparent molecular weights were 20,000 to 16,000 and 16,000, respectively. No differences were detected in their susceptibility toward reduction of disulfide bonds by beta-mercaptoethanol. Both could be obtained on a preparative scale with minimal losses in biological activity.

Topics: Binding Sites; Chromatography, Affinity; Disulfides; Hemolysis; Humans; Interferons; Leukocytes; Molecular Weight; Parainfluenza Virus 1, Human; Protein Binding; Protein Conformation; Sodium Dodecyl Sulfate

A group of proteins was readily extracted at neutrality from trichloroacetic acid precipitates of staphylococcal culture filtrate supernatants, while alpha-toxin was dissolved and activated by treating the precipitate with 8 M urea, with acidic buffers or by heating to 90-100 degrees C at neutrality. Heat activation of the precipitate produced a relatively pure alpha-toxin with a molecular weight of 39,000. alpha-Toxin was eluted together with three other proteins on hydroxyl apatite chromatography, and evidence was obtained for an association between the four proteins. On isoelectric focusing a haemolytic fraction was obtained at pH 6.2, probably due to acid activation of the precipitate formed at the cathodic end of the column. The alpha-haemolytic fractions with pI's of 7.4 and 8.6 were shown to consist of alpha-toxin only when analyzed by acrylamide electrophoresis in the presence of sodium dodecyl sulphate. The haemolytic component with a pI of 9.2 contained two additional components of molecular weights of 27,500 and 18,000. Chromatography of this material on Sephadex G-200 showed that alpha-toxin and the two proteins appeared as a high molecular complex.

Topics: Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Hemolysis; Hot Temperature; Hydrogen-Ion Concentration; Isoelectric Focusing; Molecular Weight; Proteins; Sodium Dodecyl Sulfate; Staphylococcus aureus; Toxins, Biological; Urea

A complement regulatory principle, C4b inactivator, was isolated in a partially purified form from normal human serum. The C4b inactivator, a beta1-globulin with an approximate mol wt of 88,000 daltons, and which may be identical to C3b inactivator, cleaved C4b in free solution or on the surface of cells and rendered it unable to participate in hemolytic reactions or to interact with cells, having receptors for C4b. C/b inactivator functioned by cleaving the alpha-polypeptide chain of C4b at a single site which was sufficient to dissociate the molecule into two fragments, C4c and C4d, and to inactivate it biological function. Certain structural correlates of C4 functions deriving from these studies are discussed and a model for C4 structure based on these findings is presented.

Topics: Animals; Antigen-Antibody Reactions; Beta-Globulins; Blood Protein Electrophoresis; Blood Proteins; Cattle; Chromatography; Complement Inactivator Proteins; Electrophoresis, Disc; Epitopes; Erythrocytes; Hemolysis; Immune Adherence Reaction; Immunochemistry; Immunoelectrophoresis; Iodine Radioisotopes; Protein Binding; Sodium Dodecyl Sulfate; Ultracentrifugation

The termostable direct hemolysin of Vibrio parahaemolyticus is inactivated by heating at around 55 C with an inactivating factor isolated from culture filtrates of V. parahaemolyticus. The characteristics of the temperature-dependent inactivating factor were studied by using polyacrylamide gel disc electrophoresis. It was found that when heated with the hemolysin, the inactivating factor destroyed the hemolysin and thus inactivated the hemolytic activity, suggesting that the inactivating factor has proteolytic activity. It was also demonstrated that the inactivating factor itself is heat labile, losing its activity on heating with the hemolysin at 95 C for 15 min. The inactivating factor was stimulated by the presence of NaCl or MgCl2 and showed maximal activity at around pH 8.0. The results support our previous hypothesis that the Arrhenius effect observed with crude hemolysin of V. parahaemolyticus is due to the presence of a temperature-dependent inactivating factor. The fact that the factor is activated on heating at 50 to 60 C but is inactivated on heating at 70 to 100 C explains the Arrheius effect of crude hemolysin of V. parahaemolyticus.

Topics: Cell-Free System; Electrophoresis, Disc; Erythrocytes; Hemolysin Proteins; Hemolysis; Humans; Magnesium; Sodium Chloride; Sodium Dodecyl Sulfate; Temperature; Vibrio parahaemolyticus

The molecular weight of phallolysin, the toxic haemolysin from Amanita phalloides, was established by gel chromatography to be 30000 daltons. The isoelectric point (I.P.) was found in Ampholine pH 7-10 at 8.34. In Ampholine pH 7-9 the gel chromatographically homogeneous phallolysin was separated into phallolysin A (I.P. 8.06) and phallolysin B (I.P. 7.49). Sodium dodecylsulphate-polyacrylamide gel electrophoresis indicated a molecular weight of 33000 daltons for phallolysin A. Phallolysin was thermo- and acid-labile. It was relatively stable in alkaline solutions. 8 M urea as well as 0.1% sodium dodecylsulphate caused irreversible denaturation. On the other hand, phallolysin showed resistance to diverse proteases (pepsin, trypsin, alpha-chymotrypsin, subtilisin, pronase E, bromelin, proteinase K) and also alpha-amylase and pancreatin. Treatment with proteinase K did not change the molecular weight and the isoelectric points of phallolysin. Resistance to proteases was not due to inhibition of proteases by phallolysin.

Topics: Agaricales; Amanita; Amanitins; Animals; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Female; Hemolysin Proteins; Hemolysis; In Vitro Techniques; Isoelectric Focusing; Mice; Molecular Weight; Peptide Hydrolases; Rabbits; Sodium Dodecyl Sulfate; Urea

Topics: Cell Membrane; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Molecular Weight; Proteins; Sodium Dodecyl Sulfate; Solubility; Water

Conditions are defined for the production, in high titers, of an extracellular hemolytic toxin of Aeromonas hydrophila, here termed "aerolysin." Substantial purification of the toxin was accomplished by means of salt fractionation, dialysis, and gel filtration, with a yield of 24% of the starting activity. Analysis of the product by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single, heavy protein band and a number of faint protein bands. The estimated molecular weight of the heavy band (50,000) was in close agreement with that (53,000) of the substance responsible for hemolytic activity as determined by gel filtration. Purified aerolysin is a labile substance, apparently protein. It is not inactivated by any of several proteases under the conditions employed nor is it inhibited by any of several lipids tested. About 0.1 mug administered to mice intravenously is lethal. The physical properties of aerolysin show considerable resemblance to those described for the exotoxin of Pseudomonas aeruginosa.

Topics: Aeromonas; Animals; Bacterial Proteins; Bacteriological Techniques; Calcium; Chloroform; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Hemolysis; Isoelectric Focusing; Lethal Dose 50; Magnesium; Mice; Molecular Weight; Peptide Hydrolases; RNA; Sodium Dodecyl Sulfate; Solubility; Toxins, Biological

Topics: Adenosine Triphosphatases; Biological Transport, Active; Cations, Monovalent; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Kinetics; Molecular Weight; Ouabain; Phosphorus Radioisotopes; Phosphorylases; Potassium; Pronase; Radioisotopes; Rubidium; Sodium; Sodium Dodecyl Sulfate; Solubility; Spectrophotometry; Streptomyces griseus

Topics: Antigen-Antibody Complex; Autoradiography; Chymotrypsin; Complement System Proteins; Electrophoresis, Disc; Hemolysis; Humans; Immunodiffusion; Immunoelectrophoresis; Iodine Radioisotopes; Isotope Labeling; Microbial Collagenase; Ovalbumin; Pancreatic Elastase; Sodium Dodecyl Sulfate; Trypsin; Urea

Topics: Alcohols; Basidiomycota; Benzopyrans; Clostridium perfringens; Dose-Response Relationship, Drug; Drug Antagonism; Drug Synergism; Erythrocytes; Flavonoids; Hemolysin Proteins; Hemolysis; Humans; Hydroxides; In Vitro Techniques; Ketones; Lysophosphatidylcholines; Mycotoxins; Salts; Saponins; Sodium; Sodium Dodecyl Sulfate

Topics: Animals; Antibodies; Cell Membrane; Complement System Proteins; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Immunoelectrophoresis; Isoelectric Focusing; Molecular Weight; Rabbits; Sheep; Sodium Dodecyl Sulfate

Topics: Antibodies, Viral; Antigen-Antibody Complex; Cell Fusion; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glucosamine; Glycoproteins; Hemagglutination Inhibition Tests; Hemagglutinins, Viral; Hemolysis; Measles virus; Microscopy, Electron; Peptides; Sodium Dodecyl Sulfate; Tritium; Viral Proteins

Topics: Animals; Catalase; Chromatography, DEAE-Cellulose; Chromatography, Gel; Drug Stability; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Immunoelectrophoresis; Mice; Mice, Inbred BALB C; Rabbits; Sodium Dodecyl Sulfate; Species Specificity; Spectrophotometry, Ultraviolet

Topics: Antigens, Bacterial; Binding Sites, Antibody; Carbohydrates; Centrifugation, Density Gradient; Choline; Chromatography, Gel; Chromatography, Paper; Chromatography, Thin Layer; Cross Reactions; Forssman Antigen; Hemolysis; Myeloma Proteins; Protein Binding; Sodium Dodecyl Sulfate; Streptococcus pneumoniae; Tritium

Streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) with a molecular weight of about 55,000 and an isoelectric pH of 8.55 was isolated from crude streptolysin O (SLO) preparations. NADase differed from SLO in size, charge, and immunological behavior. Streptococcal NADase is considered to have no role in the hemolytic process because it has no hemolytic activity; conversely, partially purified SLO showed no NADase activity. The hemolytic activity of crude SLO was completely inhibited by anti-tetanolysin, whereas the NADase activity in the same reaction mixture was unaffected. Experiments involving double diffusion in agar also demonstrated immunological nonidentity of the two proteins.

Topics: Bacteriological Techniques; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Hemolysis; Immunodiffusion; Isoelectric Focusing; Molecular Weight; N-Glycosyl Hydrolases; NAD; Neutralization Tests; Sodium Dodecyl Sulfate; Streptococcus pyogenes; Streptolysins; Tetanus Antitoxin

Topics: Albumins; Blood Glucose; Chromatography; Colorimetry; Creatinine; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Methods; Sodium Dodecyl Sulfate; Spectrophotometry; Time Factors

Topics: Animals; Antigens, Viral; Centrifugation, Density Gradient; Centrifugation, Zonal; Chick Embryo; Complement Fixation Tests; Culture Techniques; Deoxycholic Acid; Encephalitis Viruses; Encephalomyelitis, Equine; Ethyl Ethers; Hemagglutination Tests; Hemolysis; Immune Sera; Rabbits; Sodium Dodecyl Sulfate; Surface-Active Agents; Viral Plaque Assay; Viral Proteins; Virus Cultivation

Topics: Amino Acids; Ammonium Sulfate; Animals; Blood Proteins; Chemical Precipitation; Chromatography, Gel; Chromatography, Ion Exchange; Collagen; Complement System Proteins; Electrophoresis; Galactose; Glucose; Hemolysis; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulins; Macromolecular Substances; Molecular Weight; Rabbits; Sheep; Sodium Dodecyl Sulfate; Ultracentrifugation

Topics: Blood Proteins; Calcium; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Humans; Hydrolysis; Magnesium; Peptide Hydrolases; Sodium Dodecyl Sulfate; Trypsin

Topics: Acetylcholinesterase; Blood Proteins; Cell Membrane; Chromatography, Gel; Colloids; Densitometry; Erythrocytes; Half-Life; Hemolysis; Humans; Isoelectric Focusing; Kinetics; Macromolecular Substances; Mathematics; Models, Biological; Phosphatidylcholines; Phospholipids; Sodium Dodecyl Sulfate; Solubility; Surface Tension; Surface-Active Agents

Topics: Animals; Anura; Blood Pressure; Bone Marrow; Dogs; Electrocardiography; Haplorhini; Heart; Hemolysis; Infusions, Parenteral; Kidney; Liver; Lung; Pathology; Rabbits; Research; Sodium Dodecyl Sulfate; Sulfates; Surface-Active Agents; Toxicology

Topics: Fatty Alcohols; Hemolysis; Humans; Radioactivity; Soaps; Sodium; Sodium Dodecyl Sulfate; Sulfur

Topics: Cell Death; Erythrocytes; Hemolysis; Humans; Sodium Dodecyl Sulfate; Sulfates