Omiganan interaction with bacterial membranes and cell wall models. Assigning a biological role to saturation

Omiganan pentahydrochloride (ILRWPWWPWRRK-NH(2).5Cl) is an antimicrobial peptide currently in phase III clinical trials. This study aims to unravel the mechanism of action of this drug at the membrane level and address the eventual protective role of peptidoglycan in cell walls. The interaction of omiganan pentahydrochloride with bacterial and mammalian membrane models - large unilamellar vesicles of different POPC:POPG proportions - was characterized by UV-Vis fluorescence spectroscopy. The molar ratio partition constants obtained for the two anionic bacterial membrane models were very high ((18.9+/-1.3)x10(3) and (43.5+/-8.7)x10(3)) and about one order of magnitude greater than for the neutral mammalian models ((3.7+/-0.4)x10(3) for 100% POPC bilayers). At low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially for the most anionic systems. Membrane saturation can account for such observations and mathematical models were derived to further characterize the peptide-lipid interaction under those conditions; a possible relation between saturation and MIC was deduced; this was supported by differential quenching studies of peptide internalization. Interaction with the bacterial cell wall was assessed using Staphylococcus aureus peptidoglycan extracts as a model. A strong partition towards the peptidoglycan mesh was observed, but not as large as for the membrane models.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Synergistic Effects of the Membrane Actions of Cecropin-Melittin Antimicrobial Hybrid Peptide BP100

BP100 (KKLFKKILKYL-NH₂) is a short cecropin A-melittin hybrid peptide, obtained through a combinatorial chemistry approach, which is highly effective in inhibiting both the in vitro and in vivo growth of economically important plant pathogenic Gram-negatives. The intrinsic Tyr fluorescence of BP100 was taken advantage of to study the peptide's binding affinity and damaging effect on phospholipid bilayers modeling the bacterial and mammalian cytoplasmic membranes. In vitro cytotoxic effects of this peptide were also studied on mammalian fibroblast cells. Results show a stronger selectivity of BP100 toward anionic bacterial membrane models as indicated by the high obtained partition constants, one order of magnitude greater than for the neutral mammalian membrane models. For the anionic systems, membrane saturation was observed at high peptide/lipid ratios and found to be related with BP100-induced vesicle permeabilization, membrane electroneutrality, and vesicle aggregation. Occurrence of BP100 translocation was unequivocally detected at both high and low peptide/lipid ratios using a novel and extremely simple method. Moreover, cytotoxicity against mammalian models was reached at a concentration considerably higher than the minimum inhibitory concentration. Our findings unravel the relationships among the closely coupled processes of charge neutralization, permeabilization, and translocation in the mechanism of action of antimicrobial peptides.     

Two homologous neutrophil serine proteases bind to POPC vesicles with different affinities: When aromatic amino acids matter

Neutrophil serine proteases Proteinase 3 (PR3) and human neutrophil elastase (HNE) are homologous antibiotic serine proteases of the polymorphonuclear neutrophils. Despite sharing a 56% sequence identity they have been shown to have different functions and localizations in the neutrophils. In particular, and in contrast to HNE, PR3 has been detected at the outer leaflet of the plasma membrane and its membrane expression is a risk factor in a number of chronic inflammatory diseases. Although a plethora of studies performed in various cell-based assays have been reported, the mechanism by which PR3, and possibly HNE bind to simple membrane models remains unclear. We used surface plasmon resonance (SPR) experiments to measure and compare the affinity of PR3 and HNE for large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). We also conducted 500-nanosecond long molecular dynamics simulations of each enzyme at the surface of a POPC bilayer to map the interactions between proteins and lipids and rationalize the difference in affinity observed in the SPR experiment. We find that PR3 binds strongly to POPC large unilamellar vesicles (K_d = 9.2 × 10^− 7 M) thanks to the insertion of three phenylalanines, one tryptophan and one leucine beyond the phosphate groups of the POPC lipids. HNE binds in a significantly weaker manner (K_d > 10^− 5 M) making mostly electrostatic interactions via lysines and arginines and inserting only one leucine between the hydrophobic lipid tails. Our results support the early reports that PR3, unlike HNE, is able to directly and strongly anchor directly to the neutrophil membrane.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.     

ESR study of sublevel structure of anionic bis(phthalocyaninato)gadolinium(III)

Sublevel structure of the ⁸S_7/2 electronic ground state of anionic bis(phthalocyaninato)gadolinium(III) has been determined by simulation analysis of an ESR spectrum in frozen solution. The simplex multidimensional minimization algorithm was employed to find the zero-field-splitting parameter set giving the minimum RMS error from the observed spectrum. The parameter set {B₂⁰,B₄⁰,B₆⁰} for the potential of D_4d symmetry has been determined to be ±{(1.54 ± 0.01)× 10⁻² cm⁻¹, (0.9 ± 0.1)× 10⁻⁴ cm⁻¹, (−0.6 ± 0.9)× 10⁻⁶ cm⁻¹}. The energy difference between the lowest and highest sublevels has been found to be about 0.5 cm⁻¹.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36 h later. Sorted (S) and non-sorted (NS) doses of 1 or 15 × 10⁶ motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66 h after progestagen withdrawal. An additional treatment dose of 0.5 × 10⁶ S or NS sperm was inseminated at the 58 h time point (n = 60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1 × 10⁶ and 15 × 10⁶ sperm achieved similar pregnancy rates, regardless of sperm type, at 58 h (S1: 46 ± 9.4%; S15: 43 ± 9.3%; NS1: 41 ± 9.2%; NS15: 49 ± 9.4%). However, pregnancy rates were lower (P < 0.05) for doses of 1 than 15 × 10⁶ sperm inseminated at 50 (15 ± 6.3% vs. 36 ± 9.1%), 54 (14 ± 4.4% vs. 55 ± 7.3%), 62 (33 ± 6.9% vs. 54 ± 7.3%), and 66 h (29 ± 8.6% vs. 56 ± 9.5%). There was no difference between S and NS sperm for inseminations with 0.5 × 10⁶ motile sperm at 58 h after PR (15 ± 3.6% vs. 14 ± 3.3%), nor with 15 × 10⁶ motile sperm at all insemination times (49 ± 6.3% vs. 49 ± 6.3%). However, fertility was higher for S than NS sperm at the 1 × 10⁶ dose level (37 ± 6.1% and 16 ± 4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing C CPMAS, H, and REDOR solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca²⁺ regulator. ¹³C cross-polarization magic angle spinning spectra of ¹³C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding ¹³C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an α-helical conformation as monitored by an enhancement of the α-helical carbonyl ¹³C resonance in the corresponding NMR spectrum. ¹³C-¹⁵N REDOR solid-state NMR spectroscopic experiments revealed the distance between the ¹³C carbonyl carbon of Leu39 and the ¹⁵N amide nitrogen of Leu42 to be 4.2 ± 0.2Å indicating an α-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three ²H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11° ± 5° tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer.     

Membrane interface composition drives the structure and the tilt of the single transmembrane helix protein PMP1: MD studies

PMP1, a regulatory subunit of the yeast plasma membrane H⁺-ATPase, is a single transmembrane helix protein. Its cytoplasmic C-terminus possesses several positively charged residues and interacts with phosphatidylserine lipids as shown through both ¹H- and ²H-NMR experiments. We used all-atom molecular dynamics simulations to obtain atomic-scale data on the effects of membrane interface lipid composition on PMP1 structure and tilt. PMP1 was embedded in two hydrated bilayers, differing in the composition of the interfacial region. The neutral bilayer is composed of POPC (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylcholine) lipids and the negatively charged bilayer is composed of POPC and anionic POPS (1-palmitoyl-2-oleoyl-3-glycero-phosphatidylserine) lipids. Our results were consistent with NMR data obtained previously, such as a lipid sn-2 chain lying on the W28 aromatic ring and in the groove formed on one side of the PMP1 helix. In pure POPC, the transmembrane helix is two residues longer than the initial structure and the helix tilt remains constant at 6 ± 3°. By contrast, in mixed POPC-POPS, the initial helical structure of PMP1 is stable throughout the simulation time even though the C-terminal residues interact strongly with POPS headgroups, leading to a significant increase of the helix tilt within the membrane to 20 ± 5°.     

Interactions of an anionic antimicrobial peptide with Staphylococcus aureus membranes

The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3 mM and to induce maximal surface pressure changes of 3.8 mN m⁻¹ over 1200 s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be α-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Δν circa 0.5 cm⁻¹). These combined data show that AP1 is able to function as an α-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.     

Effects of biofouling on ion transport through cation exchange membranes and microbial fuel cell performance

This study examines the effects of biofouling on the electrochemical properties of cation exchange membranes (CEMs), such as membrane electrical resistance (MER), specific proton conductivity (SC), and ion transport number (t₊), in addition to on microbial fuel cell (MFC) performance. CEM biofouling using a 15.5 ± 4.6 μm biofilm was found to slightly increase the MER from 15.65 Ω cm² (fresh Nafion) to 19.1 Ω cm², whereas an increase of almost two times was achieved when the electrolyte was changed from deionized water to an anolyte containing a high cation concentration supporting bacterial growth. The simple physical cleaning of CEMs had little effect on the Coulombic efficiency (CE), whereas replacing a biofouled CEM with new one resulted in considerable increase of up to 59.3%, compared to 45.1% for a biofouled membrane. These results clearly suggest the internal resistance increase of MFC was mainly caused by the sulfonate functional groups of CEM being occupied with cations contained in the anolyte, rather than biofouling itself.     

Physiological roles of AQP7 in the kidney: Lessons from AQP7 knockout mice

The aquaporin7 (AQP7) water channel is known to be a member of the aquaglyceroporins, which allow the rapid transport of glycerol and water. AQP7 is abundantly present at the apical membrane of the proximal straight tubules in the kidney. In this paper, we review the physiological functions of AQP7 in the kidney. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP7 knockout mice compared to wild-type mice (AQP7, 18.0 ± 0.4 × 10⁻³ cm/s vs. wild-type, 20.0 ± 0.3 × 10⁻³ cm/s). Although AQP7 solo knockout mice did not show a urinary concentrating defect, AQP1/AQP7 double knockout mice showed reduced urinary concentrating ability compared to AQP1 solo knockout mice, indicating that the contribution of AQP7 to water reabsorption in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glycerol in their urine (AQP7, 1.7 ± 0.34 mg/ml vs. wild-type, 0.005 ± 0.002 mg/ml). This finding identified a novel pathway of glycerol reabsorption that occurs in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic-reperfusion ARF model, an increase of urine glycerol was observed (pre-treatment, 0.007 ± 0.005 mg/ml; cisplatin, 0.063 ± 0.043 mg/ml; ischemia, 0.076 ± 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.     

Kinetic studies of the reaction of heme-thiolate enzyme chloroperoxidase with peroxynitrite

The kinetics of the reaction of chloroperoxidase with peroxynitrite was studied under neutral and acidic pH by stopped-flow spectrophotometry. Chloroperoxidase catalyzed peroxynitrite decay with the rate constant, k_c, increasing with decreasing pH. The values of k_c obtained at pH 5.1, 6.1 and 7.1 were equal to: (1.96 ± 0.03) × 10⁶, (1.63 ± 0.04) × 10⁶ and (0.71 ± 0.01) × 10⁶ M⁻¹ s⁻¹, respectively. Chloroperoxidase was converted to compound II by peroxynitrite with pH-dependent rate constants: (12.3 ± 0.4) × 10⁶ and (3.8 ± 0.3) × 10⁶ M⁻¹ s⁻¹ at pH 5.1 and 7.1, respectively. After most of peroxynitrite had disappeared, the conversion of compound II into the ferric form of chloroperoxidase was observed. The recovery of the native enzyme was completed within 1 s and 5 s at pH 5.1 and 7.1, respectively. The possible reaction mechanisms of the catalytic decomposition of peroxynitrite by chloroperoxidase are discussed.     

Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Characterization of the binding of sulfonylurea drugs to HSA by high-performance affinity chromatography

Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (K_a) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high-affinity binding regions and weaker binding sites for each drug, with average K_a values of 1.3 (±0.2) × 10⁵ and 3.5 (±3.0) × 10² M⁻¹ for acetohexamide and values of 8.7 (±0.6) × 10⁴ and 8.1 (±1.7) × 10³ M⁻¹ for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high-affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug-binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with K_a values of 1.3 (±0.1) × 10⁵ and 4.3 (±0.3) × 10⁴ M⁻¹, respectively, at 37 °C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with K_a values of 5.5 (±0.2) × 10⁴ and 5.3 (±0.2) × 10⁴ M⁻¹, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug–protein interactions.