Effect of pH on the interfacial adsorption activity of pulmonary surfactant

The effect of pH on the interfacial adsorption activity of pulmonary surfactant was examined. Measurements of the surface tension were made in a Wilhelmy-like surface microbalance specially designed to assay small volumes of hypophase in thermostatically controlled conditions. Alkaline pH caused a significant decrease of the surface activity of both pulmonary surfactant and a lipid extract from surfactant (LES) (containing all of the lipids and surfactant protein-B (SP-B) and surfactant protein-C (SP-C) hydrophobic surfactant proteins, but lacking surfactant protein-A). The pK calculated from the change of surface activity versus pH was 9.18±0.26 and 9.27±0.31 for pulmonary surfactant and LES, respectively. The results from this study support the idea that electrostatic interactions between basic residues of SP-B and SP-C and negatively charged surfactant phospholipids could be important for the interfacial adsorption activity of pulmonary surfactant.     

Characterization of bovine surfactant proteins B and C by electrospray ionization mass spectrometry

Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.     

Quantitative analysis of hydrophobic pulmonary surfactant proteins by high-performance liquid chromatography with light-scattering detection

A new method for the separation and quantification of two hydrophobic lung surfactant proteins (SPs) is described. It is based on size-exclusion chromatography using the apolar stationary phase butyl silicagel with a pore size of 30 nm and isocratic elution with chloroform, methanol and trifluoroacetic acid. The samples were prepared from sheep lung lavage fluid by centrifugation and fractional extraction with butanol and chloroform–methanol. The chromatograms show three peaks in the elution order SP-B, SP-C and lipids. A small peak ahead of SP-B, which disappeared after reduction with 2-mercaptoethanol, was oligomeric SP-B. The response of the evaporative light-scattering detector was non-linear. For preparative high-performance liquid chromatography ultraviolet detection at 279 nm is recommended.     

Alzheimer amyloid beta(1-40) peptide: interactions with cationic gemini and single-chain surfactants

The aggregation of amyloid beta-peptide [Abeta(1-40)] into fibril is a key pathological process associated with Alzheimer's disease. The effect of cationic gemini surfactant hexamethylene-1,6-bis-(dodecyldimethylammonium bromide) [C(12)H(25)(CH(3))(2)N(CH(2))(6)N(CH(3))(2)C(12)H(25)]Br(2) (designated as C(12)C(6)C(12)Br(2)) and single-chain cationic surfactant dodecyltrimethylammonium bromide (DTAB) on the Alzheimer amyloid beta-peptide Abeta(1-40) aggregation behavior was studied by microcalorimetry, circular dichroism (CD), and atomic force microscopy (AFM) measurements at pH 7.4. Without addition of surfactant, 0.5 g/L Abeta(1-40) mainly exists in dimeric state. It is found that the addition of the monomers of C(12)C(6)C(12)Br(2) and DTAB may cause the rapid aggregation of Abeta(1-40) and the fibrillar structures are observed by CD spectra and the AFM images. Due to the repulsive interaction among the head groups of surfactants and the formation of a small hydrophobic cluster of surfactant molecules, the fibrillar structure is disrupted again as the surfactant monomer concentration is increased, whereas globular species are observed in the presence of micellar solution. Different from single-chain surfactant, C(12)C(6)C(12)Br(2) has a much stronger interaction with Abeta(1-40) to generate larger endothermic energy at much lower surfactant concentration and has much stronger ability to induce the aggregation of Abeta(1-40).     

Peptoids with Antibiofilm Activity against the Gram Negative Obligate Anaerobe,Fusobacterium nucleatum

Peptoids (oligo N-substituted glycines) are peptide analogues, which can be designed to mimic host antimicrobial peptides, with the advantage that they are resistant to proteolytic degradation. Few studies on the antimicrobial efficacy of peptoids have focused on Gram negative anaerobic microbes associated with clinical infections, which are commonly recalcitrant to antibiotic treatment. We therefore studied the cytotoxicity and antibiofilm activity of a family of peptoids against the Gram negative obligate anaerobe Fusobacterium nucleatum, which is associated with infections in the oral cavity. Two peptoids, peptoid 4 (NaeNpheNphe)4 and peptoid 9 (NahNspeNspe)₃ were shown to be efficacious against F. nucleatum biofilms at a concentration of 1 μM. At this concentration, peptoids 4 and 9 were not cytotoxic to human erythrocytes or primary human gingival fibroblast cells. Peptoids 4 and 9 therefore have merit as future therapeutics for the treatment of oral infections.     

Kinetics and equilibria of cis/trans isomerization of backbone amide bonds in peptoids

The biological activities of N-substituted glycine oligomers (peptoids) have been the subject of extensive research. As compared to peptides, both the cis and trans conformations of the backbone amide bonds of peptoids can be significantly populated. Thus, peptoids are mixtures of configurational isomers, with the number of isomers increasing by a factor of 2 with each additional monomer residue. Here we report the results of a study of the kinetics and equilibria of cis/trans isomerization of the amide bonds of N-acetylated peptoid monomers, dipeptoids, and tripeptoids by NMR spectroscopy. Resonance intensities indicate the cis conformation of the backbone amide bonds of the peptoids studied is more populated than is generally the case for the analogous secondary amide bond to proline residues in acyclic peptides. Rate constants were measured by inversion-magnetization transfer techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. The rate of cis/trans isomerization by rotation around the amide bonds in the peptoids studied is generally slower than that around amide bonds to proline residues and takes place on the NMR inversion-magnetization transfer time scale only by rotation around the amide bond to the C-terminal peptoid residue.     

基于光纤折射率传感原理的表面活性剂临界胶束浓度测定方法

提出并研究了一种新颖的基于光纤折射率传感原理的表面活性剂临界胶束浓度(cmc)测定方法.应用此方法测定有代表性的阴离子表面活性剂十二烷基硫酸钠(SDS)与阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)在25℃下的cmc分别为8.05×10-3和9.11×10-4mol·L-1,与文献值比较,结果相当吻合.从而证实了本方法的准确性.进一步研究了各种条件对测量表面活性剂cmc的影响,结果表明温度和无机盐NaCl的加入对本方法测量的准确性影响小,证明了本方法对测试环境的要求不苛刻,适用性好.最后对本方法进行了重复性和稳定性测试,相对标准偏差(RSD)为0.17%,与预期符合,效果良好.     

Stable helical peptoids via covalent side chain to side chain cyclization

Peptoids are oligomeric N-substituted glycines with potential as biologically relevant compounds. Helical peptoids provide an attractive fold for the generation of protein-protein interaction inhibitors. The generation of helical peptoid folds in organic and aqueous media has been limited to strict design rules, as peptoid-folding is mainly directed via the steric direction of alpha-chiral side-chains. Here a new methodology is presented to induce helical folds in peptoids with the aid of side chain to side chain cyclization. Cyclic peptoids were generated via solid-phase synthesis and their folding was studied. The cyclization induces significant helicity in peptoids in organic media, aids the folding in aqueous media, and requires the incorporation of only relatively few chiral aromatic side chains.     

Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids

The fragmentation patterns of a group of doubly protonated ([P + 2H]²⁺) and mixed protonated-sodiated ([P + H + Na]²⁺) peptide-mimicking oligomers, known as peptoids, have been studied using electron capturing dissociation (ECD) tandem mass spectrometry techniques. For all the peptoids studied, the primary backbone fragmentation occurred at the N-C_α bonds. The N-terminal fragment ions, the C-ions (protonated) and the C′-ions (sodiated) were observed universally for all the peptoids regardless of the types of charge carrier. The C-terminal ions varied depending on the type of charge carrier. The doubly protonated peptoids with at least one basic residue located at a position other than the N-terminus fragmented by producing the Z^?-series of ions. In addition, most doubly protonated peptoids also produced the Y-series of ions with notable abundances. The mixed protonated-sodiated peptoids fragmented by yielding the Z^?′-series of ions in addition to the C′-series. Chelation between the sodium cation and the amide groups of the peptoid chain might be an important factor that could stabilize both the N-terminal and the C-terminal fragment ions. Regardless of the types of the charge carrier, one notable fragmentation for all the peptoids was the elimination of a benzylic radical from the odd-electron positive ions of the protonated peptoids ([P + 2H]^?+) and the sodiated peptoids ([P + H + Na]^?+). The study showed potential utility of using the ECD technique for sequencing of peptoid libraries generated by combinatorial chemistry.

Design and facile solid-phase synthesis of conformationally constrained bicyclic peptoids

Triazine-bridged bicyclic peptoids as conformationally constrained peptidomimetics are described. Bicyclic peptoids composed of 6-12 peptoid residues (m, n = 3-6) were synthesized in excellent yields using a highly efficient solid-phase synthetic route.     

Structure and dynamics of micelles and cubic phase structures with ethoxylated phytosterol surfactant in water

The self-assembly of a sterol ethoxylate surfactant with 30 oxyethylene units in water was studied by 1H NMR self-diffusion measurements in a wide concentration range in the micellar region (0-25 wt %). The data showed that the surfactant aggregates do not interact by hard sphere interactions but rather a strong concentration dependence of the diffusion coefficient was noted which was explained by polymer scaling theory. In the cubic phase (30-65 wt %), the self-diffusion data from water, from surfactant, and from free polyoxyethylene suggest spherical micelles, although water diffusion was much restricted due to binding to the surfactant headgroup. From X-ray measurements in the cubic phase, the unit cell size was calculated, and together with surfactant self-diffusion measurements the exchange dynamics between free and aggregated surfactant was obtained.     

新型液膜振荡器

表面活性剂不仅对均相的非线性化学反应动力学研究有重要作用”－‘］,而且表面活性剂穿越油水界面扩散时形成的自发液膜振荡过程亦可作为一个简单模型,用以说明多相反应与扩散偶合所产生的复杂周期现象,特别是说明生物系统生理现象中的振荡与混饨．这些早在贺占博的博士论文l’］就已提出,但至今此方面的研究仍进展甚微,而其意义却非常重大,迫切需要进一步研究．关于液膜振荡的本质,目前有两种说法．一种是界面流体力学的Marangoni效应,另一种为化学本质的胶束一单分子膜一反胶束的表面活性剂聚集状态的周期变化l’‘．我们的实验…     

New insights into lung surfactant monolayers using vibrational sum frequency generation spectroscopy

At the air-water interface, interfacial molecular structure, intermolecular interactions, film relaxation and film respreading of model lung surfactant monolayers were studied using vibrational sum frequency generation (VSFG) spectroscopy combined with a Langmuir film balance. Chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoyl-phosphatidylglycerol (POPG), palmitic acid (PA) and tripalmitin were investigated. In the DPPC-d62-PA binary monolayer, PA showed a condensing effect on the DPPC chains. On the contrary, in the DPPC-d62-POPG binary monolayer, POPG showed a fluidizing effect on the DPPC chains. In the ternary monolayer system of DPPC-d62-POPG-PA, the balance between the fluidizing and the condensing effect was also observed. In addition, the film relaxation behavior of DPPC-d62 and the enhanced film stability of DPPC-d62 caused by the addition of tripalmitin were observed. Real-time VSFG was also employed to study the respreading properties of a complex lung surfactant mixture containing DPPC-d62, POPG, PA and KL4 (a mimic of SP-B) peptide, which revealed DPPC enrichment after film compression.     

Tuning peptoid secondary structure with pentafluoroaromatic functionality: a new design paradigm for the construction of discretely folded peptoid structures

Peptoids, or oligomers of N-substituted glycine, are an important class of non-native polymers whose close structural similarity to natural alpha-peptides and ease of synthesis offer significant advantages for the study of biomolecular interactions and the development of biomimetics. Peptoids that are N-substituted with alpha-chiral aromatic side chains have been shown to adopt either helical or "threaded loop" conformations, depending upon solvent and oligomer length. Elucidation of the factors that impact peptoid conformation is essential for the development of general rules for the design of peptoids with discrete and novel structures. Here, we report the first study of the effects of pentafluoroaromatic functionality on the conformational profiles of peptoids. This work was enabled by the synthesis of a new, alpha-chiral amine building block, (S)-1-(pentafluorophenyl)ethylamine (S-2), which was found to be highly compatible with peptoid synthesis (delivering (S)-N-(1-(pentafluorophenyl)ethyl)glycine oligomers). The incorporation of this fluorinated monomer unit allowed us to probe both the potential for pi-stacking interactions along the faces of peptoid helices and the role of side chain electrostatics in peptoid folding. A series of homo- and heteropeptoids derived from S-2 and non-fluorinated, alpha-chiral aromatic amide side chains were synthesized and characterized by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Enhancement of pi-stacking by quadrupolar interactions did not appear to play a significant role in stabilizing the conformations of heteropeptoids with alternating fluorinated and non-fluorinated side chains. However, incorporation of (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomers enforced helicity in peptoids that typically exhibit threaded loop conformations. Moreover, we found that the incorporation of a single (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomer could be used to selectively promote looped or helical structure in this important peptoid class by tuning the electronics of nearby heteroatoms. The strategic installation of this monomer unit represents a new approach for the manipulation of canonical peptoid structure and the construction of novel peptoid architectures.     

Real-time investigation of lung surfactant respreading with surface vibrational spectroscopy

The respreading of a lung surfactant monolayer at the air-water interface is investigated with broad bandwidth sum frequency generation (BBSFG) spectroscopy. The lung surfactant mixture contains chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and KL4 (a 21-residue polypeptide analogue to the surfactant protein SP-B). DPPC-d62 serves as a probe molecule for the spectroscopic investigation. The BBSFG spectra of DPPC-d62 in the lung surfactant mixture are obtained in the C-D stretching region in real-time during film compression and expansion in a Langmuir trough. The BBSFG intensity of the CD3 stretch peak from DPPC-d62 terminal methyl groups is used as a measure of the interfacial density of DPPC-d62 after careful consideration of orientation effects. For the first time, the interfacial loss of DPPC in a complex lung surfactant mixture is quantified. Spectroscopic results reveal that there is an 18% DPPC-d62 interfacial loss during film respreading. However, the surface pressure-area isotherm measurements demonstrate that there is a rather large trough area reduction (37%) during film expansion. The relatively small interfacial loss of DPPC-d62 and the rather large trough area reduction indicate that the respreading of DPPC and non-DPPC components in the lung surfactant is not uniform and a surface refinement process exists during film compression and expansion. This refinement process results in a DPPC-enriched monolayer with a significant depletion of non-DPPC components after film respreading. Implication for replacement surfactant design from this work is discussed.     

The influence of surfactant mixing ratio on nano-emulsion formation by the pit method

The formation of O/W nano-emulsions by the PIT emulsification method in water/mixed nonionic surfactant/oil systems has been studied. The hydrophilic-lipophilic properties of the surfactant were varied by mixing polyoxyethylene 4-lauryl ether (C12E4) and polyoxyethylene 6-lauryl ether (C12E6). Emulsification was performed in samples with constant oil concentration (20 wt%) by fast cooling from the corresponding HLB temperature to 25 degrees C. Nano-emulsions with droplet radius 60-70 nm and 25-30 nm were obtained at total surfactant concentrations of 4 and 8 wt%, respectively. Moreover, droplet size remained practically unchanged, independent of the surfactant mixing ratio, X(C12E6). At 4 wt% surfactant concentration, the polydispersity and instability of nano-emulsions increased with the increase in X(C12E6). However, at 8 wt% surfactant concentration, nano-emulsions with low polydispersity and high stability were obtained in a wide range of surfactant mixing ratios. Phase behavior studies showed that at 4 wt% surfactant concentration, three-liquid phases (W+D+O) coexist at the starting emulsification temperature. Furthermore, the excess oil phase with respect to the microemulsion D-phase increases with the increase in X(C12E6), which could explain the increase in instability. At 8 wt% surfactant concentration, a microemulsion D-phase is present when emulsification starts. The low droplet size and polydispersity and higher stability of these nano-emulsions have been attributed, in addition to the increase in the surface or interfacial activity, to the spontaneous emulsification produced in the microemulsion D-phase.     

Helical peptoid mimics of lung surfactant protein C

Among the families of peptidomimetic foldamers under development as novel biomaterials and therapeutics, poly-N-substituted glycines (peptoids) with alpha-chiral side chains are of particular interest for their ability to adopt stable, helical secondary structure in organic and aqueous solution. Here, we show that a peptoid 22-mer with a biomimetic sequence of side chains and an amphipathic, helical secondary structure acts as an excellent mimic of surfactant protein C (SP-C), a small protein that plays an important role in surfactant replacement therapy for the treatment of neonatal respiratory distress syndrome. When integrated into a lipid film, the helical peptoid SP mimic captures the essential surface-active behaviors of the natural protein. This work provides an example of how an abiological oligomer that closely mimics both the hydrophobic/polar sequence patterning and the fold of a natural protein can also mimic its biophysical function.     

Extraordinarily robust polyproline type I peptoid helices generated via the incorporation of α-chiral aromatic N-1-naphthylethyl side chains

Peptoids, or oligomers of N-substituted glycines, are a class of foldamers that have shown extraordinary functional potential since their inception nearly two decades ago. However, the generation of well-defined peptoid secondary structures remains a difficult task. This challenge is due, in part, to the lack of a thorough understanding of peptoid sequence-structure relationships and, consequently, an incomplete understanding of the peptoid folding process. We seek to delineate sequence-structure relationships through the systematic study of noncovalent interactions in peptoids and the design of novel amide side chains capable of such interactions. Herein, we report the synthesis and detailed structural analysis of a series of (S)-N-(1-naphthylethyl)glycine (Ns1npe) peptoid homo-oligomers by X-ray crystallography, NMR spectroscopy, and circular dichroism (CD) spectroscopy. Four of these peptoids were found to adopt well-defined structures in the solid state, with dihedral angles similar to those observed in polyproline type I (PPI) peptide helices and in peptoids with α-chiral side chains. The X-ray crystal structure of a representative Ns1npe tetramer revealed an all cis-amide helix, with approximately three residues per turn, and a helical pitch of approximately 6.0 ?. 2D-NMR analysis of the length-dependent Ns1npe series showed that these peptoids have very high overall backbone amide K(cis/trans) values in acetonitrile, indicative of conformationally homogeneous structures in solution. Additionally, CD spectroscopy studies of the Ns1npe homo-oligomers in acetonitrile and methanol revealed a striking length-dependent increase in ellipticity per amide. These Ns1npe helices represent the most robust peptoid helices to be reported, and the incorporation of (S)-N-(1-naphthylethyl)glycines provides a new approach for the generation of stable helical structure in this important class of foldamers.     

Sequence and Structure of Peptoid Oligomers Can Tune the Photoluminescence of an Embedded Ruthenium Dye

The understanding of structure–function relationships within synthetic biomimetic systems is a fundamental challenge in chemistry. Herein we report the direct correlation between the structure of short peptoid ligands—N-substituted glycine oligomers incorporating 2,2′-bipyridine groups—varied in their monomer sequence, and the photoluminescence of Ru^II centers coordinated by these ligands. Based on circular dichroism and fluorescence spectroscopy we demonstrate that while helical peptoids do not affect the fluorescence of the embedded Ru^II chromophore, unstructured peptoids lead to its significant decay. Transmittance electron microscopy (TEM) revealed significant differences in the arrangements of metal-bound helical versus unstructured peptoids, suggesting that only the latter can have through-space interactions with the ruthenium dye leading to its quenching. High-resolution TEM enabled the remarkable direct imaging of singular ruthenium-bound peptoids and bundles, supporting our explanation for structure-depended quenching. Moreover, this correlation allowed us to fine-tune the luminescence properties of the complexes simply by modifying the sequence of their peptoid ligands. Finally, we also describe the chiral properties of these Ru–peptoids and demonstrate that remote chiral induction from the peptoids backbone to the ruthenium center is only possible when the peptoids are both chiral and helical.     

Mass spectrometry strategies applied to the characterization of proline-rich peptides from secretory parotid granules of pig (Sus scrofa)

Basic proline-rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline-rich peptides (SP-A and SP-B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss-Prot data bank: Q95JC9-1, Q95JC9-2 and Q95JC9-3). Together the coding regions for SP-A and SP-B, which are repeated many times, account for 52-70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP-HPLC-ESI-IT-MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline-rich peptides derived from the three proproteins. Together with the coding regions for SP-A and SP-B already identified it was possible to assign 68-75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases.