Surface-induced unfolding of human lactoferrin

We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 angstroms on the air side and a bottom diffuse layer of some 50 angstroms into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm(-3), close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 x 10(-8) mol m(-2) in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 x 10(-8) mol m(-2). Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 angstroms and a length of 105 angstroms in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 angstroms and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption.     

Adsorption behavior of statherin and a statherin peptide onto hydroxyapatite and silica surfaces by in situ ellipsometry

The salivary protein statherin is known to adsorb selectively onto hydroxyapatite (HA), which constitutes the main mineral of the tooth enamel. This adsorption is believed to be crucial for its function as an inhibitor of primary (spontaneous) and secondary (crystal growth) precipitation of calcium phosphate salts present in saliva. A fragment corresponding to the first 21 N-terminus amino acids of statherin (StN21) was previously found to reduce the rate of demineralization of HA. Therefore, the interfacial properties of this peptide and statherin onto silica, hydrophobized silica and HA discs was studied by in situ ellipsometry. Their reversibility induced by dilution and elutability induced by buffer and sodium dodecyl sulfate (SDS) was also determined. The results revealed that statherin adsorbed at a greater extent onto the HA as compared to StN21, suggesting that the hydrogen bonding between the uncharged polar residues at the C-terminal region of statherin and HA contributes to its adsorption. However, on both silica surfaces the peptide adsorption appeared to proceed in a similar way. Onto the hydrophobized silica the adsorption of both peptides was suggested to occur either via multilayer formation or adsorption of aggregates from solution, while onto the hydrophilic silica adsorption of peptide aggregates from solution was the suggested mechanism. Further, both peptides were observed to be strongly adsorbed onto HA, even after SDS treatment, in comparison to the layers adsorbed onto hydrophobized silica. Both peptide layers were found to be weakly adsorbed onto the hydrophilic silica surface as they were totally removed by buffer dilution.     

Adsorption of lysozyme on a hemodialysis sulfonated polyacrylonitrile membrane, with and without preadsorbed poly(ethyleneimine) on the external faces

We present the influence of pH, from pH 4 to 10, with a focus on the neutral range, on the adsorption of lysozyme (isoelectric point pI=11) on a sulphonated membrane and the same membrane pre-treated with poly(ethyleneimine) (PEI). We found a steep increase of the adsorbed amount above pH 6 in phosphate buffer. The adsorbed amount was about twice as low in Tris buffer, around the neutral pH. The difference between the two types of buffer is attributed to their different ionic composition. High interfacial concentration in phosphate buffer is especially linked to the phosphate divalent anions. In the presence of divalent sulphate anions, we measured the same level of interfacial concentration than with phosphate buffer. With the PEI pre-treated membrane, we observed, on the time scale of our experiments (15–20 h), similar adsorbed amounts than on the raw membrane, showing that the PEI layer does not constitute a true barrier to the penetration of lysozyme into the membrane core. However, its presence leads to a slower adsorption rate in a system where convection does not occur through the membrane.     

Adsorption of copolymers aggregates: from kinetics to adsorbed layer structure

We examined the adsorption, on hydrophobic and hydrophilic surfaces, of 4 rake-type poly(dimethyl siloxane) (PDMS) copolymers varying the amount of poly(ethylene glycol) (PEG) graft arms from 41 to 72%. The copolymers formed large aggregates in solution, complicating their adsorption kinetics and layer structures. We found the adsorption process always to be dominated by the adsorption of large aggregates, with strongly bound layers resistant to rinsing in adsorbing buffer. Adsorbed amounts were nearly independent of the substrate. However, subtleties in the adsorption kinetics suggested different layer structures for the different systems. On hydrophilic silica, aggregates adsorbed at the transport limited rate until surface saturation, and associated interfacial structures were likely retained. On the hydrophobic surface, a subset of the copolymers exhibited retarded late stage adsorption kinetics suggestive of brush formation. This work demonstrates how subtle differences in adsorption kinetics provide insight into potential interfacial layer structures.     

Application of bare gold nanoparticles in open-tubular CEC separations of polyaromatic hydrocarbons and peptides

In this study, bare gold nanoparticles (GNPs) immobilized in the sol-gel-pretreated fused-silica (FS) capillary as a stationary phase for open-tubular capillary electrochromatography (OT-CEC) are for the first time shown to be able to separate both hydrophobic polyaromatic hydrocarbons (PAHs) as well as hydrophilic cationic antimicrobial peptides. Model mixture of four PAHs, naphthalene, fluorene, phenanthrene, and anthracene, was resolved by OT-CEC in the GNP-modified FS capillaries using the hydro-organic background electrolyte (BGE) composed of 20 mmol/L sodium phosphate buffer, pH 7, modified with ACN at 8:2 v/v ratio. On the other hand, three synthetic analogues of an antimicrobial peptide mastoparan PDD-B, basic tetradecapeptides INWKKLGKKILGAL-NH(2), INSLKLGKKILGAL-NH(2) and NWLRLGRRILGAL-NH(2), were separated in aqueous acidic BGEs, pH 2.1-3.1, composed of weak acids (formic and acetic) or amphoteric amino or imino acids (aspartic or iminodiacetic), utilizing the advantage of a slow reversed (anodic) EOF and slightly positive charge of the GNP-modified FS capillary suppressing the adsorption of cationic peptides on the inner capillary wall and improving their resolution.     

Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption

Control over selective recognition of biomolecules on inorganic nanoparticles is a major challenge for the synthesis of new catalysts, functional carriers for therapeutics, and assembly of renewable biobased materials. We found low sequence similarity among sequences of peptides strongly attracted to amorphous silica nanoparticles of various size (15-450 nm) using combinatorial phage display methods. Characterization of the surface by acid base titrations and zeta potential measurements revealed that the acidity of the silica particles increased with larger particle size, corresponding to between 5% and 20% ionization of silanol groups at pH 7. The wide range of surface ionization results in the attraction of increasingly basic peptides to increasingly acidic nanoparticles, along with major changes in the aqueous interfacial layer as seen in molecular dynamics simulation. We identified the mechanism of peptide adsorption using binding assays, zeta potential measurements, IR spectra, and molecular simulations of the purified peptides (without phage) in contact with uniformly sized silica particles. Positively charged peptides are strongly attracted to anionic silica surfaces by ion pairing of protonated N-termini, Lys side chains, and Arg side chains with negatively charged siloxide groups. Further, attraction of the peptides to the surface involves hydrogen bonds between polar groups in the peptide with silanol and siloxide groups on the silica surface, as well as ion-dipole, dipole-dipole, and van-der-Waals interactions. Electrostatic attraction between peptides and particle surfaces is supported by neutralization of zeta potentials, an inverse correlation between the required peptide concentration for measurable adsorption and the peptide pI, and proximity of cationic groups to the surface in the computation. The importance of hydrogen bonds and polar interactions is supported by adsorption of noncationic peptides containing Ser, His, and Asp residues, including the formation of multilayers. We also demonstrate tuning of interfacial interactions using mutant peptides with an excellent correlation between adsorption measurements, zeta potentials, computed adsorption energies, and the proposed binding mechanism. Follow-on questions about the relation between peptide adsorption on silica nanoparticles and mineralization of silica from peptide-stabilized precursors are raised.     

Interface of covalently bonded phospholipids with a phosphorylcholine head: characterization, protein nonadsorption, and further functionalization

Surface anchored poly(methylhydrosiloxane) (PMHS) thin films on oxidized silicon wafers or glass substrates were functionalized via the SiH hydrosilylation reaction with the internal double bonds of 1,2-dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2 Cis). The surface was characterized by X-ray photoelectron spectroscopy, contact angle measurements, atomic force microscopy, and scanning electron microscopy. These studies showed that the PMHS top layer could be efficiently modified resulting in an interfacial high density of phospholipids. Grafted phospholipids made the initially hydrophobic surface (θ = 106°) very hydrophilic and repellent toward avidin, bovine serum albumin, bovine fibrinogen, lysozyme, and α-chymotrypsin adsorption in phosphate saline buffer pH 7.4. The surface may constitute a new background-stable support with increased biocompatibility. Further possibilities of functionalization on the surface remain available owing to the formation of interfacial SiOH groups by Karstedt-catalyzed side reactions of SiH groups with water. The presence of interfacial SiOH groups was shown by zeta potential measurements. The reactivity and surface density of SiOH groups were checked by fluorescence after reaction of a monoethoxy silane coupling agent bearing Alexa as fluorescent probe.     

Electrochemical Study on the Interaction Between Neutral Red and DNA

IntroductionDeoxyribonucleic acid( DNA) is the most im-portant germ plasma of most organisms.It playsan importantrole in the process ofstoring,copyingand transmitting germ messages.There have beenmany papers studying on the interaction betweensmall molecules and DNA since the1 960′s.Nowthe researches have become a field of common in-terest[1] .Those researches have contributed to theunderstanding of the way of the interaction be-tween DNA and protein.What is more,those re-searches are he…     

Electrochemical Study on the Interaction Betwwen Neutral Red and DNA

A voltammetric study of the interaction of neutral Red(NR) with DNA at a gold electrode in a phosphate buffer solution is described. After adding DNA in an NR solution, the reduction peak current of NR decreases. The binding mechahisms of NR to DNA in different pH ranges are different. The reduction peak potential of NR in a pH 7.0 phosphate buffer solution in the presence of DNA shifts positively, indicating that the binding of NR to DNA is intercalation action, but at pH=6.0 the reduction peak potential of NR shifts negatively, indicating that the binding of NR to DNA is electrostatic action. The formed complexes are DNA-NR when [NR]/[DNA]<0.18 and DNA-3NR when [NR]/[DNA]>0.35, respectively.     

Structure of DNA-cationic surfactant complexes at hydrophobically modified and hydrophilic silica surfaces as revealed by neutron reflectometry

In this article, we discuss the structure and composition of mixed DNA-cationic surfactant adsorption layers on both hydrophobic and hydrophilic solid surfaces. We have focused on the effects of the bulk concentrations, the surfactant chain length, and the type of solid surface on the interfacial layer structure (the location, coverage, and conformation of the DNA and surfactant molecules). Neutron reflectometry is the technique of choice for revealing the surface layer structure by means of selective deuteration. We start by studying the interfacial complexation of DNA with dodecyltrimethylammonium bromide (DTAB) and hexadecyltrimethylammonium bromide (CTAB) on hydrophobic surfaces, where we show that DNA molecules are located on top of a self-assembled surfactant monolayer, with the thickness of the DNA layer and the surfactant-DNA ratio determined by the surface coverage of the underlying cationic layer. The surface coverages of surfactant and DNA are determined by the bulk concentration of the surfactant relative to its critical micelle concentration (cmc). The structure of the interfacial layer is not affected by the choice of cationic surfactant studied. However, to obtain similar interfacial structures, a higher concentration in relation to its cmc is required for the more soluble DTAB surfactant with a shorter alkyl chain than for CTAB. Our results suggest that the DNA molecules will spontaneously form a relatively dense, thin layer on top of a surfactant monolayer (hydrophobic surface) or a layer of admicelles (hydrophilic surface) as long as the surface concentration of surfactant is great enough to ensure a high interfacial charge density. These findings have implications for bioanalytical and nanotechnology applications, which require the deposition of DNA layers with well-controlled structure and composition.     

pH and ionic strength effect on single fibrinogen molecule adsorption on mica studied with AFM

Although several investigations have been reported on the effect of pH or ionic strength on protein adsorption, most of them have been carried out with protein monolayers and not with single molecules. We have used atomic force microscopy to image, in phosphate buffer, single fibrinogen molecules adsorbed on mica and compare the surface coverage at variable pH (7.4, 5.8, 3.5) or ionic strength (15, 150, 500 mM) conditions. The images obtained and the statistical analysis of the surface coverage indicate adsorption enhancement at the IEP of fibrinogen (pH 5.8) and minimum adsorption at pH 3.5. On the other hand, more protein was adsorbed when the salt concentration of the buffer at pH 7.4 was increased from 15 to 150 mM. However, further increase of salt concentration up to 500 mM resulted in decreased adsorption. To confirm the aforementioned results an approaching bare Si(3)N(4) tip was used as an electrostatic analogue to a protein molecule and interaction force curves between it and the substrate were recorded. The results were in consistence with the double layer theory which justifies the screening of electrostatic repulsion as the salt concentration increases.     

Properties and structure of interfacial layers formed by hydrophilic silica dispersions and palmitic acid

The properties and structure of different types of interfacial layers obtained from aqueous dispersions of nanometric silica and palmitic acid (PA) have been studied and characterized by different diagnostics and measurements. The investigations concern PA monolayers spread on the silica dispersions, dispersions in contact with PA solutions in oil and silica dispersions containing PA, aiming at elucidating the role of the PA interaction with the particles and investigating the surface-activity of the originated silica-PA complexes. Drop shape tensiometry was utilized to measure the dynamic surface and interfacial tension while a Langmuir trough apparatus was used to obtain compression isotherms of the spread PA layers and to measure the dilational viscoelasticity according to the oscillating barrier method. Brewster angle microscopy and ellipsometry were utilized to investigate the lateral and vertical structure of the interfacial layers. From this multifold approach emerges a complex picture of the features of these interfacial layers that can be rationalized on the basis of the adsorption of PA on the particle surface. The results evidence a threshold in PA adsorption above which particles change from hydrophilic to partially hydrophobic, promoting their incorporation into the interfacial layer.     

溶菌酶在裸金电极和疏基乙酸或正十二疏烷基醇修饰电极上的吸附

电化学石英晶体阻抗系统;疏基乙酸;溶菌酶在裸金电极和疏基乙酸或正十二疏烷基醇修饰电极上的吸附     

Dynamic adsorption and surface tension of aqueous dilauroylphosphatidylcholine dispersions under physiological conditions

The dynamic surface tension and equilibrium adsorption behavior of DLPC dispersions in phosphate buffer saline at 37 and 25 degrees C was studied with tensiometry, infrared reflection--absorption spectroscopy (IRRAS), and ellipsometry. The results are compared with those in water (Pinazo et al. Langmuir 2002, 18, 8888). Even though the pH and salinity have no apparent effect on the equilibrium surface tension and the surface pressure--area isotherm, they affect the dynamic surface tension by decreasing the adsorption rate and increasing the dynamic tension minima at a pulsating area of 20 or 80 cycles per minute. Moreover, IRRAS and ellipsometry results show that the adsorbed layers and the spread monolayers have larger area per molecule, or looser packing, in buffer than in water. A new hypothesis is proposed to elucidate the effect of pH/salinity on this zwitterionic surfactant: there is some specific interaction or binding between the ions from the buffer saline with the polar headgroups of DLPC. This interaction induces stronger intermolecular repulsions of the surfactant layer in buffer than that in water, despite the expected electrostatic screening effect, and causes higher dynamic surface tensions. The results have implications in designing lung surfactant replacement formulations.     

In-situ investigation of the adsorption of globular model proteins on stimuli-responsive binary polyelectrolyte brushes

Binary brushes constituted from two incompatible polymers can be used in the form of ultrathin polymeric layers as a versatile tool for surface engineering to tune physicochemical surface characteristics such as wettability, surface charge, chemical composition, and morphology and furthermore to create responsive surface properties. Mixed brushes of oppositely charged weak polyelectrolytes represent a special case of responding surfaces that are sensitive to changes in the pH value of the aqueous environment and therefore represent interesting tools for biosurface engineering. The polyelectrolyte brushes used for this study were composed of two oppositely charged polyelelctrolytes poly(2-vinylpyridine) (P2VP) and poly(acrylic acid) (PAA). The in-situ properties and surface characteristics such as as surface charge, surface tension, and extent of swelling of these brush layers are functions of the pH value of the surrounding aqueous solution. To test the behavior of the mixed polylelctrolyte brushes in contact with biosystems, protein adsorption experiments with globular model proteins were performed at different pH values and salt concentrations (confinement of counterions) of the buffer solutions. The influence of the pH value, buffer salt concentration, and isoelectric points (IEP) of the brush and protein on the adsorbed amount and the interfacial tension during protein adsorption as well as the protein adsorption mechanism postulated in reference to recently developed theories of protein adsorption on polyelectrolyte brushes is discussed. In the salted regime, protein adsorption was found to be similar to the often-described adsorption at hydrophobic surfaces. However, in the osmotic regime the balance of electrostatic repulsion and a strong entropic driving force, "counterion release", was found to be the main influence on protein adsorption.     

Analysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non‐aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit® gave the best results, providing the separation of the four major protamine peptides within 25 min.     

Phosphate and arsenate electro-insertion processes into a N,N,N′,N′-tetraoctylphenylenediamine redox liquid

The electro-insertion of ions is a well-known phenomenon, which allows the transfer of anions or cations across phase boundaries to be monitored and driven electro-chemically. Extremely hydrophilic anions, such as phosphate and arsenate, are not usually observed to undergo electro-insertion. It is shown here that at organic redox liquid|water|electrode triple interfaces these anions can be forced electro-chemically to transfer into organic media.The transfer process of phosphate anions from aqueous buffer solutions into organic microdroplets of the redox liquid N,N,N^′,N^′-tetraoctylphenylenediamine (TOPD) is pH and concentration sensitive. It is shown that phosphate is transferred in the form of PO₄HK⁻ in the presence of phosphate buffer. Two distinct potential regions are identified and attributed to (i) interfacial redox processes at the liquid|liquid interface associated with deprotonation and (ii) bulk redox processes associated with anion transfer from the aqueous to the organic phase.The comparison of phosphate and arsenate electro-insertion processes suggests that arsenate is less hydrophilic and transferred into the organic phase preferentially.     

Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTipPPY-C18) based on hybrid electrostatic, Π–Π stacking and hydrophobic interactions for mass spectrometric analysis

Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and Π–Π stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip_PPY-C18 can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip_PPY-C18 should be able to facilitate the exploration of large scale phosphoproteome.     

Study of the selectivity,retention mechanisms and performance of alternative silica-based stationary phases for separation of ionised solutes in hydrophilic interaction chromatography

The separation of a mixture of neutral, strongly acidic and strongly basic compounds was studied in hydrophilic interaction chromatography using a bare silica phase, and bonded silica phases with diol, zwitterionic, amide and hydrophilic/hydrophobic groups. The mobile phase was acetonitrile–ammonium formate buffer at low pH. Differences in selectivity between these various columns indicate that the stationary phase cannot function merely as an inert support for a water layer into which the solutes partition from the bulk mobile phase. Attempts to fit the retention data to equations which describe either partition or adsorption mechanisms were inconclusive. Ion exchange was a significant contributor to the retention of ionised bases on all columns studied. Van Deemter plots indicated that the efficiency as a function of flow rate varied between the columns, which might be attributable in part to the presence of either monomeric or polymeric bonded phase layers.