Photoprocesses of Xanthene Dyes Bound to Lysozyme or Serum Albumin

The ground and excited state processes of eosin, erythrosin and rose bengal in aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). Noncovalent protein-dye binding was analyzed by circular dichroism (CD), fluorescence and UV–Vis absorption spectroscopy. The effects of protein concentrations and pH were studied. Fluorescence quenching of the dye takes place due to binding to lysozyme and fluorescence enhancement due to low loading to BSA. The effects of proteins on the xanthene triplet state and its precursor were observed by time-resolved 530 nm photolysis. The triplet lifetime is quenched by lysozyme and prolonged by loading to BSA. Light-induced damages on both the dyes and proteins were observed under exclusion of oxygen. Photo-oxidation is efficient for lysozyme and lower for BSA. The CD signal of the eosin/BSA system is maximum at pH 4, where the photo-oxidation is minor.     

Modification of proteins with cyclodextrins prevents aggregation and surface adsorption and increases thermal stability

This work describes a general approach for preventing protein aggregation and surface adsorption by modifying proteins with β-cyclodextrins (βCD) via an efficient water-driven ligation. As compared to native unmodified proteins, the cyclodextrin-modified proteins (lysozyme and RNase A) exhibit significant reduction in aggregation, surface adsorption and increase in thermal stability. These results reveal a new chemistry for preventing protein aggregation and surface adsorption that is likely of different mechanisms than that by modifying proteins with poly(ethylene glycol).     

Assessment of interactions between four proteins and benzothiazole derivatives by DSC and CD

The thermal denaturation of ovalbumin, lysozyme, myoglobin and fibrinogen at different BTS concentrations have been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Thermodynamic parameters: melting temperatures (T_m), calorimetric enthalpy (ΔH), van’t Hoff enthalpy (ΔH_v) were obtained for all the systems under study. Thermal denaturation of the four proteins was completely irreversible. Changes in the protein conformation due to the adsorption of BTS molecules have been monitored by using UV-CD spectra. Greater changes in α-helical contents correspond with the BTS higher concentrations. The lysozyme denaturation temperature increases at low concentrations BTS indicating that BTS acts as a structure stabilizer; meanwhile it acts as a destabilizer at higher concentrations in all the proteins studied. The major effect is observed in the case of myoglobin, the protein with the highest α-helical secondary structure (75%).     

Enhanced refolding of lysozyme with imidazolium-based room temperature ionic liquids: Effect of hydrophobicity and sulfur residue

The expression of recombinant proteins in microorganism frequently leads to the formation of insoluble aggregates, inclusion bodies (IBs). Thus, the additional in vitro protein refolding process is required to convert inactive IBs into water-soluble active proteins. This study investigated the effect of sulfur residue and hydrophobicity of imidazolium-based room temperature ionic liquids (RTILs) on the refolding of lysozyme as a model protein in the batch dilution method which is the most commonly used refolding method. When lysozyme was refolded in the refolding buffer containing [BF4]-based RTILs with a systematic variety of alkyl chain on cations varying from two to eight, less hydrophobic imidazolium cations having shorter alkyl chains were effective to facilitate lysozyme refolding. Compared to the conventional refolding buffer, 2 times higher lysozyme refolding yield was obtained in 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) containing refolding buffer. The refolding yield of lysozyme was even more increased by 2.5 times when 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MS]) containing sulfur residue on anion was used. The sulfur residue in [BMIM][MS] is supposed to improve the refolding yield of lysozyme which has 4 intramolecular disulfide bonds. For dilution-based refolding of lysozyme, the optimum concentrations of RTILs in refolding buffer were found to be 1.0 M [EMIM][BF4] and 0.5 M [BMIM][MS], respectively. The optimum temperate for dilution-based refolding of lysozyme with RTILs was 4 °C.     

Adsorption behavior of acidic and basic proteins onto citrate-coated Au surfaces correlated to their native fold, stability, and pI

The adsorption of eight different proteins (alpha-lactalbumin (types I and III), bovine serum albumin, hemoglobin, myoglobin, cytochrome c, alpha-casein, and lysozyme) onto a model anionic surface was performed at equivalent bulk (solvent, ionic strength, pH) and surface conditions. Adsorption was monitored on a quartz crystal microbalance with dissipation monitoring (QCM-D) with citrate-coated gold surfaces as adsorbents and has been correlated to native fold stability determined from near- and far-UV circular dichroism (CD) measurements. The proteins studied here were chosen based on their pI and documented knowledge about their structural stability and flexibility. Protein adsorption was found to be independent of global protein charge. Rather, binding occurs through oppositely charged patches on protein and surface. Moreover, data indicate that there is a correlation between secondary and tertiary structure stability and the adsorption characteristics at interfaces. Also, protein surface coverage, layer thickness, and flexibility can be tuned as a function of deposition method. This is discussed in terms of adsorption/spreading kinetics and intermolecular (protein-surface and protein-protein) interactions. Adsorption to surfaces can induce formation of supramolecular structures such as micelles (in the case of alpha-Cas) and multilayers (as for Hb). In the case of alpha-casein, this phenomenon depends on the deposition method and protein concentration. When ranking the surface coverage for proteins added in excess, the order is Lyz < Cyt c < Mb < BSA < alpha-La I < alpha-Cas < alpha-La III < Hb, which can be correlated to the proteins ability to form supramolecular structures (alpha-Cas, Hb), overall conformational flexibilities, and ability to form stable intermediates.     

Protic ionic liquids with fluorous anions: physicochemical properties and self-assembly nanostructure

A series of 11 new protic ionic liquids with fluorous anions (FPILs) have been identified and their self-assembled nanostructure, thermal phase transitions and physicochemical properties were investigated. To the best of our knowledge this is the first time that fluorocarbon domains have been reported in PILs. The FPILs were prepared from a range of hydrocarbon alkyl and heterocyclic amine cations in combination with the perfluorinated anions heptafluorobutyrate and pentadecafluorooctanoate. The nanostructure of the FPILs was established by using small- and wide-angle X-ray scattering (SAXS and WAXS). In the liquid state many of the FPILs showed an intermediate range order, or self-assembled nanostructure, resulting from segregation of the polar and nonpolar hydrocarbon and fluorocarbon domains of the ionic liquid. In addition, the physicochemical properties of the FPILs were determined including the melting point (T(m)), glass transition (T(g)), devitrification temperature (T(c)), thermal stability and the density ρ, viscosity η, air/liquid surface tension γ(LV), refractive index n(D), and ionic conductivity κ. The FPILs were mostly solids at room temperature, however two examples 2-pyrrolidinonium heptafluorobutyrate (PyrroBF) and pyrrolidinium heptafluorobutyrate (PyrrBF) were liquids at room temperature and all of the FPILs melted below 80 °C. Four of the FPILs exhibited a glass transition. The two liquids at room temperature, PyrroBF and PyrrBF, had a similar density, surface tension and refractive index but their viscosity and ionic conductivity were very different due to dissimilar self-assembled nanostructure.     

The role of glycerol and phosphatidylcholine in solubilizing and enhancing insulin stability in reverse hexagonal mesophases

The potential of reverse hexagonal mesophases based on monoolein (GMO) and glycerol (as cosolvent) to facilitate the solubilization of proteins, such as insulin was explored. H(II) mesophases composed of GMO/decane/water were compared to GMO/decane/glycerol/water and GMO/phosphatidylcholine (PC)/decane/glycerol/water systems. The stability of insulin was tested, applying external physical modifications such as low pH and heat treatment (up to 70°C), in which insulin is known to form ordered amyloid-like aggregates (that are associated with several neurodegenerative diseases) with a characteristic cross β-pleated sheet structure. The impact of insulin confinement within these carriers on its stability, unfolding, and aggregation pathways was studied by combining SAXS, FTIR, and AFM techniques. These techniques provided a better insight into the molecular level of the "component interplay" in solubilizing and stabilizing insulin and its conformational modifications that dictate its final aggregate morphology. PC enlarged the water channels while glycerol shrank them, yet both facilitated insulin solubilization within the channels. The presence of glycerol within the mesophase water channels led to the formation of stronger hydrogen bonds with the hosting medium that enhanced the thermal stability of the protein and remarkably affected the unfolding process even after heat treatment (at 70°C for 60 min).     

Sequential adsorption of bacteriophage T4 lysozyme stability variants at solid–water interfaces

The sequential adsorption of the wild type T4 lysozyme and one of its structural stability variants was studied, using ellipsometry and ¹²⁵I radioisotope labeling techniques. The mutant lysozyme was produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability. The mutant protein was more resistant to surfactant-mediated elution, and apparently adsorbed at the interfaces with a greater interfacial area/molecule than the wild typeT4 lysozyme. However, the results of each type of experiment suggested that sequential adsorption and exchange of proteins occurred only in the case of the less stable mutant followed by the wild type. This suggests that, in these exchange reactions, properties of the adsorbing protein (e.g. its ability to adsorb when a relatively small amount of unoccupied area is present) were more important than the apparent binding strength of the adsorbed protein molecules.     

Effect of Stereoregular Polyelectrolyte on Protein Thermal Stability

Myoglobin was used as a model protein to study the effect of polyelectrolyte on protein thermal stability for solutions. Stereoregular polystyrene sulfonate was used to investigate the effect of chain properties on protein polyion binding affinity. Turbidity measurement indicate stronger binding to protein of atactic polystyrene sulfonate than isotactic polystyrene sulfonate, an effect that might be due to the higher chain flexibility of the atactic form. Differential scanning calorimetry (DSC) and small angle x-ray (SAXS) scattering indicate the presence of the polyelectrolyte has a destabilizing effect on the protein. The results showed that, although the presence of polyelectrolytes has no effect on myoglobin structure at room temperature at pH 7.4, myoglobin stability is reduced as the temperature is elevated. This effect is linked to the binding of the protein to the polylectrolyte. This binding is probably driven by a combination of electrostatic and hydrophobic interactions, the latter of which are enhanced at higher temperatures.     

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by ¹H nuclear magnetic resonance (¹H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV–vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV–vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins.     

Equilibrium clusters in concentrated lysozyme protein solutions

We have studied the structure of salt-free lysozyme at 293 K and pH 7.8 using molecular simulations and experimental SAXS effective potentials between proteins at three volume fractions, ?=0.012, 0.033, and 0.12. We found that the structure of lysozyme near physiological conditions strongly depends on the volume fraction of proteins. The studied lysozyme solutions are dominated by monomers only for ?≤0.012; for the strong dilution 70% of proteins are in a form of monomers. For ?=0.033 only 20% of proteins do not belong to a cluster. The clusters are mainly elongated. For ?=0.12 almost no individual particles exits, and branched, irregular clusters of large extent appear. Our simulation study provides new insight into the formation of equilibrium clusters in charged protein solutions near physiological conditions.     

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).     

A thermosensitive monolithic column as an artificial antibody for the on-line selective separation of the protein

The main objective of this study was to develop a new methodology for the preparation of a protein (antigen) that is a molecularly imprinted polymer (MIP, an artificial antibody) modified onto the surface of a silica skeleton in which the resulting stationary phase is thermosensitive. The silica monolithic skeleton with vinyl groups was synthesized in a stainless-steel column by using a mild one-step sol-gel process with two types of precursor: methyltrimethoxysilane (MTMS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, three types of the thermosensitive protein MIP were anchored onto the surface of the silica skeleton to prepare the MIP monoliths, which were systematically investigated for back pressure and separation ability at different temperatures to establish good imprinting conditions. Under the optimized imprinting conditions, the chromatographic behavior of the thermosensitive MIP monolith exhibited strong retention ability for the lysozyme template (target antigen) in relation to the nonimprinting monolith (NIP monolith). The imprinting factor (IF) for lysozyme reached 3.48 at 20 °C. Moreover, this new type of artificial antibody displayed favorable binding characteristics for lysozyme over competitive proteins and was further evaluated to selectively separate lysozyme in a real sample by using an on-line method. The run-to-run and column-to-column repeatability measurements of the thermosensitive MIP monoliths were also satisfactory.     

Modulation of structure and dynamics by disulfide bond formation in unfolded states

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.     

The role of peptides and proteins in melanoidin formation

High‐molecular‐weight (HMW) coloured compounds called melanoidins are widely distributed, particularly in foods. It has been proposed that they originate through the Maillard reaction, a non‐enzymatic browning reaction, due to the interaction between protein or peptide amino groups and carbohydrates. The melanoidin structure is not definitively known, and they have been generally defined as HMW nitrogen‐containing brown polymers. In order to gain information on the nature of melanoidins, a simple in vitro model was chosen to investigate the products of the reactions between sugars and peptide/proteins. This approach would elucidate whether melanoidin formation is due to the binding of different sugar units to a peptide/protein or vice versa. With this aim, the reactivity of two different peptides, EPK177 and physalaemin, and a low‐molecular‐weight (LMW) protein, lysozyme, was tested towards different saccharides (glucose, maltotriose (MT), maltopentaose and dextran 1000) in aqueous solutions at different temperatures. The incubation mixtures were analysed at different reaction times by MALDI/MS. Furthermore, in order to verify the possible role of sugar pyrolysis products in melanoidin formation, the products arising from the thermal treatment at 200 °C of MT were incubated with lysozyme, and the reaction products were analysed by the same MS approach. The obtained results allowed the establishment of some general views: melanoidins cannot simply originate by reactions of sugar moieties with proteins. In fact, the reaction easily occurs, but it does not lead to any coloured product, as melanoidins have been described to be; melanoidins cannot originate from the thermal degradation products of glycated proteins. In fact, the thermal treatment of glycated lysozyme leads to a severe degradation of the protein with the formation of LMW species, far from the view of melanoidins as HMW compounds; experimental evidence has been gained on the melanoidin formation through reaction of intact protein with the pyrolysis products of MT. This hypothesis has been supported either from MALDI measurements or from spectroscopic data that show an absorption band in the range 300–600 nm, typical of melanoidins. Copyright © 2009 John Wiley & Sons, Ltd.     

Thermal stability of lysozyme Langmuir-Schaefer films by FTIR spectroscopy

Fourier transform infrared spectroscopy has been applied to study the thermal stability of multilayer Langmuir-Schaefer (LS) films of lysozyme deposited on silicon substrates. The study has confirmed previous structural findings that the LS protein films have a high thermal stability that is extended in a lysozyme multilayer up to 200 degrees C. 2D infrared analysis has been used here to identify the correlated molecular species during thermal denaturation. Asynchronous 2D spectra have shown that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown that, at the first drying stage, up to 100 degrees C, only the water that is not fully hydrogen bonded is removed. Moreover, the amine intensity band does not change up to 200 degrees C, the temperature at which the structural stability of the multilayer lysozyme films ceases.     

The effect ofpH on thermal stability of globular proteins

In this study we try to re-analyze thepH dependence of thermal stability of small globular proteins. From the thermodynamic point of view a long series of calorimetric and spectroscopic investigations has shown that the decreased stability in very acidic conditions can be ascribed to entropic effects. The same conclusion is reached, from a microscopic point of view, by assuming that a binding of protons on equal and noninteracting sites takes place as a consequence of unfolding process. By linking the conformational unfolding equilibrium to the proton binding equilibrium, a model is developed that is able to describe the dependence on thepH of the thermal denaturation processes of small globular protiens. The application of the model to hen lysozyme and T4 lysozyme correctly accounts for the experimental results.     

Calorimetric and structural investigation of the interaction of lysozyme and bovine serum albumin with poly(ethylene oxide) and its copolymers

This work reports investigations aiming at verifying the occurrence of specific interactions between lysozyme or bovine serum albumin (BSA) and poly(ethylene oxide) and its copolymers with poly(propylene oxide). Thermal stability of these proteins, followed by means of high sensitivity DSC, was found to be mostly unaffected by the presence of these polymers. Chromatographic experiments (reverse-phase HPLC and size exclusion chromatrography) did not reveal any sign of specific interaction for these mixtures, either. Isothermal titration calorimetry revealed an increase in enthalpy for the mixtures, represented by a positive enthalpy of transfer for these proteins from buffer to polymer solutions. Moreover, SAXS analyses confirmed that at ambient temperatures these polymers do not affect lysozyme structure. In summary, no evidence is found to support earlier suggestions that some kind of complex could be formed between these proteins and poly(ethylene oxide) or its copolymers, but the present results suggest the occurrence of entropically driven hydrophobic effects.     

Fibril formation of lysozyme upon interaction with sodium dodecyl sulfate at pH 9.2

Fibril formation seems to be a general property of all proteins. Its occurrence in hen or human lysozyme depends on certain conditions, namely acidic pHs or the presence of some additives. This paper studies the interaction of lysozyme with sodium dodecyl sulfate (SDS) at pH 9.2, using UV-visible spectrophotometry, circular dichroism (CD) spectropolarimetry, electron microscopy (EM) and chemometry. Based on observations such as the strange increase in absorbance at 650nm (pH 9.2) and the presence of intermediates, it is assumed that lysozyme fibrils have been formed at pH 9.2 in the presence of SDS as an anionic surfactant. Thioflavin T emission fluorescence and an EM image confirmed this assumption. beta-cyclodextrin was then used as a turbidity inhibitor to establish its effect on the distribution of intermediates that participate in fibril formation.     

Circular dichroism and fluorescence spectroscopy studies of the effect of cyclodextrins on the thermal stability of chicken egg white lysozyme in aqueous solution

Circular dichroism spectroscopy revealed that the thermal stability of chicken egg white lysozyme in an aqueous buffer solution is significantly lowered by the addition of 6-O-α-d-glucosyl-β-cyclodextrin (G1-β-CD), whereas it is raised by the addition of methyl α-d-glucopyranoside. The α- and γ-cyclodextrin also lowered the thermal stability, although the effects were less prominent than that of G1-β-CD. Fluorescence spectroscopy suggested that cyclodextrins include the side chains of tryptophan residues within their internal cavities to lower the thermal stability of lysozyme. The fluorescence intensity of a sample, re-cooled to 25 °C after thermal denaturation at 75 °C in the presence of G1-β-CD, was stronger than that observed for native lysozyme. The fact that the fluorescence intensity of the re-cooling sample was stronger than that of the native one indicates that G1-β-CD persists in binding to the side chains of tryptophan residues of the re-cooled lysozyme.