Thermal Denaturation Of Lysozyme Research Articles

Taurine Stabilizing Effect on Lysozyme.

Taurine is an important organic osmolyte in mammalian cells, and it weakens inflammation and oxidative stress mediated injuries in some diseases. Recently, taurine has been demonstrated to play a therapeutic role against neurodegenerative disorders, although its parallel involvement in several biochemical mechanisms makes not clear taurine specific role in these diseases. Furthermore, the stabilizing effect of this molecule in terms of protein stability is known, but not deeply investigated. In this work we explore by Circular Dichroism the stabilizing impact of taurine in lysozyme thermal denaturation and its influence in lysozyme aggregation into amyloid fibrils. Taurine even at low concentration modifies protein-protein interactions in lysozyme native state, as revealed by Small Angle X-ray Scattering experiments, and alters the amyloid aggregation pattern without completely inhibiting it, as confirmed by UV/Vis spectroscopy with Congo Red and by Atomic Force Microscopy. Evaluation of the cytotoxicities of the amyloid fibrils grown in presence or in absence of taurine is investigated on SH-SY5Y neuroblastoma cells.

Monitoring of lysozyme thermal denaturation by volumetric measurements and nanoDSF technique in the presence of N-butylurea

The results of thermal studies of denaturation of hen egg white lysozyme (HEWL) in water and an aqueous solution of N-butylurea (BU) are presented. High-precision densimetric measurements were used to characterize and analyze the changes of the specific volume, v, during temperature elevation. The temperature of the midpoint of protein denaturation was also determined by nanoDSF technique (differential scanning fluorimetry). The densities of lysozyme solutions were measured at temperatures ranging from 298.15 to 353.15 K with an interval of 5 K at atmospheric pressure (0.1 MPa). The concentration of the protein covered the range from 2 to 20 mg per 1 ml of the solution. The optimal range of the concentration for the densimetric measurements was roughly estimated. In the transition region, the structural changes of the protein are accompanied by the biggest increase of ν values with temperature. Our measurements show that this effect can be monitored from volumetric data without precise determination of protein concentration. The results prove that a two-state model of denaturation could be used for data interpretation. Contrary to common misconception, the volumetric measurements suggest that the denatured protein does not necessarily need to be in a fully extended state. In this way, the ‘protein volume paradox’ could be explained. The surface area of the protein remains unchanged and thus the increase of the specific volume of the protein is relatively small. Additionally, the self-stabilizing effect of the protein in BU solution was reported. For the HEWL in pure water, this phenomenon was not observed.

A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme

The influence of HPβCD on the thermal denaturation of lysozyme was analyzed mainly from microcalorimetry and Raman investigations carried out in the molecular fingerprint and the low-frequency regions. It was shown that Raman spectroscopy investigations performed on a wide spectral range give the opportunity to describe the influence of HPβCD on the mechanism of protein denaturation. Using D2O as solvent allowed us to show that HPβCD mainly destabilizes the tertiary structure of lysozyme by enhancing the protein flexibility and thus inducing the destabilization of the secondary structure. Principal components analysis (PCA) was used for spectra treatment, providing important information about inclusion complex formation between protein hydrophobic residues and CDs molecules. Combining PCA and classical technics (curve fitting) of data analysis allowed a better understanding of the influence of HPβCD on the protein denaturation that seems to be related to the CDs capacity to form inclusion complex. It was observed that these interactions prevent the formation of new strong H-bonds between β-sheet structures thereby inhibiting protein aggregation. This study reveals that CDs are promising systems for inhibiting protein aggregation without protein denaturation, only if designing derivative CDs is carefully controlled.

The effect of cations on reversibility and thermodynamic stability during thermal denaturation of lysozyme

Abstract Differential scanning calorimetry (DSC) has been used to investigate the role that cations of different salts (CaCl2, MgCl2, NH4Cl, NaCl and KCl) play on the thermodynamic stability and reversibility of the lysozyme’s folding-unfolding process. In this work, thermodynamic parameters such as enthalpy change ΔΗ, apparent heat capacity change (ΔCp) and the melt transition temperature (Tm) were obtained in order to explore the effects of various cations on the thermodynamic stability and thermostability of lysozyme, reflected on the Gibbs free energy change and the melting temperature of the transition. Furthermore, the reversibility index (RI) has been calculated in order to study the reversibility of the process and the effect of cations on protein aggregation. Under all conditions studied, lysozyme undergoes reversible thermal unfolding that can be well represented by a reaction of the form N ⇌ U indicating that the unfolding is a two-state process for the first and the second heating runs that have been used in current experimental analysis. The results of those experiments clearly show that cations destabilize the lysozyme and increase the reversibility of the unfolding process.

Carbonyl-based blue autofluorescence of proteins and amino acids.

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method

ABSTRACTThermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

Thermal Denaturation of Lysozyme in Aqueous Solution with Ionic Liquids

Thermal Denaturation of Lysozyme in Aqueous Solution with Ionic Liquids

Analysis of Bulk and Hydration Water During Thermal Lysozyme Denaturation Using Raman Scattering

We describe a method for analyzing protein hydration by Raman spectroscopy on the model protein lysozyme. The analysis of the protein hydration shell is made possible by dissolving the protein in D2O, providing via isotopic exchange the uncoupled O – H stretching spectrum of water molecules early bound to the protein, which are thereafter spread into the solvent. The spectrum of the hydration water can be obtained by subtracting the spectrum of the contribution of D2O from that of the aqueous lysozyme solution in the intramolecular O – D stretching vibrations region (2,200–2,800 cm−1). Raman investigations were simultaneously carried out in the amide I region (1,500–1,800 cm−1) and in the O – D/H stretching spectrum (3,200–3,800 cm−1) during thermal denaturation of lysozyme, to analyze structural changes of the protein in relation to the physical properties of hydration water. It was found that the H-bond network of hydration water is slightly distorted compared to the bulk water at room temperature, with a loss of the tetrahedral local order. The difference between hydration and bulk water is significantly enhanced at T = 90 °C in the denaturated state of the protein. The quantification of water molecules in direct interaction with the protein provides the temperature dependence of the solvent-accessible surface area during the denaturation process. Both kinds of information on hydration water and protein structure lead to a detailed description and overall understanding of the mechanism of protein denaturation.

Structural studies on the interaction of nano-SiO2 with lysozyme

The interaction between nano-SiO2 and lysozyme was investigated by the method of UV-Visible detection and fluorescence spectroscopic techniques. The thermal denaturation of lysozyme has been investigated in the presence and absence of nano-SiO2 over the temperature range (293-373) K in different buffers and pH values, using temperature scanning spectroscopy. The presence of nano-SiO2 caused the destabilization of lysozyme resulting in a decrease in the temperature of unfolding with an increase in nano-SiO2 concentration.

Anomalous behavior of Brillouin light scattering at thermal denaturation of lysozyme

The thermal denaturation of lysozyme heated from 293 to 355 K has been studied using Brillouin light scattering. An anomalous temperature behavior of the velocity and damping of hypersound, which is accompanied by a decrease in the intensity of the Brillouin components in the experiments with the 180° scattering geometry and almost complete their disappearance in the case of the 90° scattering geometry, has been observed at the sol-gel transition in the vicinity of 343 K. Such anomalies in the light scattering spectra are absent for a sodium acetate buffer used to prepare protein solutions. A mechanism describing the behavior of the intensities of the Brillouin components has been proposed.

Spectroscopic Studies on the Interaction of Nano-TiO2 with Lysozyme

In the present study, the interaction between nano-TiO2 and lysozyme was investigated by the method of UV-Vis detection and fluorescence spectroscopic techniques. The thermal denaturation of lysozyme has been investigated in the presence and absence of nano-TiO2 over the temperature range (293-373) K in different buffer and pH, using temperature scanning spectroscopy. The presence of nano-TiO2 caused the destabilization of lysozyme resulting in a decrease in the temperature of unfolding with an increase in nano-TiO2 concentration.

Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to examine the structure of lysozyme and its charge dependence on pH. Student groups are then assigned one of four 45 mM sodium acetate buffers: pH 3.6 with either 0 or 60 mM NaCl or pH 4.6 with either 0 or 60 mM NaCl. Using their assigned buffers, student groups determine the second virial coefficient of lysozyme using laser light scattering and gel permeation chromatography. Student groups also determine the van't Hoff enthalpy of unfolding by UV absorbance thermal denaturation and compare their results to the van't Hoff enthalpy and calorimetric enthalpy of unfolding determined by differential scanning calorimetry. We stress the use of complementary techniques to determine these thermodynamic variables for student validation of experimental results. Students present their results in a peer reviewed, journal-style report and, using the data from the entire class, must determine the dependence of the lysozyme second virial coefficient and unfolding enthalpies on pH and ionic strength. Upon completion of these experiments, students are anticipated to appreciate the pH dependence of protein charge and screening of protein electrostatic repulsion with ionic strength.

Study on Thermal Denaturation of Lysozyme by DSC

The thermal denaturation of solid lysozyme and the effects of denaturants and their concentrations on denaturation of lysozyme in aqueous solutions were studied by differential scanning calorimetry (DSC). The results showed that both denaturation temperature and denaturation enthalpy of lysozyme decreased as the existence of water and the addition of urea and guanidine hydrochloride (GuHCl). In addition, denaturation temperature and denaturation enthalpy decreased as the concentrations of urea and GuHCl increased. As a denaturant, GuHCl is more effective than urea due to its added ability of electrostatic interaction.

Analysis of Sugar Bioprotective Mechanisms on the Thermal Denaturation of Lysozyme from Raman Scattering and Differential Scanning Calorimetry Investigations

Sugar-induced thermostabilization of lysozyme was analyzed by Raman scattering and modulated differential scanning calorimetry investigations, for three disaccharides (maltose, sucrose, and trehalose) characterized by the same chemical formula (C(12)H(22)O(11)). This study shows that trehalose is the most effective in stabilizing the folded secondary structure of the protein. The influence of sugars on the mechanism of thermal denaturation was carefully investigated by Raman scattering experiments carried out both in the low-frequency range and in the amide I band region. It was determined that the thermal stability of the hydrogen-bond network of water, highly dependent on the presence of sugars, contributes to the stabilization of the native tertiary structure and inhibits the first stage of denaturation, that is, the transformation of the tertiary structure into a highly flexible state with intact secondary structure. It was found that trehalose exhibits exceptional capabilities to distort the tetra-bonded hydrogen-bond network of water and to strengthen intermolecular O-H interactions responsible for the stability of the tertiary structure. Trehalose was also observed to be the best stabilizer of the folded secondary structure, in the transient tertiary structure, leading to a high-temperature shift of the unfolding process (the second stage of denaturation). This was interpreted from the consideration that the transient tertiary structure is less flexible and inhibits the solvent accessibility around the hydrophobic groups of lysozyme.

Sugar bioprotective effects on thermal denaturation of lysozyme: Insights from Raman scattering experiments and molecular dynamics simulation

The bioprotective properties of trehalose, sucrose and maltose on lysozyme protein are studied using Raman spectroscopy and molecular dynamics simulation. The temperature dependencies of the α-helical unfolding processes are determined in presence of different sugars and at various concentrations using the amide I band. The energies of stabilization and acting forces are discussed. The problem of the threshold sugar concentration needed to protect proteins is addressed. The results point out that the three sugars exhibit very similar bioprotective behaviors in the investigated temperature and concentration ranges and suggest that the difference between their respective effects arise mostly from the different denaturated states of lysozyme in the three sugar solutions. Indeed, trehalose gives rise to a significantly more important shift of the denaturation temperature Tm and of the Gibbs net free energy Δ(ΔGND) of stabilization of lysozyme with respect to the two other sugars.

Thermodynamic study of solvation of some amino acids, diglycine and lysozyme in aqueous and mixed aqueous solutions

Apparent molar adiabatic compressibilities and viscosities of glycine, dl-α-alanine, dl-α-amino- n-butyric acid, l-valine, l-leucine and diglycine have been determined in aqueous and mixed aqueous solutions of m B =1.0, 2.0, 3.0, 4.0 and 5.0 aqueous n-propanol solutions at 298.15 K. From these data the partial molar adiabatic compressibilities and viscosity B-coefficients have been evaluated to calculate the corresponding transfer functions. The partial molar adiabatic compressibilities of transfer at infinite dilution Δ tr K° 2,S for all the studied model compounds are positive and increase with the concentration of n-propanol. Positive and negative B-coefficients of transfer Δ tr B have been observed for the studied amino acids in lower and in higher concentration of n-propanol, respectively. The activation free energy for viscous flow in aqueous and mixed aqueous n-propanol solutions has been calculated from B-coefficient and partial molar volume data. Hydration numbers and interaction coefficients have also been calculated from these data. These parameters have been discussed in terms of solute–cosolvent interactions. Thermal denaturation of lysozyme has also been studied using UV-visible spectrophotometer in aqueous and in mixed aqueous solutions of n-propanol, 1,2-propandiol and glycerol. The thermodynamic parameters accompanying the thermal denaturation have been evaluated. The results have been explained on the basis of competing patterns of interactions of the cosolvents with the native↔denatured reaction. The preferential interaction parameters have been calculated from these thermodynamic data and by correlating the surface tension data of n-propanol and 1,2-propandiol to the surface area of the protein. Some parallelism in the patterns of interactions has been observed for the studied model compounds and protein in the aqueous solutions of these solvents.

The Endothermic Effects during Denaturation of Lysozyme by Temperature Modulated Calorimetry and an Intermediate Reaction Equilibrium

To gain insight into the thermodynamics of protein denaturation, the complex heat capacity, Cp* (= Cp‘ − iCp‘ ‘) of lysozyme−water system has been measured at pH 2.5 in the 293−368 K range by using temperature-modulated scanning calorimetry (TMSC), a technique in which the thermally reversible enthalpy changes are measured separately and simultaneously with the thermally irreversible enthalpy changes. The plot of Cp‘ against the temperature T shows a broad peak, which is similar to that observed in Cp,DSC, measured here and elsewhere by differential scanning calorimetry (DSC), a technique which gives the sum of both the reversible and irreversible contributions in the apparent heat capacity value. This peak in Cp,DSC has been generally attributed to endothermic heat absorption on reversible and irreversible unfolding processes and irreversible thermal denaturation. It is shown that the observed Cp‘ peak results from heat absorption when the equilibrium constant between the native lysozyme state and a conformationally different intermediate state increases with T. The plot of Cp‘ versus T is subdivided into four regions, corresponding to the dominance of a certain process. Thermal denaturation of lysozyme was found to occur according to a scheme, native state ↔ unfolded (intermediate) state → denatured state. This conclusion is consistent with the general view that the first step of denaturation of small one-domain globular protein like lysozyme is a reversible conformational (unfolding) transition, and the second step is irreversible denaturation. It is shown that when kept isothermally at T > 341 K, i.e., within the transition temperature range, Cp‘ of lysozyme decreases. This decrease is exponential in time and corresponds to a rate constant, which varies according to the Arrhenius-type equation, with a preexponential factor of 5 × 1020 s-1 and energy of 167 kJ/mol. The overall kinetics of the denaturation reaction is of the first order.

Thermodynamic Analysis of Proteins Adsorbed on Silica Particles: Electrostatic Effects

Electrostatic effects on protein adsorption were investigated using differential scanning calorimetry (DSC) and adsorption isotherms. The thermal denaturation of lysozyme, ribonuclease A (RNase), and α-lactalbumin in solution and adsorbed onto silica nanoparticles was examined at three concentrations of cations: 10 and 100 mM of sodium and 100 mM of sodium to which 10 mM of calcium was added. The parameters investigated were the denaturation enthalpy (ΔH), the temperature at which the denaturation transition was half-completed (Tm), and the temperature range of the denaturation transition.For lysozyme and RNase, adsorption isotherms depend strongly on the ionic strength. At low ionic strength both proteins have a high affinity for the silica particles and adsorption is accompanied by a 15–25% reduction in ΔH and a 3–6°C decrease in Tm, indicating that the adsorbed state of the proteins is destabilized. Also, an increase in the width of the denaturation transition is observed, signifying a larger conformational heterogeneity of the surface bound proteins. At higher ionic strengths, both with and without the addition of calcium, no significant adsorption-induced alteration in ΔH was observed for all three proteins. The addition of calcium, however, decreases the width of the denaturation transition for lysozyme and RNase in the adsorbed state.

Synergetic effect of polyols with tetrabutylammonium bromide and urea on the thermal stability of lysozyme

Abstract The thermal denaturation of lysozyme was studied in 2.0 molal aqueous solutions of polyols at pH 2.50 and varying concentration of glycerol at pH 2.50 and 6.00 using differential scanning calorimetry (DSC). The transition temperature, heat capacity, enthalpy, entropy and free energy of stabilization have been determined by a least square fit of the excess heat capacity data to the two-state model. Polyols are found to stabilize lysozyme and the stabilization increases with an increase in the number of hydroxyl groups. The stabilization increases with an increasing concentration of glycerol. The stabilization has been explained in terms of preferential hydration or due to the strengthening of the water structure which in turn intensify the hydrophobic interactions of the protein. The calorimetric studies have been done on the thermal denaturation of lysozyme in the presence of 0.5 m tetrabutylammonium bromide (Bu 4 NBr) + 2.0 m polyols at pH 2.50 and 0.5 m Bu 4 NBr or urea + varying concentration of glycerol (0.0–10.0m) at pH 2.50 and 6.00. The effect is found to be nearly additive in the case of Bu 4 NBr and polyols. The results on the combined systems of Bu 4 NBr and varying concentration of glycerol shows that effect is additive at low pH but not at high pH. This has been explained in terms of predominance of the enthalpic contribution of glycerol in comparison to the entropic contribution of Bu 4 NBr. The comparison of the combined effect of 0.5 m urea + glycerol and 0.5 m Bu 4 NBr + glycerol indicates that Bu 4 NBr is a stronger destabilizer than urea.

Effect of Aqueous Alcohol Solutions on the Thermal Transition of Lysozyme: A Calorimetric Study

Thermal denaturation of lysozyme has been studied at pH = 3 in water/ethanol and water/tert-butyl alcohol mixtures in the water rich region of composition (mole fraction of cosolvent x2 < 0.12) by high-sensitivity differential scanning calorimetry. The results show that on increasing alcohol concentration, the enthalpy and entropy of denaturation of lysozyme first reach a maximum at an intermediate composition x2 = typical for each alcohol ( ≅ 0.06 for ethanol and ≅ 0.02 for tert-butyl alcohol) and then decrease with increasing x2. In addition, two enthalpy−entropy compensation patterns each having its own compensation temperature (Tc) clearly appear from the data: a compensation data line obtained with rising x2 in the 0− range (with Tc = 281 ± 6 K) followed by a compensation line with Tc = 403 ± 14 K after x2 passes the value. The value of is close to that at which a change in the nature of solvent component interaction occurs as inferred from compressibility and IR absorption measurements. The data have been interpreted on the basis of the assumption that the addition of short chain alcohols affects the thermal transition of proteins, modifying the extent of enthalpy and entropy contribution associated with structural reorganization of water in the unfolding process.