Denaturation Of Lysozyme Research Articles

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.

Effect of DMSO on Denaturation of Hen Egg-white Lysozyme in Aqueous Solution

A study on thermal denaturation of hen egg-white lysozyme(HEWL)was carried out by differential scanning calorimetry(DSC)and modulated temperature differential scanning calorimetry(MDSC).It was found that DSC profiles of HEWL were dependent on heating rates and concentrations of enzyme as well as cosolvent.The enthalpy of denaturation(ΔHm)increased with increasing enzyme concentration in aqueous solution.In aqueous solution containing dimethylsolfoxide(DMSO),denaturation temperature(Tm)shifted toward lower temperature,and the transition peak became shallower and broader with increasing volume proportion of DMSO.When the content of DMSO was more than 70%,only a declining smooth line could be observed instead of a single endothermic transition curve.Interestingly,from MDSC curves of HEWL in aqueous solution without DMSO,a small and symmetrical peak in front of the main transition(I)was observed marginally,which was regarded as a pre-transition(II).Specially,this small and marginal peak could not be found in aqueous solution containing DMSO.In addition,with increasing amount of protein,Tm(II)shifted toward lower temperature,and ΔHm(II)decreased along with the temperature gap between two transitions lengthened.The results were considered to be due to the reversible dissociation of HEWL dimers in aqueous solution.

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.

Solvent‐induced lysozyme gels: Effects of system composition and temperature on structural and dynamic characteristics

The gelation process of lysozyme in water/tetramethylurea in the presence of salt was investigated as a function of temperature and system composition by rheology, infrared spectroscopy, and microcalorimetry. Times and temperatures of gelation were determined from the variation of the storage (G') and loss (G'') moduli. It was found that gelation times follow exponential decays with both protein and tetramethylurea (TMU) concentrations and with temperature. The activation energy for the overall process shows a linear dependence on TMU mass fraction. A strongly increased beta-sheet content and reduced alpha-helix occur with the increase of TMU concentration in the binary solvent. Also, a linear decrease of lysozyme denaturation temperature and enthalpy on TMU concentration is found for the TMU mass fraction up to 0.5, above which no denaturation signal can be detected.

Hydration of Thermally Denatured Lysozyme Studied by Sorption Calorimetry and Differential Scanning Calorimetry

We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90 degrees C, in the other procedure a sample that contained 20% of water was denatured at 150 degrees C. The protein denatured at 90 degrees C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150 degrees C were studied at several temperatures in the range of 25-60 degrees C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.

Rewetting Drops Containing Surface Active Agents Improve the Clinical Performance of Silicone Hydrogel Contact Lenses

The purpose of this study was to investigate the impact of using a rewetting drop (RWD) containing surface active agents (OPTI-FREE RepleniSH; Alcon, Fort Worth, TX) on the clinical performance and protein deposition when using a continuous-wear (CW) silicone hydrogel (SH) contact lens. Subjects wore lotrafilcon A SH lenses on a 30-day CW basis for two consecutive 1-month periods while inserting either 0.9% unpreserved unit-dose saline (control) or multidose OPTI-FREE RepleniSH (test RWD). Subjective comfort and symptoms were assessed after 2 and 4 weeks with each product. After 1 month of wear with each product, lenses were collected and analyzed in the laboratory for total protein, total lysozyme, and percentage of denatured lysozyme. Symptoms of dryness and comfort varied across the day regardless of drop type (p < 0.001) with dryness being maximal on waking, least in the middle of the day, and increased towards the evening. The test RWD provided greater comfort on insertion (p = 0.02), better visual quality (p < 0.01), and less mucous discharge on waking (p = 0.02) than the control product. Lysozyme deposition was significantly reduced after the use of the test RWD as compared to saline (0.73 +/- 0.5 microg/lens vs. 1.14 +/- 0.7 microg/lens; p < 0.001) as was total protein deposition (1.17 +/- 0.7 microg/lens vs. 1.86 +/- 0.8 microg/lens; p < 0.001). Lysozyme denaturation was also reduced with the use of the test RWD compared with the control (76 +/- 10% vs. 85 +/- 7%; p < 0.01). The use of a RWD containing surface active agents provided greater subjective satisfaction, reduced lysozyme and total protein deposition, and reduced denatured lysozyme than a RWD containing saline alone.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Denaturation of Lysozyme and Myoglobin in Laser Spray

In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO(2) laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of approximately 2 W and a focal spot size ( approximately 0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was approximately 3 x 10(3) W/cm(2). This resulted in a quiescent and smooth vaporization of aqueous solutions. This "evaporation-mode" laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to approximately 2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds.

Effect of starch on the thermal kinetics and transmittance properties of lysozyme

Lysozyme is being tested for use as a preservative in the food industry. The interaction between starch and lysozyme will help in recommending starch or starch fractions as carriers for lysozyme. The effect of starch fractions on the folding and unfolding of lysozyme was estimated by differential scanning calorimetry (DSC), kinetics and transmittance turbidometry. Lysozyme was unfolded (heated to 90 °C) and folded (cooled to 20 °C) five times in the presence of starch fractions. Starch was added at 1 and 2%. Overall, a trend of higher onset temperature (To) values occurred at 2% addition of all starch fractions except amylose. The increase in the number of cycles influenced the effect of starch on lysozyme denaturation. The percentage of lysozyme's ΔH values decreased as a new heating and cooling cycle was performed (ie 74.4% of the ΔH remained from the first cycle). The effect of amylose (AM) and amylopectin (AP) on the kinetics of lysozyme unfolding and folding was found to be different based on the assumption that the peak DSC temperature is the fastest step of the reaction. The unfolding showed higher activation energy (Ea) in the presence of both AM and AP, while the folding was not significantly changed. The turbidity of the solution containing lysozyme and potato starch showed transmittance in between that of lysozyme and starch. Stirring of the blend kept the transmittance unchanged while an increase in the transmittance was noticed when stirring ceased. Copyright © 2004 Society of Chemical Industry

Effect of hydrocolloids on the thermal denaturation of proteins

The thermal denaturation of bovine serum albumin (BSA), lysozyme and whey protein isolate (WPI) in the presence of hydrocolloids (pectin, guar gum, ι-carrageenan) was investigated. A decrease in the thermal stability of lysozyme was observed in the mixture of protein with ι-carrageenan. The increase in the enthalpy of denaturation (Δ H) of BSA and lysozyme in the presence of hydrocolloids was attributed to the protection of globular proteins against aggregation through blockage of their hydrophobic binding sites by the bulky polysaccharide moeity. Biopolymers had a stabilizing effect on WPI. The thermal stability was the highest in the presence of pectin, whereas the lowest transition temperature was observed in the presence of guar gum. A single transition peak was observed for pure WPI. However, WPI exhibited two transition temperatures when together with pectin and i-carrageenan. WPI was stable against heat denaturation at acidic pH values (pH 4.0), while it was denatured at a low temperature at an alkaline pH (pH 9.0) in the presence of pectin. This was attributed to the formation of extra hydrogen bonding. The increase in the concentration of pectin has little affect on the heat stability of WPI; however, it reduces the cooperativity of transition.

Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme.

The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation.

Interaction of 2,2,2-trifluoroethanol with proteins: calorimetric, densimetric and surface tension approach

The thermal denaturation of hen egg-white lysozyme was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using micro differential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions were evaluated. It is observed that thermal unfolding of lysozyme in the presence of TFE upto a concentration of 4.0 mol dm −3 follows a two-state denaturation mechanism as indicated by the equality of van't Hoff and calorimetric enthalpies. The finer details of interaction were studied by measuring the partial molar volume of some constituent amino acids and glycine peptides from water to aqueous TFE at 298.15 K. The physico-chemical properties of aqueous TFE: apparent molar heat capacities, apparent molar volumes and surface tension were measured to understand the intrinsic properties of the cosolvent as well. From the correlation among the thermal unfolding data on lysozyme in aqueous TFE, calculated preferential interaction parameters, physico chemical properties of aqueous TFE and partial molar volumes of transfer, it is concluded that both solvent mediated effect and direct interaction constitute the mechanism of TFE–protein interactions.

Influence of urea on thermal denaturation of lysozyme investigated by optical and dielectric spectroscopies

Different structural probes following the unfolding process of a protein may evidence the existence of stable intermediates from the differences in the unfolding curves. In this work, we analyze the thermal unfolding of chicken egg lysozyme in the presence of 8 M urea at pH 5 combining dielectric spectroscopy in the radiofrequency region, UV absorbance and circular dichroism in the far and near UV regions. The data are consistent with a two-state model of unfolding equilibrium and do not provide support for the presence of a significantly populated intermediate state.

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.

Preferential interactions of urea with lysozyme and their linkage to protein denaturation

The interactions involved in the denaturation of lysozyme in the presence of urea were examined by thermal transition studies and measurements of preferential interactions of urea with the protein at pH 7.0, where it remains native up to 9.3 M urea, and at pH 2.0, where it undergoes a transition between 2.5 and 5.0 M urea. The destabilization of lysozyme by urea was found to follow the linear dependence on urea molar concentration, M u , Δ G u o =Δ G w o −2.1 M u , over the combined data, where Δ G u o and Δ G w o are the standard free energy changes of the N⇌D reaction in urea and water, respectively. Combination with the measured preferential binding gave the result that the increment of preferential binding, δΓ 23=Γ 23 D −Γ 23 N , is also linear in M u . A temperature dependence study of preferential interactions permitted the evaluation of the transfer enthalpy, Δ H ̄ 2, tr o , and entropy, Δ S ̄ 2, tr o of lysozyme from water into urea in both the native and denatured states. These values were found to be consistent with the enthalpy and entropy of formation of inter urea hydrogen bonds (Schellman, 1955; Kauzmann, 1959), with estimated values of Δ H ̄ 2, tr o =ca. −2.5 kcal mol −1 and Δ S ̄ 2, tr o =ca. −7.0 e.u. per site. Analysis of the results led to the conclusion that the stabilization of the denatured form was predominantly by preferential binding to newly exposed peptide groups. Combination with the knowledge that stabilizing osmolytes act by preferential exclusion from peptide groups (Liu and Bolen, 1995) has led to the general conclusion that both the stabilization and destabilization of proteins by co-solvents are controlled predominantly by preferential interactions with peptide groups newly exposed on denaturation.

Ac nanocalorimeter for measuring heat capacity of biological macromolecules in solution

A precise calorimeter has been developed to measure the heat capacity of a small amount of liquid using a novel ac calorimetric method in which the effect of heat loss from a sample cell is corrected using the phase of the ac temperature. The sample cell is made of a fine glass tube, whose outer surface is plated with a nickel film as an ac heater. The ac temperature of the sample is detected precisely with a microbead thermistor attached to the middle of the tube. The resistance of the thermistor is measured with an ac Wheatstone bridge which is composed of resistors with a low temperature coefficient of ±1 ppm/K. The unbalance ac signal of the bridge is measured with a lock-in amplifier. To reduce the drift of measured values caused by the variation of room temperature, the amplifier and measuring instruments with temperature coefficients of &amp;gt;1 ppm/K are kept at constant temperature. Moreover, the gain of the amplifier is calibrated at every measuring point. Consequently, the heat capacity of 10 μl liquid can be determined with an extremely high sensitivity of ±0.001%, which corresponds to heat capacity changes of ±300 nJ/K. A test of the performance was made of the heat capacity measurement during thermal denaturation of lysozyme dissolved in buffered solution. This method is particularly useful for studying thermal properties of biological macromolecules in solution, since the heat capacity of macromolecules can be measured with a 10 μg sample.

Antimicrobial Properties of Lysozyme in Relation to Foodborne Vegetative Bacteria

The purpose of this review is to describe the antibacterial properties and mode of action of lysozyme against Gram-positive and Gram-negative bacteria, and to provide insight in the underlying causes of bacterial resistance or sensitivity to lysozyme. Such insight improves our understanding of the role of this ubiquitous enzyme in antibacterial defense strategies in nature and provides a basis for the development and improvement of applications of this enzyme as an antibacterial agent. The bactericidal properties of lysozyme are primarily ascribed to its N-acetylmuramoylhydrolase enzymic activity, resulting in peptidoglycan hydrolysis and cell lysis. However, an increasing body of evidence supports the existence of a nonenzymic and/or nonlytic mode of action. Because Gram-negative bacteria, including some major foodborne pathogens, are normally insensitive to lysozyme by virtue of their outer membrane that acts as a physical barrier preventing access of the enzyme, several strategies have been developed to extend the working spectrum of lysozyme to Gram-negative bacteria. These include denaturation of lysozyme, modification of lysozyme by covalent attachment of polysaccharides, fatty acids and other compounds, attachment of C-terminal hydrophobic peptides to lysozyme by genetic modification, and the use of outer membrane permeabilizing agents such as EDTA or polycations or permeabilizing treatments such as high hydrostatic pressure treatment.

The free energy of denaturation of lysozyme: An undergraduate experiment in biophysical chemistry*

AbstractAn undergraduate laboratory experiment to measure the free energy of denaturation of the enzyme lysozyme is described. The fluorescence of the protein in solutions of varying concentrations of urea and guanidine hydrochloride is measured. The data are analyzed graphically assuming a two‐state model for the denaturation.

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.