Denaturation Enthalpy Research Articles

Effect of crosslinking method on biological properties of grass carp skin collagen sponges

In this study,the effect of crosslinking method on biological properties of grass carp skin collagen sponge was discussed.Collagen was extracted from skin of grass carp(Ctenopharyngodon idellus)and collagen sponge was prepared from this collagen.Then,this collagen sponge was crosslinked with different methods,such as UV,dehydrothermal,EDC/NHS and glutaraldehyde crosslinking processes.At the same time,the biological and mechanical properties of those collagen sponges,including degree of crosslinking,denaturation temperature,tensile strength and enzymatic sensitivity in vitro,were evaluated and compared.Experiment results indicated that the grass carp skin collagen was type Ⅰ collagen.The degree of crosslinking of different crosslinking methods decreased in the order of glutaraldehyde(72.0%)EDC/NHS(32.5%)dehydrothermal(29.9%)UV(15.6%).Compared with control collagen sponges,the denaturation temperature(67.4 ℃),tensile strength(125.6 kPa)and enzymatic sensitivity in vitro of collagen sponges crosslinked by glutaraldehyde were significantly improved(P0.05);EDC/NHS crosslinking could lead to obvious increasing in denaturation enthalpy(6.86 J/g)and moderate improving on tensile strength(98.6 kPa)and enzymatic sensitivity for collagen sponge(P0.05).The changes of biological and mechanical properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were not notable.The results of FTIR showed that glutaraldehyde crosslinking could improve properties of collagen sponges by forming new covalent bond in triple helix structure of collagen,while EDC/NHS crosslinking do that by forming new hydrogen bonds among collagen molecules.This research shows that glutaraldehyde and EDC/NHS crosslinking could lead to obvious improvement in properties of collagen sponges but the influences on properties of collagen sponges after being crosslinked by dehydrothermal and UV crosslinking processes were limited.

Catalytic activity of copper ions in the amyloid fibrillation of β-lactoglobulin

The self-assembly of proteins and polypeptides in amyloid fibrillar aggregates is rapidly emerging as a promising route towards the fabrication of nano-objects with controlled morphologies and properties. Transition metal ions are known to play an important but elusive role in the amyloid fibrillation associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We have considered the effect of copper ions Cu2+ on the nanoscale morphology and fibrillation kinetics of β-lactoglobulin (βLG), a model protein not related to amyloid diseases, which denatures and self-assembles in nanofibrils upon heating at low pH and ionic strength. We found that an increasing level of Cu2+ decreases the enthalpy of denaturation and significantly increases the rate of fibril nucleation, also producing a small increase of the fibril elongation rate. Cu2+ acts as a catalytic agent during protein denaturation and fibrillation, without binding to βLG before or after heating, and produces only minor changes in fibril morphology. Beside possible implications for amyloid pathologies in vivo, our results suggest that transition metal ions can be used to control the self-assembly of protein-based nano-objects in vitro.

Physicochemical and Functional Properties of Dehydrated Japanese Quail (<i>Coturnix japonica</i>) Egg White

Physicochemical, functional and digestibility analyses were done of dehydrated quail egg white to determine its possible practical applications. Quail egg white was dehydrated by air convection using one of two temperatures and times: M1 (65℃, 3.5 h), M2 (65℃, 5.0 h), M3 (70℃, 3.5 h) and M4 (70℃, 5.0 h). Lyophilized quail egg white was used as a standard. All four air-dried treatments had good protein levels (92.56% to 93.96%), with electrophoresis showing the predominant proteins to be lysozyme, ovalbumin and ovotransferin. Denaturation temperatures ranged from 81.16℃ to 83.85℃ and denaturation enthalpy values from 5.51 to 9.08 J/g. Treatments M1-4 had lower water-holding (0.90 - 2.95 g/g) and oil-holding (0.92 - 1.01 g/g) capacities than the lyophilized treatment (4.5 g/g, 1.95 g/g, respectively). Foaming capacity was pH-dependent in all five treatments, with the lowest values at alkaline pH and the highest (153% to 222%) at acid pH (pH 2). Foam stability was lowest at acid pH and highest at alkaline pH. Emulsifying activity in the air-dried treatments was highest at pH 8 (41% - 46%). Emulsion stability was pH-dependent and highest in M3 between pH 2 and 4 (96.16% to 95.74%, respectively). In the air-dried treatments, in vitro protein digestibility was as high as 83.02% (M3).

La radiothérapie post-chirurgicale précoce augmente la survie sans progression tumorale des patients présentant un méningiome de grade 2

Chitin deacetylase (CDA) with a molecular mass of 53 kDa was purified from Penicillium oxalicum SAEM-51. Optimal temperature and pH of the purified enzyme were 50 °C and 9.0 respectively. Activation energy (Ea,d), free energy (ΔGd*), enthalpy (ΔHd*) and entropy (ΔSd*) for enzyme denaturation at optimal temperature were 114.72 kJ mol−1, 97.86 kJ mol−1, 112.04 kJ mol−1, 43.93 J mol−1 K−1, respectively. Enzyme had the half life of 693.10 min at its optimum temperature. It had notably deacetylated glycol chitin and chitin oligomers having degree of polymerization of more than four. Increased enzyme activity was observed with metal ions mainly Cu2+ and Co2+. No inhibition of the enzyme was observed by the end product i.e. acetate (0–60 mM). Far-UV CD spectroscopic analysis revealed presence of 56.26% α and 15.63% β-helical structures. Significant homology of the enzyme was observed with CDAs from fungal and yeast strains.

The effect of heat treatment on γ-glutamyl transferase activity in non-bovine and bovine milk – A comparative kinetic and thermodynamic investigation

The activities and degree of inactivation of γ-glutamyl transferase (GGT) in raw milk from three species (goat, sheep and cow) were measured in relation to the heating process in order to determine the sustainability of this enzyme as a marker for the evaluation of pasteurization. GGT showed the highest activity in the whole milk, in the following order: cow > sheep > goat milk. Kinetic and thermodynamic studies were carried out in the temperature range of 60–77 °C and showed that the thermal inactivation followed the first-order kinetics. Based on the thermal death time model, decimal reduction time D and inactivation rate constant k values decreased and increased respectively, with increasing temperature, indicating a more rapid inactivation at higher temperatures in whole milk than in skimmed milk. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius and thermal death time models. The corresponding z-values for skimmed and whole milk were 8.02 ± 0.23 °C and 7.09 ± 0.09 °C for goat milk, 5.97 ± 0.08 °C, 5.88 ± 0.027 °C for sheep milk and 5.80 ± 0.05 °C and 5.83 ± 0.01 °C for cow milk respectively. The calculated values for activation energy and change in enthalpy of denaturation suggested that the enzyme is more stable toward thermal denaturation in goat milk.

Structural perturbation of diphtheria toxoid upon adsorption to aluminium hydroxide adjuvant

Aluminium-containing adjuvants are often used to enhance the potency of vaccines. In the present work we studied whether adsorption of diphtheria toxoid to colloidal aluminium hydroxide induces conformational changes of the antigen. Diphtheria toxoid has a high affinity for the aluminium hydroxide particles based on a high adsorption degree, adsorption rate and adsorptive capacity. The conformation and stability of diphtheria toxoid in solution and adsorbed to aluminium hydroxide adjuvant were characterized using five physicochemical techniques: intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, infrared spectroscopy and differential scanning calorimetry. Diphtheria toxoid adsorbed to aluminium hydroxide resulted in a minimal shift of the tryptophan fluorescence spectrum, whereas a large increase in the emission of the Bis-ANS probe was observed, indicating that hydrophobic sites of the protein became accessible due to adsorption. In addition, circular dichroism and infrared spectroscopy revealed that adsorption to aluminium hydroxide caused an increase of β-sheet content and a decrease of α-helix content in diphtheria toxoid. Differential scanning calorimetry demonstrated a major decrease in the enthalpy of denaturation upon adsorption. In conclusion, the adsorption of diphtheria toxoid to aluminium hydroxide adjuvant leads to substantial conformational changes in the antigen. Since physicochemical methods can be used to monitor these conformational changes, these analytical methods might offer a tool in regulatory required vaccine quality control by demonstrating consistency in production.

Trivalent chromium and aluminum affect the thermostability and conformation of collagen very differently

Ultrasensitive differential scanning calorimetry (US-DSC) was used to directly measure the thermal transition temperature and energy change of acid soluble collagen in the presence of Cr3+ and Al3+ sulfates. The behavior of Cr3+ was analogous to kosmotropes in the cation Hofmeister series and increased the stability of collagen in dilute solutions. Meanwhile, the denaturational enthalpy change (ΔH) of collagen was substantially reduced with change to increasing Cr3+ concentration. This is likely due to the uni-point binding of Cr3+ with carboxyl groups of collagen side chains that could decrease the hydrogen-bonding in collagen and result in the increase of protein hydrophobicity. In the case of Al3+, the interactions between the ions and collagen showed very different properties: at low and medium ion concentrations, the stability of the collagen was decreased; however, a further increase of Al3+ concentration led to a salting-out effect of collagen, indicating the Al3+ is a typical chaotropic ion. This striking difference of the two ions in the stabilization of collagen can be explained in terms of the different interactions between the cations and the carboxyl groups of collagen side chains. Additionally, we studied metal ion induced conformational change by the combination of circular dichroism (CD) and atomic force microscopy (AFM). CD measurements revealed that neither metal ion interactions of collagen with Cr3+ nor Al3+ ions destroyed the triple-helical backbone structure of collagen in the solution. AFM results further confirmed that the dehydration of collagen by Cr3+ is more significant than Al3+, thus inducing the aggregation of collagen fibrils.

Thermal stability of yeast hydrolysate as a novel anti-obesity material

We examined the thermal stability of yeast hydrolysates before and after ultrafiltration (UF) in vitro, and the anti-obesity activity of yeast hydrolysates before and after heat treatment in vivo. Yeast hydrolysate after UF showed significantly higher thermal stability than before UF. Yeast hydrolysates before and after UF showed 3 and 4 thermal transition peaks in their thermograms, respectively, and the total thermal denaturation enthalpies of yeast hydrolysates before and after UF were 69.5 and 36.5J/g, respectively. For the anti-obesity activity study, yeast hydrolysates before and after heating were administered ad libitum with water to 7-week-old male SD rats. The administration of yeast hydrolysate (YH-control; no heat treatment, YH-1; heat treatment at 140°C, and YH-2; heat treatment at 160°C) significantly increased mRNA expression of cocaine- and amphetamine-regulated transcript (CART) compared with control rats (saline administration). However, there was no significant difference between the heat-treated groups and YH-control and there was no significant difference in neuropeptide Y expression between the heat-treated groups and YH-control. These results suggest that yeast hydrolysate can be use an anti-obesity material after heat treatment.

Effect of high pressure treatment on ovotransferrin

High pressure processing of ovotransferrin was carried out to study the structural and physiochemical changes of ovotransferrin under various pressure levels. At pH 8 and pressures higher than 200MPa, a decrease in total sulfhydryl groups and an increase in surface hydrophobicity were observed along with a partial aggregation. A gradual shift of denaturation peak towards higher temperature was noticed up to 500MPa, leading to a total loss of the enthalpy of denaturation at pressures of 600 and 700MPa, where a significant decrease in intrinsic fluorescence was also observed. At pH 3, the ovotransferrin adopted a molten globule state, associated with a significant increase in surface hydrophobicity and reactive sulfhydryl content; structurally, no clear denaturation peaks in differential scanning calorimetry (DSC) were detected at any level of pressure treatment whereas a noticeable decrease in intrinsic fluorescence was evidenced up to 600MPa and then increased at 700MPa pressure treatment. Fourier transform infrared spectroscopy (FT-IR) revealed that the conformational structure were changed from helices, sheets, turns, and aggregated strand to mostly intermolecular β-sheets or aggregated strands at pH 8 at 200MPa but switched back to original structure at higher pressures.

Rheological properties and protein quality of UV-C processed liquid egg products

Rheological properties define the functionality and consequently, the technological quality, of food proteins. This study deals with the effects of short-wave ultraviolet treatments (dynamic or static UV-C) on the dynamic viscosity, flow behaviour, temperature-dependent viscosity, thermal properties and electrophoretic patterns of liquid egg products. Remarkably, none of the investigated parameters have been significantly influenced by UV-C treatments, static or dynamic. Rheological data, and thermal and electrophoretic properties indicate that the flow behaviour of UV-C treated egg white, whole egg or egg yolk, has not varied, and relevant protein denaturation or aggregation could be discarded. On the contrary, heat pasteurizations caused an increase on the viscosity-shear rate dependency, and also a certain degree of coagulation could be observed in the flow behaviour diagrams. Pasteurization additionally caused a reduction on the denaturation enthalpy observed in temperature-dependence viscosity measurements, and in DSC thermograms. The electrophoretic profiles also confirmed a certain degree of denaturation after pasteurization, with a reduction in the amount and intensity of the observed proteins. These results indicate that UV-C treatments do not affect significantly the rheological properties and the protein profile of liquid egg fractions, and could be used as an alternative to pasteurization.

Mechanical and thermal processing effects on protein integrity and peptide fingerprint of pea protein isolate

Abstract Pea protein is considered to be an emerging alternative for the formulation of numerous food products, including functional foods, because of its purported hypolipidemic, anti-hypertensive and hypoglycaemic activities. The present investigation was designed to evaluate the effects of thermal and mechanical treatments on an industrial pea protein isolate (Pisane®) from Pisum sativum L. As a preliminary step, the main protein components of P. sativum seed were identified, using the canonical proteomic approach, including 2D-separation and mass spectrometry. Most of the main spots were assigned to the major pea storage proteins: legumin, vicilin and convicilin. Differential scanning calorimetry and proteomic techniques were used to investigate the effects of processing on the protein profile and to assess the availability of stable peptides. After prolonged treatments, no proteins were present in their native forms and the protein solubility was greatly decreased; however, some intense spots were still present on 2D-gels. The spot identity was confirmed by HPLC-Chip-MS/MS, showing that the vicilin 30 kDa fragment and legumin acidic subunits were resistant and some specific peptides remained intact. DSC thermograms showed that strong treatments decreased the denaturation enthalpy. The mechanical treatments, instead, did not modify (in a relevant way) either the protein profile or DSC thermograms.

Kinetic and thermodynamic study of cloned thermostable endo-1,4-β-xylanase from Thermotoga petrophila in mesophilic host

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.

Role of pH on stability and mechanical properties of gelatin films

The effect of the film-forming solution pH on the triple-helix content, thermal stability, and mechanical properties of gelatin films was investigated. The films were prepared from solutions at different pHs of type A pigskin gelatin, and their mechanical characteristics were determined. At pHs higher than 9 and lower than 5, Young’s modulus, E, and the stress at break, σb, of the films decreased significantly. Cross-linking with genipin reduced deformation at break, ϵb, and increased Young’s modulus. The intensity of the 1.1-nm X-ray diffraction reflection and the denaturation enthalpy decreased at these pHs, indicating that the triple helix reduced. Preliminary in vitro tests on the cross-linked samples indicated good cell proliferation and viability.

Effect of peanut protein isolate on functional properties of chicken salt-soluble proteins from breast and thigh muscles during heat-induced gelation

In this study, we investigated the impact of peanut protein isolate (PPI) on the functional properties of chicken salt-soluble protein (SSP) prepared from breast and thigh muscles during heat-induced gelation. The addition of PPI increased the water-holding capacity, gel strength and elasticity of heat-induced chicken SSP mixed gel. Breast and thigh SSP had the best gel properties at the addition of 2.5% and 3.5% PPI, respectively. Rheology indicated that thigh SSP showed higher storage modulus (G′) than breast SSP. Differential scanning calorimetry showed that the addition of PPI changed transition temperatures (Tmax) and enthalpy of denaturation (ΔH) of chicken SSP. Scanning electron microscopy indicated that the PPI-treated SSP gels had more compact ultrastructures than controls. The results suggested that PPI may be a potential protein additive for improve the characteristics of SSP gelations.

Effects of temperature and relative humidity on fibrillar collagen in parchment: A micro differential scanning calorimetry (micro DSC) study

Micro DSC measurements were used to investigate the synergistic effects of temperature and relative humidity on deterioration of parchment. Samples were obtained by exposing new parchments to various temperature and relative humidity atmospheres for increasing times in controlled test chambers. The impact of this accelerated ageing was assessed by measuring the thermodynamic parameters associated with the thermal denaturation of fibrillar collagen: variation of denaturation temperature, Tmax, indicated changes in collagen thermal stability, increases of DSC peak half-width, ΔT1/2, revealed greater thermal heterogeneity and structural disorder, whereas parallel decrease of denaturation enthalpy, ΔH, and DSC peak maximum height, Cpex max, measured the loss of fibrillar structure. Deconvolution of DSC denaturation peaks provided a valuable illustration of both the dynamics and the pattern of the deterioration of fibrillar collagen. Impact of temperature and relative humidity on parchment integrity and stability is discussed in terms of their implications for conservation science.

Protein disulphide isomerase-mediated grafting of cysteine-containing peptides onto over-bleached hair

The ability of Protein disulphide isomerase (PDI) to promote the grafting of two cysteine-containing peptides onto hair was investigated in order to develop an alternative treatment for over-bleached hair. The studied peptides were designed based on human keratin and human lung surfactant proteins and were linked to a fluorescent dye to facilitate visualisation of the grafting process and to assess hair penetration. The ability of the peptides to restore mechanical and thermal properties lost by repeated bleaching treatments was also studied. After eight bleaching treatments, hair samples displayed 42% less mechanical resistance, coupled with a decrease in α-helix denaturation enthalpies and temperatures. Hair surface damage following bleaching was visualized by scanning electron microscopy. Addition of PDI to the treatment formulations promoted peptide attachment to the hair via disulphide bonds, facilitating their penetration into the hair cortex, as observed by fluorescence microscopy. The proposed peptide treatment resulted in an increase in α-helix denaturation enthalpy in over-bleached hair, as well as an increase in both Young's modulus and tensile strength. Thus, mechanical and thermal properties were improved after the peptide treatment in the presence of PDI; suggesting that the formulations presented in this work are promising candidates for hair-care applications.

Unusual thermodynamic properties of a compact state of IgG3 Kuc and Sur pFh fragments (Hinge region)

The pFh fragments from the hinge region of human IgG3 Kuc and Sur are able to fold into a compact structure, thereby giving proteins with secondary (supersecondary) structure, mainly represented by the left-handed polyproline II helix. It has been shown that thermal denaturation of a compact pFh from the hinge region of IgG3 Kuc and Sur comprises two stages. At the first stage, the compact protein structure unfolds according to the all-or-none model with retention of the secondary structure. At the second stage, the left-handed polyproline II helix, composed of four separate cooperative blocks formed of strands with a high content of proline residues, melts itself. A polyproline conformation of the secondary structure and a large number of disulfide bonds between the chains determine a high specific enthalpy of denaturation and a high thermal stability, respectively.

Cryoprotective effect of trehalose on washed chicken meat.

The cryoprotective effects of trehalose (w = 0-10%) on washed chicken meat (WCM) were investigated. WCM was produced from broiler, frozen and stored for 360days on -30°C. Myofibrillar protein functional stability was monitored by salt extractable protein (SEP) and differential scanning calorimetry (DSC). Salt extractable protein (SEP) showed that the addition of trehalose caused smaller decrease in protein solubility during frozen storage. Peak thermal transition (T p) and denaturation enthalpy (ΔH) of myofibrillar proteins were evaluated. Differential scanning calorimetry (DSC) revealed a shift in peak thermal transition temperature (T p) of myosin and actin to higher temperature as the mass fraction of trehalose increases. The transitions enthalpies of myosin and actin of WCM samples showed higher increase with the increase of mass fraction of trehalose. Since the value of denaturation enthalpy is directly related to amount of native proteins, higher values of ΔH indicates to the higher cryoprotective effects of trehalose.

Thermal denaturation and structural changes of α-helical proteins in keratins

To gain insight into the thermal stability of intermediate filaments and matrix in the biological composite structure of α-keratins, the thermal denaturation performance of human hair fibers was investigated by Modulated Differential Scanning Calorimetry (MDSC) in the dry and the wet state. Denaturation enthalpy ΔH(D) in water was found to be independent of heating rate (11.5J/g) and to be approximately double as high as in the dry state (5.2J/g). The lower enthalpy (dry) and its dependency on heating rate are attributed to effects of pyrolysis. The stepwise change of reversing heat capacity ΔC(p) marks the denaturation process as a classic two-stage transition. The increase of ΔC(p) with heating rate reflects a continuous shift of the nature of the denaturation of the α-helical material, first, into random coil and then towards random β-materials for lower heating rates. Denaturation temperatures follow Arrhenius relationships with heating rate, yielding activation energies of 416kJ/mol (dry) and 263kJ/mol (wet), respectively. A decrease of activation energy (wet) for high heating rates supports the hypothesis of systematic changes of the pathway of denaturation.

Physicochemical Properties of Soy Protein: Effects of Subunit Composition

Soy protein elastomer (SPE) exhibits elastic, extensible, and sticky properties in its native state and displays great potential as an alternative to wheat gluten. The objective of this study was to better understand the roles of soy protein subunits (polypeptides) contributing to the functional properties of SPE. Six soy protein samples with different subunit compositions were prepared by extracting the proteins at various pH values on the basis of the different solubilities of conglycinin (7S) and glycinin (11S) globulins. Soy protein containing a large amount of high molecular weight aggregates formed from α' and α subunits exhibited stronger viscoelastic solid behavior than other soy protein samples in terms of dynamic elastic and viscous modules. Electrophoresis results revealed that these aggregates are mainly stabilized through disulfide bonds, which also contributed to higher denaturation enthalpy as characterized by DSC and larger size protein aggregates observed by TEM. Besides, the most viscoelastic soy protein sample exhibited flat and smooth surfaces of the protein particles as observed by SEM, whereas other samples had rough and porous particle surfaces. It was proposed that the ability of α' and α to form aggregates and the resultant proper protein-protein interaction in soy proteins are the critical contributions to the continuous network of SPE.