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Objective: Sound, natural dentin collagen can be stabilized against enzymatic degradation through exogenous crosslinking treatment for durable bonding; however, the effect on denatured dentin (DD) collagen is unknown. Hence, the ability of different crosslinkers to enhance/restore the properties of DD collagen was assessed. Methods: Demineralized natural and DD collagen films (7 mm × 7 mm × 7 µm) and beams (0.8 mm × 0.8 mm × 7 mm) were prepared. DD collagen was experimentally produced by heat or acid exposure, which was then assessed by various techniques. All specimens were then treated with 1 wt% of chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide (EDC/NHS) and two structurally different flavonoids—theaflavins (TF) from black tea and type-A proanthocyanidins from cranberry juice (CR) for either 30 s or 1 h. The controls were untreated. Dentin films were assessed for chemical interaction and cross-linking effect by FTIR, biostability against exogenous collagenase by weight loss (WL) and hydroxyproline release (HYP), and endogenous matrix metalloproteinases (MMPs) activity by confocal laser microscopy. Dentin beams were evaluated for tensile properties. Data were analyzed using ANOVA and Tukey's test (α = 0.05). Results: Compared with natural collagen, DD collagen showed pronounced structural changes, altered biostability and decreased mechanical properties, which were then improved to various degrees that were dependent on the crosslinkers used, with EDC/NHS being the least effective. Surprisingly, the well-known MMP inhibitor EDC/NHS showed negligible effect on or even increased MMP activity in DD collagen. As compared with control, cross-linking induced by TF and CR significantly increased collagen biostability (reduced WL and HYP release, p < 0.05), MMP inhibition (p < 0.001) and mechanical properties (p < 0.05), regardless of denaturation. Conclusions: DD collagen cannot or can only minimally be stabilized via EDC/NHS crosslinking; however, the challenging substrate of DD collagen can be enhanced or restored using the promising flavonoids TF and CR. [ABSTRACT FROM AUTHOR]

In order to select the suitable characterization method for the denaturation degree of shrimp head protein, the mixed protein extracted from shrimp head was heated at 100 ℃ for a certain time. The denaturation degree of protein was characterized and compared by solubility method, SDS-PAGE method, circular dichroism and fluorescence spectrum. The results showed that solubility method, SDS-PAGE method, circular dichroism and fluorescence spectroscopy were consistent in characterizing the denaturation degree of shrimp head protein during heating. The denaturation degree of shrimp head protein with different concentrations showed an S-shaped upward trend during heating, but there was a difference in denaturation rate between heating 1~4 min. Among them, the denaturation degree of protein solubility was the most sensitive to heating time. This showed that solubility was more convenient to choose as a method to characterize the denaturation degree of shrimp head protein. The results provide a method reference for characterizing the degree of protein denaturation in food processing.

Objective: Sound, natural dentin collagen can be stabilized against enzymatic degradation through exogenous crosslinking treatment for durable bonding; however, the effect on denatured dentin (DD) collagen is unknown. Hence, the ability of different crosslinkers to enhance/restore the properties of DD collagen was assessed. Methods: Demineralized natural and DD collagen films (7 mm × 7 mm × 7 µm) and beams (0.8 mm × 0.8 mm × 7 mm) were prepared. DD collagen was experimentally produced by heat or acid exposure, which was then assessed by various techniques. All specimens were then treated with 1 wt% of chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide (EDC/NHS) and two structurally different flavonoids—theaflavins (TF) from black tea and type-A proanthocyanidins from cranberry juice (CR) for either 30 s or 1 h. The controls were untreated. Dentin films were assessed for chemical interaction and cross-linking effect by FTIR, biostability against exogenous collagenase by weight loss (WL) and hydroxyproline release (HYP), and endogenous matrix metalloproteinases (MMPs) activity by confocal laser microscopy. Dentin beams were evaluated for tensile properties. Data were analyzed using ANOVA and Tukey’s test (α = 0.05). Results: Compared with natural collagen, DD collagen showed pronounced structural changes, altered biostability and decreased mechanical properties, which were then improved to various degrees that were dependent on the crosslinkers used, with EDC/NHS being the least effective. Surprisingly, the well-known MMP inhibitor EDC/NHS showed negligible effect on or even increased MMP activity in DD collagen. As compared with control, cross-linking induced by TF and CR significantly increased collagen biostability (reduced WL and HYP release, p < 0.05), MMP inhibition (p < 0.001) and mechanical properties (p < 0.05), regardless of denaturation. Conclusions: DD collagen cannot or can only minimally be stabilized via EDC/NHS crosslinking; however, the challenging substrate of DD collagen can be enhanced or restored using the promising flavonoids TF and CR.

Summary: Pea protein is a promising option for plant protein‐based emulsification, but commercial proteins have limited functional properties related to their poor physicochemical properties such as solubility. To address this issue, this research investigated the effects of high‐pressure homogenisation and extraction conditions on the solubility and emulsification properties of pea protein. Heating during extraction can denature pea protein and simulate the reduced functional properties of commercial proteins. Homogenising at 10 000 psi combined with the heating process during extraction significantly improved the solubility and emulsification properties of pea protein. Freeze drying at pH 9.0 improved the solubility of pea protein and lowered the droplet size of resultant emulsions compared to pH 7.0. Extracting the protein in propylene glycol to inhibit hydrophobic interactions slightly improved solubility but decreased the emulsification properties. The combination of homogenisation during heating and freeze drying at an elevated pH produced the optimum solubility and emulsification properties. These results suggest that high‐pressure homogenisation and drying at elevated pH levels can be effective tools to limit the functionality‐reducing effects of denaturation that occurs during the commercial extraction of pea protein. This information can be used to design better production methods for pea protein and improve its functional properties. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine the foaming properties of sodium caseinates (Na-cas) extracted from bovine and camel fresh milks after heating at 70, 80, 90 and 100°C for 30 min at laboratory scale. Experimental results indicated that greater foam was obtained with camel Na-cas than with bovine Na-cas at all heating temperatures due to the higher β-casein content in camel caseins (~53.4% of total proteins, RP-HPLC results). Increasing heat-treatment temperature to 100°C significantly enhanced the foaming properties by raising surface hydrophobicity and decreasing electronegative charge as well as interfacial tension values upon heating. This study concluded that camel Na-cas has important foaming properties in agricultural and food industries. [ABSTRACT FROM AUTHOR]

The thermal stability of myosin and the protective effect of F-actin on myosin in kuruma prawn myofibrils were investigated from the thermal inactivation rates at 25 °C of Ca-ATPase in myosin and myofibrils at various concentrations of KCl. The thermal inactivation rate constant (kD) of myofibrillar Ca-ATPase increased with increasing KCl concentration; however, the thermal inactivation followed a biphasic first-order reaction regardless of the KCl concentration: a relatively fast inactivation rate in the earlier phase (kDe) followed by a slower inactivation rate in the later phase (kDl) of the heat treatment. The thermal inactivation of myosin at various concentrations of KCl or sorbitol also followed a biphasic first-order reaction, and the differences between the kDe and kDl of myosin were always about twofold. kDe and kDl of myosin at 0.1 M KCl were decreased to 1/6 and 1/13, respectively, by the binding action of F-actin. These results suggest that the thermal stability of myosin and the protective effect of F-actin affect the stability and thermal inactivation of kuruma prawn myofibrils at physiological ionic strength. [ABSTRACT FROM AUTHOR]

[Display omitted] • Macromolecular crowding transforms acid-denatured cyt c III to MG MC -state. • Size and shape of crowders control thermodynamic stabilization of MG MC -state. • Macromolecular crowding increases thermodynamic stability of U A -state. • Δ Γ W tracks molecular size/shape of crowders to refolding of U A -state to MG MC -state. • MG MC -state is comparatively less thermodynamically stable than the A-state. The macromolecular crowding effect transforms the acid-denatured ferricytochrome c (cyt c III) (U A -state) to molten-globule (MG MC -state) at pH 1.85. Crowding-induced stabilization free energy (ΔΔG) and preferential hydration Δ Γ W were estimated for the U A → MG MC transition. The magnitudes of ΔΔ G and Δ Γ W were found to be decreased as dextran 70 (D70) > dextran 40 (D40) > ficoll 70 (F70), which demonstrates that ΔΔ G and Δ Γ W track the molecular size and shape of the crowder towards refolding and stabilization of U A -state to MG MC -state. Analysis of effects of crowders (D40, D70, F70) on thermal and chemical-denaturations of acid-denatured cyt c III provided several important information, (i) macromolecular crowding increased the thermodynamic stability of acid-denatured cyt c III, (ii) concentration, size and shape of crowder control the crowding-induced thermodynamic stabilization of MG MC -state, (iii) crowding effect increased the thermal-denaturation midpoint (T m) with a slight change in enthalpy (Δ H m), suggesting that the steric-excluded volume effect contributes to the crowding-induced increased thermal stability of the acid-denatured protein. Analysis of entropy − enthalpy plots for D40, D70, and F70 reveals that in addition to the steric-excluded volume effect, the enthalpic contribution is also added to the macromolecular crowding-induced stabilization of acid-denatured cyt c III. The dilute-medium, compound-crowder, purely entropic-crowder and purely enthalpic-crowder curves were obtained for acid-denatured cyt c III for D70, D40 and F70. The crossover temperature, T x was calculated from the dilute and compound-crowder curves. The T x values measured for D40, D70, and F70 were found to be ∼ 250.15 K, 272.15 K, and 275.15 K, respectively, which suggests that the T x value depends on the size and shape of the crowder. Furthermore, the observation of a lower value of T x and a minor enthalpic component for D40, D70, and F70 is likely due to the formation of weaker soft interactions of acid-denatured cyt c III with D40, D70, and F70. [ABSTRACT FROM AUTHOR]

The antibody is one of the most well-studied multi-domain proteins because of its abundance and physiological importance. In this article, we describe the effect of the complex, multi-domain structure of the antibody on its denaturation by heat. Natural antibodies are composed of 6 to 70 immunoglobulin fold domains, and are irreversibly denatured at high temperatures. Although the separated single immunoglobulin fold domain can be refolded after heat denaturation, denaturation of pairs of such domains is irreversible. Each antibody subclass exhibits a distinct heat tolerance, and IgE is especially known to be heat-labile. IgE starts unfolding at a lower temperature compared to other antibodies, because of the low stability of its CH3 domain. Each immunoglobulin domain starts unfolding at different temperatures. For instance, the CH3 domain of IgG unfolds at a higher temperature than its CH2 domain. Thus, the antibody has a mixture of folded and unfolded structures at a certain temperature. Co-existence of these folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate which causes the inactivation of the antibody. [ABSTRACT FROM AUTHOR]

The effect of high pressure homogenization (HPH) on the structure of β-lactoglobulin (β-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from β-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified β-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at C β-lg  = 0.1 wt%. However, no modification of the interfacial properties was observed at C β-lg  = 1 wt%. At this protein concentration, the prior denaturation of β-lg by HPH did not modify the droplet size of nanoemulsions prepared with these β-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent. [ABSTRACT FROM AUTHOR]

A newly developed method of analysis of difference-UV spectra was successfully implemented in the study of the effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate. It was found that whey proteins exhibit their highest stability against heat denaturation at pH 3.75. At very low pH values, i.e. 2.5, they exhibited considerable cold denaturation, while after heating at this pH value, the supplementary heat denaturation rate was lower compared to that at neutral pH. The highest heat denaturation rates were observed at pH values higher than neutral. High power sonication on whey proteins, previously heated at 90 °C for 30 min, resulted in a rather small reduction of the fraction of the heat denatured protein aggregates. Finally, when ethanol was used as a cosolvent in the concentration range 20–50%, a sharp increase in the degree of denaturation, compared to the native protein solution, was observed. [ABSTRACT FROM AUTHOR]

A novel approach in the analysis of difference-UV spectrophotometric data for determining the heat denaturation degree of bovine serum albumin (BSA) was assessed. Five different parameters of difference-UV spectra were obtained by subtracting spectra of unheated and denatured protein solutions at different temperature-time combinations. BSA was found to exhibit a maximum degree of heat denaturation of about 17% compared to the complete unfolding caused by 6 M guanidine hydrochloride. This low degree of heat denaturation is probably caused by the aggregation of the initially unfolded protein molecules. The kinetic analysis exhibited discontinuities in the Arrhenius plots, distinguishing the unfolding and aggregation phases of the denaturation process, whereas such a discrimination could not be obtained by differential scanning calorimetry analyses. The proposed method is accurate, fast, simple and sensitive enough to detect changes in the protein heat denaturation even at short temperature-time intervals. [ABSTRACT FROM AUTHOR]

The role of heat-denatured sarcoplasmic proteins in water-holding is not well understood. Here we propose a new hypothesis that in PSE-like conditions denatured sarcoplasmic proteins aggregate within and outside myofilaments, improving the water-holding of denatured myofibrils. The process is compartmentalized: 1) within the filaments the denatured sarcoplasmic proteins shrink the lattice space and water is expelled; and 2) between the myofibrils and in the extracellular space, the coagulated sarcoplasmic proteins trap the expelled water from interfilamental space. The effect of sarcoplasmic proteins on the water-holding of myofibrils following incubation for 1 h at 21 to 44 °C was investigated. Our results were consistent with the new hypothesis. Myofibrils without sarcoplasm had the poorest water-holding. With increasing amount of denatured sarcoplasmic proteins, the water-holding of heat-denatured myofibrils improved proportionally. X-ray diffraction was used to measure the lattice space between the filaments. Precipitated sarcoplasmic proteins shrank ( P < 0.001) the lattice spacing by 6.3% at 44 °C. [ABSTRACT FROM AUTHOR]

A simple and green approach was developed to produce a novel nanogel via self-assembly of modified soy protein and dextran, to efficiently deliver riboflavin. First, modified soy protein was prepared by heating denaturation at 60 °C for 30 min or Alcalase hydrolysis for 40 min. Second, modified soy protein was mixed with dextran and ultrasonicated for 70 min so as to assemble nanogels. The modified soy protein-dextran nanogels were characterized by Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) and ζ-potential studies to confirm the formation of NGs. Transmission electron microscopy (TEM) revealed the NGs to be spherical with core-shell structures, in the range of 32–40 nm size. The nanogels were stable against various environmental conditions. Furthermore, the particle size of the nanogels hardly changed with the incorporation of riboflavin. The encapsulation efficiency of nanogels was found to be up to 65.9% at a riboflavin concentration of 250 μg/mL. The nanogels exhibited a faster release in simulated intestine fluid (SIF) compared with simulated gastric fluid (SGF). From the results obtained it can be concluded that modified soy protein-dextran nanogels can be considered a promising carrier for drugs and other bioactive molecule delivery purposes. [ABSTRACT FROM AUTHOR]

Energetics was analyzed for Trp-cage miniprotein in water to elucidate the solvation effect in heat denaturation. The solvation free energy was computed for a set of protein structures at room and high temperatures with all-atom molecular dynamics simulation combined with the solution theory in the energy representation, and its correlations were investigated against the intramolecular (structural) energy of the protein and the average interaction energy of the protein with the solvent water. It was observed both at room and high temperatures that the solvation free energy is anticorrelated to the structural energy and varies in parallel to the electrostatic component of the protein-water interaction energy without correlations to the van der Waals and excluded-volume components. When the set of folded structures sampled at room temperature was compared with the set of unfolded ones at high temperature, it was found that the preference order of the two sets is in correspondence to the van der Waals and excluded-volume components in the sum of the protein intramolecular and protein-water intermolecular interactions and is not distinguished by the electrostatic component. [ABSTRACT FROM AUTHOR]

This work aims to determine changes at molecular level of plasma proteins provoked by adding cysteine (Cys, 0.025% to 0.35% w/v) as a reducing agent and their relationship with the heat-induced gel properties obtained when subsequently the solutions were submitted to a thermal treatment. Results show that adding Cys to plasma solutions at concentrations ≥0.15% actually entails modifications in the secondary structure of their main proteins, that is, serum albumin-α-helix rich-and globulin fraction-β-sheet rich. Basically, a reduction of the intensity of the infrared (IR) bands assigned to both structures takes place concomitant to an increase of extended structures that seem to act as intermediates for the subsequent protein aggregation process through nonnative intermolecular β-sheets. Cleavage of disulfide bonds is also evidenced at Cys concentrations ≥0.15% by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the effects being directly proportional to Cys concentration. However, beneficial effects on gel hardness are gradually obtained at Cys concentrations ≤0.15%, that is, when the effects at molecular level are at most just budding, while not more improvements on this textural parameter are obtained at higher Cys concentrations. By contrast, water retention capacity is gradually diminishing as Cys concentration increases, but with a significant reduction only obtained at the highest tested concentration. These results suggest a negative effect of Cys on gel microstructure at high concentrations, which probably can be attributed to protein aggregation taking place at room temperature. [ABSTRACT FROM AUTHOR]

Abstract: Blood and plasma are unique resources and access to these products save life. In this study, albumin demand and surplus plasma makes it possible to use local experiences in plasma industry for preparation of albumin so plasma was heated after stabilization; afterward denatured proteins were precipitated and separated by continuous centrifuge system. The supernatant contained albumin was filtrated, diafiltrated, ultrafiltrated, formulated and pasteurized. Albumin preparation in pilot scale with heat denaturation was performed for the first time in Iran. This method using surplus plasma is recommended for all countries that have no access to plasma fractionation industry. Therefore with more attention it has potential for use in the production of safe plasma derived products and thereby it can be used as a safe product in clinic. [Copyright &y& Elsevier]

Abstract: Native proteins usually undergo structural modification upon adsorption at interface. Heat treatments are commonly applied at the industrial scale and lead to aggregation of proteins. We characterized nanometric aggregates of β-lactoglobulin by infrared spectroscopy in solutions, in hexadecane oil-in-water emulsions and at the air–water interface at low and high (0.1 M) ionic strengths and at pH 7. In solutions, on the contrary to native β-lactoglobulin, all aggregates prepared with or without salt possessed intermolecular β-sheets evidenced by two strong absorption bands at 1614 cm−1 and 1682 cm−1. In emulsions, at low ionic strength, they lose their intermolecular β-sheets once they are adsorbed at the oil–water interface. At high ionic strength, most of aggregates are localized at the interfaces where they lose their intermolecular β-sheets in direct contact with the surface and only partially when they are farther from the interface. The loss of intermolecular β-sheets was similarly observed at the air–liquid interface. [Copyright &y& Elsevier]

Abstract: Often in protein design research, one desires to generate thermally stable variants of a protein or domain. One route to identifying mutations that yield domains that remain folded and active at a higher temperature is through the use of directed evolution. A library of protein domain variants can be generated by mutagenic PCR, expressed on the surface of bacteriophage M13, and subjected to heat, such that the unfolded forms of the domain, showing reduced or no binding activity, are lost during subsequent affinity selection, whereas variants that still retain binding to their target are selected and enriched with each subsequent round of affinity selection. This approach takes advantage of the fact that bacteriophage M13 particles are heat stable and resistant to many proteases and protein denaturants. We present the application of this general approach to generating thermally stable variants of a eukaryotic peptide-binding domain. The benefits of producing such variants are that they typically express at high levels in Escherichia coli (30–60mg/L shake flask) and remain soluble in solution at higher concentrations for longer periods of time than the wild-type form of the domain. The process of library generation and screening generally requires about one month of effort, and yields variants with >10°C increase in thermal stability, as measured in a simple fluorescence-based thermal shift assay. It is anticipated that thermally stable variants will serve as excellent scaffolds for generating affinity reagents to a variety of targets of interest. [Copyright &y& Elsevier]

SUMMARY Aim In many countries, there is surplus plasma which is thrown out. Due to the high cost of albumin as a pharmaceutical product, it is not widely available for all patients. Therefore, we considered direct preparation of albumin in the plasma bag. Background Human plasma has many unique features compared with prepared pharmaceutical products because of its human origin. In countries which do not have a plasma fractionation industry, shortages for plasma products are commonplace. Methods Albumin stabiliser was added to the plasma in the bag to stabilised albumin molecule and make it resistant to heat. When the plasma bag was heated, other plasma proteins are irreversibly precipitated and can be separated by centrifugation. Finally, albumin is filtered and pasteurized in plasma bag. Results The purity of prepared albumin is >99% and the yield is 21 g per liter of plasma. Polymer content of pasteurized albumin measuring with High Performance Liquid Chromatography (HPLC) was less than 1%. Conclusions Since albumin preparation in plasma bag is a simple and cheap technology, it has the potential for use in the production of a safe plasma derived product, although suitability for human use would require appropriate clinical assessment for safety. Using this method, albumin could be prepared from all types of plasma. This method is suggested as a possible method of albumin production for all countries which do not have a plasma fractionation industry and even for the countries with low plasma supplies. [ABSTRACT FROM AUTHOR]

Abstract: Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85°C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin. [Copyright &y& Elsevier]

Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. Dt values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf. [ABSTRACT FROM AUTHOR]

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