Denaturation Behavior Research Articles

Effect of starch and peanut oil on physicochemical and gel properties of myofibrillar protein: Amylose content and addition form

Starch and peanut oil (PO) were widely used to improve the gel properties of surimi, however, the impact mechanism of addition forms on the denaturation and aggregation behavior of myofibrillar protein (MP) is not clear. Therefore, the effect of starch, PO, starch/PO mixture, and starch-based emulsion on the physicochemical and gel properties of MP was investigated. The results showed that amylose could accelerate the aggregation of MP, while amylopectin was conducive to the improvement of gel properties. The addition of PO, starch/PO mixture, or starch-based emulsion increased the turbidity, solubility, sulfhydryl content of MP, and improved the gel strength, whiteness, and texture of MP gel. However, compared with starch/PO mixture group, the gel strength of MP with waxy, normal and high amylose corn starch-based emulsion increased by 22.68 %, 10.27 %, and 32.89 %, respectively. The MP containing emulsion had higher storage modulus than MP with starch/PO mixture under the same amylose content. CLSM results indicated that the oil droplets aggregated in PO or starch/PO mixture group, while emulsified oil droplets filled the protein gel network more homogeneously. Therefore, the addition of starch and PO in the form of emulsion could effectively play the filling role to improve the gel properties of MP.

Thermal properties of glycinin in crowded environments

Crowded environments, commonly found in the food system, are utilized to enhance the properties of soybean proteins. Despite their widespread application, little information exists regarding the impact of crowded environments on the denaturation behaviors of soybean proteins. In this study, we investigated how crowding agents with varying molecular weights, functional groups, and topology affect the denaturation behavior of glycinin under crowded conditions. The results reveal that thermal stability in PEG crowded environments is mainly influenced by both preferential hydration and binding. The stabilization is primarily enthalpy-driven, with aggregation contributing additional entropic stabilization. Specifically, ethylene glycol and diethylene glycol exhibit temperature-dependent, bilateral effects on glycinin stability. At the denaturation temperature, hydrophobic interactions play a predominant role, decreasing glycinin's thermal stability. However, at a molecular weight of 200 g/mol, there is a delicate balance between destabilizing and stabilizing effects, leading to no significant change in thermal stability. With the addition of PEG 400, 1000, and 2000, besides preferential hydration, additional hard-core repulsions between glycinin molecules enhance thermal stability. Methylation modification experiments demonstrated that 2-methoxyethyl ether exerted a more pronounced denaturing effect. Additionally, the cyclization of PEG 1000 decreased its stabilizing effect.

The possibility of chemical transformation of glucose in choline chloride/glucose deep eutectic solvent with thermal instability.

Deep eutectic solvents (DESs), characterized by their low volatility, non-toxicity, and biodegradability, have gained attention as green solvents due to their minimal environmental impact and sustainability. The choline chloride/glucose DES, composed solely of biomass, is notable for its high biocompatibility and ability to be prepared at low cost. However, it is also known for its low thermal stability and tendency to denature when heated. In this study, we approached the choline chloride/glucose DES, with its thermal denaturation properties, as a unique chemical conversion medium entirely constituted from biomass. We investigated the thermal denaturation and reaction behaviors of the DES when subjected to prolonged heating. It was found that the choline chloride/glucose DES was relatively thermally stable at around 100 °C, but underwent thermal denaturation at 130 °C, enabling the production of 5-HMF and seven types of rare sugars derived from glucose. The yield of disaccharides containing seven types of rare sugars and 5-HMF relative to the weight of glucose was as high as approximately 70% and 5%, respectively. This study thus reveals that simply heating a liquid composed exclusively of biomass under mild conditions can generate a range of high-value compounds.

Locally Denatured DNA Compaction by Divalent Cations.

The denaturation of DNA is a critical process in biology and has many biotechnological applications. We investigated the compaction of locally denatured DNA by a chemical denaturation agent, dimethyl sulfoxide (DMSO), using magnetic tweezers (MTs), atomic force microscopy (AFM), and dynamic light scattering (DLS). Our results show that DMSO not only is capable of denaturing DNA but also able to compact DNA directly. When the DMSO concentration is above 10%, DNA condensation occurs due to the reduction in the persistence length of DNA and excluded volume interactions. Meanwhile, locally denatured DNA is easily condensed by divalent cations, such as magnesium ions (Mg2+), contrasting with no native DNA condensation by the classical divalent cations. Specifically, the addition of more than 3 mM Mg2+ to a 5% DMSO solution leads to DNA condensation. The critical condensing force (FC) increases from 6.4 to 9.5 pN when the Mg2+ concentration grows from 3 to 10 mM. However, FC decreases gradually with a further increase in Mg2+ concentration. For 3% DMSO solution, above 30 mM Mg2+ is needed to compact DNA and a weaker condensing force was measured. With increasing Mg2+ concentration, the morphology of the DMSO partially denatured DNA complex changes from loosely random coils to a dense network structure, even forming a spherical condensation nucleus, and finally to a partially disintegrated network. These findings show that the elasticity of DNA plays an important role in its denaturation and condensation behavior.

Soluble complexes of ovalbumin with fucoidan: Energetics of binding, protein stability and functional properties

The interaction between ovalbumin (OVA) and fucoidan (FVF), a branched sulfated polysaccharide of seaweeds, was studied by turbidimetry, velocity sedimentation, and high-sensitivity differential scanning calorimetry (HS-DSC). The boundary conditions of the existence of soluble OVA−FVF complexes were determined as a function of pH and/or OVA−FVF mixture composition. The yield of soluble OVA−FVF complexes was determined from the velocity sedimentation data at pH 4.0 and 5.0 in 5 mM NaCl. It achieved a maximal value of ∼90% at pH 5.0 and the OVA/FVF weight ratio of about 6. According to HS-DSC data, the denaturation behavior of OVA bound to FVF changed significantly as compared with that of the free protein. Depending on pH and OVA−FVF mixture composition, one or two heat capacity peaks were observed on the thermograms assigned either to the free or to bound protein. The denaturation temperature of the bound OVA was about 20 °C lower than that of the free OVA. Based on the HS-DSC results, the OVA to FVF binding curves were derived at pH 5.0 and 5.5. Both were well approximated by the Langmuir equation with n=66±3 and Kb=105.2±0.1 M−1 at pH 5.0, and n=1.1±0.2 and Kb=104.6±0.2 M−1 at pH 5.5. Some emulsifying and foaming properties of the OVA−FCD complexes were characterized and compared with those of OVA alone. The complexes showed a significantly lower emulsifying threshold. The foaming ability of the complexes could exceed that of OVA by more than twice. These results imply a high potential of the OVA−FCD complexes as emulsion and foam stabilizers.

Denaturation behavior of inclusions in industrial pure iron by calcium treatment

Denaturation behavior of inclusions in industrial pure iron by calcium treatment

Low-temperature in situ CO2 enhanced oil recovery

The new generation in situ CO2 enhanced oil recovery (ICE), which delivers CO2 by injection of a CO2 generating agent solution, was proven to offer robust recovery performances and potential cost and workflow benefits. However, the developed ICE systems are either formulated with a complex fluid recipe or a simplified version of a fluid system limited by high-temperature requirements. This work focused on removing the requirement of high reservoir temperature in our previous work while maintaining the simplicity of the single fluid injection system. Urease is studied as the catalyst for urea hydrolysis and evaluated experimentally. The denaturation behavior of urease and the reaction kinetics of the catalyzed urea hydrolysis process are tested at different urea concentrations, urease concentrations, and temperatures. Four sets of one-dimensional sand pack flowthrough experiments are conducted to verify the tertiary recovery potential of the newly developed low-temperature ICE system with different lithology. The extent of wettability alteration and the recovery mechanism for different lithologies was determined through core sample imbibition experiments and direct contact angle measurements. From the experimental results, urease-catalyzed urea hydrolysis is proven to be effective in tertiary oil recovery applications below the 50 °C reservoir temperature range with urea conversion ratio up to 68.7 %. The selected low-temperature ICE system (10 wt% urea solution with 31 U/g urease) had superior tertiary recoveries(Etr) in the flowthrough test for sandstone(Etr = 31.3 %) and carbonate(Etr = 27.5 %) than the corresponding high-temperature cases. Imbibition tests of this improved formulation with various porous media show distinct wettability reversal trends towards a more water-wet state post imbibition. This trend is associated with the produced NH3 and CO2 due to urea hydrolysis. Both oil-aged sandstone and carbonate imbibition tests show noticeable water wetness improvement. The observations of this work significantly expand the operational envelopes of the current ICE system.

Thermal analysis of protein stability and ligand binding in complex media

Screening of ligands that can bind to biologic products of in vitro expression systems typically requires some purification of the expressed biologic target. Such purification is often laborious and time consuming as well as a limiting challenge. What is required, representing an enormous advantage, is the ability to screen expressed proteins in the crude lysate stage without purification. For that purpose, we explore here, the utility of differential scanning calorimetry (DSC) measurements for detecting the presence of specific proteins and their interactions with ligands in the complex media where they were prepared, i.e. crude lysates. Model systems were designed to mimic analogous conditions comparable to those that may be encountered in actual in vitro expression systems. Results are reported for several examples where DSC measurements distinctly showed differences in the thermal denaturation behaviors of the crude lysate alone, proteins and proteins plus binding ligands added to the crude lysate. Results were obtained for Streptavidin/Biotin binding in E. coli lysate, and binding of Angiotensin Converting Enzyme 2 (ACE2) by captopril or lisinopril in the lysate supernatant derived from cultured Human Kidney cells (HEK293). ACE2 binding by the receptor binding domain (RBD) of SARS-CoV-2 was also examined. Binding of ACE2 by RBD and lisinopril were similar and consistent with the reported ACE2 inhibitory activity of lisinopril.

Study in the stabilization of proteins encapsulated in PLGA delivery system: Effects of additives on protein encapsulation, release, and stability

Therapeutic proteins/peptides face severe stability impairment in biotechnology drug delivery systems prepared by the W/O/W solvent evaporation method. Screening of protein stabilizers for poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles can improve the low encapsulation efficiency and undesirable release behavior of proteins caused by PLGA carriers, which is a crucial step to achieve a high proportion of continuous release of encapsulated protein in the target time and give full play to the therapeutic effect. Therefore, model proteins were selected for stability testing to simulate the specific destabilization pathways. Through the comprehensive study of protein conformation, recovery rate, encapsulation rate, and drug release behavior, the effects of additives on the stability of protein were synthetically characterized, and their action links and mechanisms were also further studied. The results showed that different denaturation environments were intertwined during the preparation and release of peptide-loaded NPs, jointly inducing protein denaturation and incomplete release behavior. After optimization with stabilizers, urea and lactose increased the encapsulation efficiency of proteins to more than 95% (P < 0.001). Except for the P188, all other stabilizers showed great potential for optimizing the release and stability of the encapsulated protein as a whole, increasing the release proportion of protein from 44% to 60 %-75% (P < 0.0001). The improvement of the drug release curve demonstrated the long-term stabilizing effect of the above-mentioned co-solubilizing additives on the therapeutic protein.

Extension of "Interfacial Adsorption Denaturation" Behavior Interpretation Based on Gibbs Monolayer Formation by Biomolecules.

Using glucose oxidase and salmon testis-derived DNA molecules, we sought to extend the recently proposed idea of interfacial adsorption denaturation. The surface pressure-time (π-t) isotherm of the glucose oxidase Gibbs monolayer exhibited a rapid increase in surface pressure and a relatively prompt transition to a liquid condensed film. The appearance of this rapid liquid expansion phase occurred much earlier than that previously identified for lysozyme, trypsin, cytochrome C, and luciferase. This experimental finding was linked to the number of hydrophobic residues in the constituent unit, and the number of hydrophobic residues in glucose oxidase was the highest among these biomolecules. On the other hand, DNA molecules do not have such hydrophobic groups, or present a positive surface on the π-t curve. However, interfacial adsorption occurred, and the existence of molecules at the air/water interface was confirmed, even in the two-dimensional gas phase state. Furthermore, it was confirmed that an increase in surface pressure was detected during the formation of a mixed film of DNA molecules and biomolecules, forming a stable Gibbs monolayer. This mimic the behavior of mixed monolayer formation with matrix molecules in Langmuir monolayers. Moreover, it was clarified that the interfacial adsorption denaturation behavior changed when pH dependence was evaluated considering the isoelectric point of the biomolecular group.

Denaturation Behavior and Kinetics of Single- and Multi-Component Protein Systems at Extrusion-Like Conditions.

In this study, the influence of defined extrusion-like treatment conditions on the denaturation behavior and kinetics of single- and multi-component protein model systems at a protein concentration of 70% (w/w) was investigated. α-Lactalbumin (αLA) and β-Lactoglobulin (βLG), and whey protein isolate (WPI) were selected as single- and multi-component protein model systems, respectively. To apply defined extrusion-like conditions, treatment temperatures in the range of 60 and 100 °C, shear rates from 0.06 to 50 s⁻1, and treatment times up to 90 s were chosen. While an aggregation onset temperature was determined at approximately 73 °C for WPI systems at a shear rate of 0.06 s⁻1, two significantly different onset temperatures were determined when the shear rate was increased to 25 and 50 s⁻1. These two different onset temperatures could be related to the main fractions present in whey protein (βLG and αLA), suggesting shear-induced phase separation. Application of additional mechanical treatment resulted in an increase in reaction rates for all the investigated systems. Denaturation was found to follow 2.262 and 1.865 order kinetics for αLA and WPI, respectively. The reaction order of WPI might have resulted from a combination of a lower reaction order in the unsheared system (i.e., fractional first order) and higher reaction order for sheared systems, probably due to phase separation, leading to isolated behavior of each fraction at the local level (i.e., fractional second order).

Green synthesis of magnetic nanoparticles via Cinnamomum verum bark extract for biological application

The green synthesis of magnetic Fe nanoparticles (NPs) was prepared via Cinnamomum Verum bark extract. The UV–vis analysis explained the formation of Fe NPs with SPR band at 288 nm. The powder X-ray diffraction analysis (XRD) explicated the mean crystallite size was at 36 nm. The Fourier Transform Infra-Red analysis (FTIR) revealed the functional groups in the prepared Fe NPs. The Scanning Electron Microscope (SEM) and High-Resolution Transmission Electron Microscope (HR-TEM) analyses notified the circular and spherical shaped Fe NPs with the size at 20−50 nm. The Energy dispersive X-ray spectroscopy (EDS) and mapping analyses validated the formation of Fe NPs with purity. The Vibrating Sample Magnetometer (VSM) revealed the paramagnetic behavior of prepared Fe NPs. The phytochemical analysis described the phytochemicals compounds which present in Cinnamomum Verum bark extract. The Antibacterial assay described the much higher inhibition zone of prepared Fe NPs upon human pathogenic bacteria. The Antioxidant (DPPH) assay described the efficient scavenging behavior of Fe NPs with 89 % at 80 μg/mL concentration. The Anti-inflammatory assay explicated the potential protein denaturation behavior of Fe NPs with 87 % at 500 μg/mL concentration. The Anti-diabetic assay reported the much higher potential efficiency of prepared Fe NPs with 84 % at 500 μg/mL concentration.

Amino acids, polyols and soluble fibres as sugar replacers in bakery applications: Egg white proteins denaturation controlled by hydrogen bond density of solutions

In this paper we demonstrate that the denaturation behavior, i.e. Tden, of egg white proteins in sugar and sugar replacer solutions is explained by the volumetric density of hydrogen bonds in the solutions, i.e. nOH,eff. The validity of the presented approach is demonstrated using 18 solutions comprising single compounds as well as 7 ternary/quaternary mixtures. Different classes of plasticizers are used at various concentrations and at various ratio with proteins. Sweet amino acids such as l-proline and glycine are included as novel alternatives to polyols. The experimental data are modelled with the Flory-Huggins (FH) theory for biopolymer melting. For such purpose, solutions are treated as a single solvent, which is described by the effective volume fraction of the solvent Φw,eff (⁓nOH,eff). Overall, the FH model can well describe the denaturation behavior of egg white proteins in sugar and sugar replacer solutions up to 30% concentration. Deviations from the model become particularly evident at high sugar concentrations (i.e. 50%), which relate to conditions of phase separation in a protein-rich and sugar-rich domain. In such conditions, Φw,eff does not reflect the composition of the solvent around the proteins. An elevation in Tden is observed due to a reduction in hydrogen bond density in the protein-rich domain. The results indicate that phase separation is driven by both the concentration and the molar volume density of effective hydroxyl groups NOH,s/vs of the plasticizers or plasticizer mixtures. Finally, the proposed approach can predict key phase-transitions which result in protein network formation in pound cake baking.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Investigation on the influence of high protein concentrations on the thermal reaction behaviour of β-lactoglobulin by experimental and numerical analyses

In this study, treatments at various temperature–time profiles were performed for β-lactoglobulin samples at different concentrations (50–70%) using a special rheometer as processing device. Rheological measurements, offline protein chemical analyses, and molecular dynamics analyses were performed to investigate the influence of high protein concentrations and treatment temperature on the denaturation and aggregation behaviour of β-lactoglobulin. Under these conditions, the degree of denaturation and aggregation decreased with increasing protein concentration. This corresponded to a strongly decreased diffusion and increased stability of exposed surface protein regions at high concentrations. Irreversible denaturation was observed for temperatures above 60 °C. Increasing thermal treatment intensity resulted in an increase of aggregation. Depending on the thermal treatment conditions, different protein–protein interactions were measured. By increasing the treatment temperature, the resulting aggregates were increasingly stabilised by covalent bonds. In addition to disulphide bonds, non-disulphide covalent cross-links were formed at temperatures above 100 °C.

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

Molar mass dependence of structure of xanthan thermally denatured and renatured in dilute solution

Double helical polysaccharide, xanthan samples with varying molar mass were thermally denatured and renatured in dilute solutions. Both the weight average molar mass, which was determined by size exclusion chromatography (SEC), and viscosity average molar mass decreased after denaturation and renaturation for samples with an initial molar mass of 106 g mol−1, but those for the samples with an initial molar mass of 105 or 107 g mol−1 decreased only slightly. Although the double helices of xanthan only partially dissociated into single chains, the molar mass distribution estimated by SEC did not broaden drastically by the denaturation and renaturation. These results can be explained by the formation of hairpin structures by the single chains, which are restricted to xanthan with a molar mass of 105 g mol−1 by the strain of the hairpin loop, and for xanthan molar masses of 107 g mol−1 by the incomplete dissociation of its long polymer chains. Double helical polysaccharide, xanthan samples with varying molar mass were thermally denatured and renatured in dilute solutions. The molar mass decreased after denaturation and renaturation for samples with an initial molar mass of 106 g mol−1, but those for the samples with an initial molar mass of 105 or 107 g mol−1 decreased only slightly. A model explaining this molar mass dependence of the denaturation and renaturation behaviors was proposed based on the experimental results.

Application of ultrasound pretreatment and glycation in regulating the heat-induced amyloid-like aggregation of β-lactoglobulin

Ultrasonication and glycation are widely used in the food industry. In this study, we evaluated the independent and combined effects of ultrasound pretreatment (UP) and glycation on the heat-induced amyloid-like aggregation of β-lactoglobulin (β-Lg) by investigating both the fibrillation kinetics and aggregate conformations. Both UP and glycation respectively were found to accelerate the fibrillation process by promoting the denaturation and unfolding behavior of β-Lg according to both the thioflavine-T binding assay. Glycation reduced the fibrillation capacity of β-Lg by reducing the content of β-sheet secondary structures in β-Lg on the basis on circular dichroism spectral measurements. Based on the results of transmission electron microscopy, glycation derived from glucose and lactose suppressed the elongation of fibrils, and β-Lg incubated with maltodextrin was shown to form spherical particle micelles. When applied jointly, UP was found to limit the glycation process by promoting aggregation during the initial heating, which may bury a portion of the glycation sites. According to these findings, UP and glycation are promising methods that can be used independently or jointly to regulate both fibrillation kinetics and fibril conformation in the food industry.

Multiple spectroscopic studies of the interaction between a quaternary ammonium-based cationic Gemini surfactant (as a carrier) and human erythropoietin

Erythropoietin (EPO) is a hematopoietic growth factor. This substance, as a strong cell protector, can increase cell maintenance during different damages of central nervous system. Since the brain-blood barrier prevents the entrance of large proteins similar to EPO into the brain, its systemic delivery gets limited. The aim of this study was to find an alternative approach for EPO delivery into the brain to skip the blood-brain barrier prevention. So, a new quaternary ammonium-based cationic Gemini surfactant has been used to study the interaction of the cationic Gemini surfactant (as a carrier) with EPO using various spectroscopic techniques of (fluorescence and circular dichroism (CD)) and thermal denaturation. Fluorescence spectroscopy studies show the formation of Gemini-EPO complex and also static quenching of protein upon this interaction. The binding parameters of number of binding sites, binding affinity, Gibbs free energy, enthalpy, and entropy were calculated according to fluorescence quenching studies. Also, CD results have further represented that the content of regular secondary structure of EPO did not show any significant alterations by increasing the Gemini concentration. Finally, thermal denaturation behavior of EPO results indicates decreasing the thermal stability of protein in the presence of Gemini. In conclusion, the obtained results proposed that Gemini as a cationic surfactant can bind to EPO without any significant diverse effects on the structure of this drug (EPO) which can be considered as a candidate for EPO delivery in future.

Effect of pH on the reaction mechanism of thermal denaturation and aggregation of bovine β-lactoglobulin

New insights in the aggregation mechanisms of the whey protein β-lactoglobulin were gained by detailed investigation of the denaturation behavior (80–120 °C) at variable pH values (5.1, 6.8, 8.0). Denaturation followed a 1.5 order kinetic and reaction velocity was depending on pH and temperature. Characterization of aggregates formed at 80 °C was correlated to the denaturation progress and comprised particle size measurement, quantification of free thiol groups and analysis of quaternary structures as well as stabilizing forces by various types of gel electrophoresis. Due to low electrostatic repulsion at pH 5.1, a fast and steady aggregate growth was observed, where intermolecular disulfide bonds gain more importance with increasing degree of denaturation. Contrary to that, denaturation at pH 6.8 and 8.0 yielded lower reaction rate constants and a steady increase of the amount of exposed thiol groups, resulting in non-native oligomers which were mainly non-covalently connected to small aggregates.