Onset Temperature Of Denaturation Research Articles

Quantifying Long-Time Hydrogen-Deuterium Exchange of Bovine Serum Albumin with Hydrogen-Deuterium Exchange Small-Angle Neutron Scattering.

Hydrogen-deuterium exchange (HDX) measured by small-angle neutron scattering (HDX-SANS) is used to measure HDX in bovine serum albumin (BSA) under different temperatures and formulation conditions. HDX-SANS measurements are performed at 40, 50, and 60 °C in D2O after storing proteins at 4 °C for 1 week to pre-exchange the readily accessible hydrogens. This enables us to probe the long-time HDX of protons at the core of the BSA proteins, which is more challenging for solvent molecules to access. The HDX kinetics are observed to follow an Arrhenius behavior with an apparent activation energy of 81.4 ± 1 kJ/mol, which is composed of the energy for protein conformational fluctuations and that for exchanging an amide hydrogen. Adding a tonicity agent of 150 mM NaCl has only a very slight effect on the HDX kinetics. Interestingly, we also observed that the formulation with faster HDX kinetics has a lower onset temperature of denaturation. This observation is qualitatively consistent with a previous study of HDX-SANS on a monoclonal antibody (mAb), despite the large difference of the secondary structure between BSA, dominated by alpha helices, and mAb, which is predominantly composed of β-sheets.

Exploring the rheological and thermal behavior of cowpea protein concentrate: Impact of pH and concentration

This study confronts challenges from global population growth, focusing on protein supply strains and the environmental impact of traditional animal sources. Delving into unexplored territory, this study investigates the combined effects of pH, concentration, and temperature on the rheological and thermal properties of cowpea protein concentrate (CPC). Steady-state and oscillatory rheological tests, along with differential scanning calorimetric analysis, were conducted to unveil the behavior of CPC solutions. The results revealed sensitivity to protein concentration and shear rate, especially at higher concentrations (>10%), and pH emerged as a critical factor, significantly influencing shear stress and viscosity. The elastic and viscous components of alkaline CPC solutions exhibit weak gel behavior, influenced by both pH and frequency. Temperature-induced changes in viscosity and gelation highlight the interaction between thermal conditions and protein structures. The thermal cycle helps explain changes in molecular interactions induced by temperature, with pH playing a role in influencing gelatinization and denaturation. The denaturation temperature (Td) in the thermograms corresponds to vicilin fractions and emphasizes the prominence of denaturation and gelation at neutral pH, where Td was pH 7 > pH 9 > pH 11. Alkaline pH induced significant structural changes, resulting in a gelation onset temperature below the denaturation onset temperature. This study provides a deeper understanding into the rheological and thermal properties of CPC solutions, emphasizing the roles of concentration, pH, and temperature, useful for various steps of industrial processing, before and after thermal gelation. These findings are important for promoting the use of cowpea proteins in the food industry in response to the evolving needs of a growing global population.

Planococcus maritimus VITP21 synthesizes (2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-β-d-fructofuranose under osmotic stress: a novel protein stabilizing sugar osmolyte

A halotolerant bacterium, Planococcus maritimus VITP21 isolated from a saltern region in Kumta along the Arabian Sea Coast of India was found to have increased cellular levels of sugars (up to 2.3-fold) under osmotic stress when grown in minimal medium with glucose as the sole carbon and energy source supplemented with 10% w/v NaCl. The major sugar osmolyte which increased with the concentration of NaCl in the growth medium was purified and characterized using various nuclear magnetic resonance spectroscopy techniques. The sugar was found to be similar to sucrose but with the C-2 hydroxyl group of the glucose ring substituted with acetamido group, which is not previously reported for its natural synthesis by any other organism. This novel sugar, (2-acetamido-2-deoxy-α-d-glucopyranosyl)-(1→2)-β-d-fructofuranose, exhibited stabilizing effect on a model protein α-amylase by increasing the apparent midpoint transition, onset temperature of denaturation, and free energy of thermal unfolding.

Freeze–Thaw Induced Biomechanical Changes in Arteries: Role of Collagen Matrix and Smooth Muscle Cells

Applications involving freeze-thaw, such as cryoplasty or cryopreservation can significantly alter artery biomechanics including an increase in physiological elastic modulus. Since artery biomechanics plays a significant role in hemodynamics, it is important to understand the mechanisms underlying these changes to be able to help control the biomechanical outcome post-treatments. Understanding of these mechanisms requires investigation of the freeze-thaw effect on arterial components (collagen, smooth muscle cells or SMCs), as well as the components' contribution to the overall artery biomechanics. To do this, isolated fresh swine arteries were subjected to thermal (freeze-thaw to -20 degrees C for 2 min or hyperthermia to 43 degrees C for 2 h) and osmotic (0.1-0.2 M mannitol) treatments; these treatments preferentially altered either the collagen matrix (hydration/stability) or smooth muscle cells (SMCs), respectively. Tissue dehydration, thermal stability and SMC functional changes were assessed from bulk weight measurements, analyses of the thermal denaturation profiles using Fourier transform infrared (FTIR) spectroscopy and in vitro arterial contraction/relaxation responses to norepinephrine (NE) and acetylcholine (AC), respectively. Additionally, Second Harmonic Generation (SHG) microscopy was performed on fresh and frozen-thawed arteries to directly visualize the changes in collagen matrix following freeze-thaw. Finally, the overall artery biomechanics was studied by assessing responses to uniaxial tensile testing. Freeze-thaw of arteries caused: (a) tissue dehydration (15% weight reduction), (b) increase in thermal stability (approximately 6.4 degrees C increase in denaturation onset temperature), (c) altered matrix arrangement observed using SHG and d) complete SMC destruction. While hyperthermia treatment also caused complete SMC destruction, no tissue dehydration was observed. On the other hand, while 0.2 M mannitol treatment significantly increased the thermal stability (approximately 4.8 degrees C increase in denaturation onset), 0.1 M mannitol treatment did not result in any significant change. Both 0.1 and 0.2 M treatments caused no change in SMC function. Finally, freeze-thaw (506+/-159 kPa), hyperthermia (268+/-132 kPa) and 0.2 M mannitol (304+/-125 kPa) treatments all caused significant increase in the physiological elastic modulus (Eartery) compared to control (185+/-92 kPa) with the freeze-thaw resulting in the highest modulus. These studies suggest that changes in collagen matrix arrangement due to dehydration as well as SMC destruction occurring during freeze-thaw are important mechanisms of freeze-thaw induced biomechanical changes.

Effects of transglutaminase treatment on the thermal properties of soy protein isolates

Abstract The effects of covalent cross-linking of microbial transglutaminase (MTGase) on the thermal properties of soy protein isolates (SPI), including the thermal denaturation and glass transition were investigated by conventional and modulated differential scanning calorimetry (DSC). The MTGase treatment significantly increased the thermal denaturation temperatures (including the on-set temperature of denaturation, T m and the peak temperature of denaturation, T d ) of glycinin and β-conglycinin components of SPI ( P ⩽ 0.05), and the thermal pretreatment of SPI further increased the extent of this improvement. The MTGase treatment also improved the ability of SPI to resist the urea-induced denaturation. Modulated DSC analysis showed that there were two glass transition temperatures ( T g ) in the reversible heat flow signals of native SPI (about 5% moisture content), approximately corresponding to 45 and 180 °C, respectively. These T g values of SPI were significantly decreased by the MTGase treatment (at 37 °C for more than 2 h) ( P ⩽ 0.05). The improvement in the hydration ability of protein and the formation of high molecular biopolymers may account for the changes of thermal properties of soy proteins caused by the MTGase cross-linking.

The effects of pH, NaCl and CaCl 2 on thermal denaturation characteristics of intramuscular connective tissue

The research was conducted with two different experiments on intramuscular connective tissue obtained from Longissimus dorsi muscle of 4-year-old beef carcasses. Differential scanning calorimetry (DSC) was used to determine the denaturation onset temperature ( T o), denaturation peak temperature ( T p), and denaturation enthalpy (Δ H D) of intramuscular connective tissue. In the first experiment, equilibration of the collagen in citrate buffers in the pH range 2.9–6.5 resulted in the decline of T o and T p with decreasing pH, but statistically significant differences were not recorded for Δ H D. In the second experiment, NaCl and CaCl 2 solutions at 0.34, 0.68 and 1.02 ionic strength at pH 3.7–5.7 were studied. Increasing NaCl resulted in the increase of T o and T p, whilst T o and T p was decreased with increasing CaCl 2.

Influence of weak organic acids and salts on the denaturation characteristics of intramuscular connective tissue. A differential scanning calorimetry study

Intramuscular connective tissue obtained from Longissimus dorsi muscle of a 4-year-old beef carcass was treated with NaCl solutions of 2, 4, and 6% (w/v), and CaCl 2 solutions of 50, 100, and 150 mM, and citric and lactic acid solutions of 0.5, 1.0, and 1.5% for three different marinating periods (24, 46, and 72 h). Changes in denaturation characteristics were investigated using differential scanning calorimetry and it was found that the denaturation onset temperature ( T o) and the denaturation peak temperature ( T p) increased as the NaCl concentration increased but decreased as the CaCl 2 concentration increased, irrespective of marinating time. Lactic and citric acid decreased T o to about 39°C, from over 60°C breaking the structure of fibrils.

Studies on genetic variants of α‐lactalbumin and β‐lactoglobulin from milk of native Portuguese ovine and caprine breeds

Summary α‐Lactalbumin (α‐La) and β‐lactoglobulin (β‐Lg) fractions were obtained from Portuguese native breeds of ewes and goats by preparative gel filtration and further purified by ion exchange; their genetic variants were characterized by isolectric focusing, and β‐Lg isolated was further characterized by differential scanning calorimetry. Separation of β‐Lg and α‐La by molecular exclusion from native whey was relatively easy, whereas β‐Lg from both breeds accounted for a single peak via ion exchange under various gradients of NaCl. Isoelectric focusing has indicated that α‐La from ovine and caprine wheys appears as a single variant in each case, as well as β‐Lg from caprine whey; however, β‐Lg from ovine whey appears as two peaks, tentatively denoted as β‐Lg A and B. Further comparison with bovine whey made it possible to rank whey proteins by increasing value of pI as follows: bovine β‐Lg A, bovine α‐La, bovine β‐Lg B, ovine and caprine α‐La, ovine β‐Lg A, and finally ovine β‐Lg B and caprine β‐Lg. β‐Lg from goat's whey showed the highest onset temperature of denaturation in the presence (78–97 °C) or absence (90–100 °C) of NaCl for every pH tested; when NaCl was present, a good correlation between pI and onset temperature of denaturation was obtained for pH values in the range 3.5–7.0.

Comparative Characterization of Whey Protein Concentrates from Ovine, Caprine and Bovine Breeds

Whey protein concentrates (WPC) obtained from ovine, caprine and bovine wheys were studied in terms of chemical composition (concentration of lactose, protein, moisture, ash, calcium, sodium and potassium) and physical properties (onset denaturation temperature and viscoelastic behaviour). Ovine, caprine and bovine WPC exhibited decreasing protein content and increasing lactose content, in this order. The differences in ash content were relatively small, but ovine and caprine WPC were quite different from bovine WPC in terms of calcium and potassium. Important differences in protein content have also been observed; ovine WPC showed the highest concentration in the proteinβ-lactoglobulin, bovine WPC the highest concentration in the proteinα- lactalbumin and caprine the highest concentration in the protein immunoglobulin G. No significant differences existed in terms of denaturation onset temperature between the three types of WPC; however, differences were found within WPC types between those cases where NaCl was present or absent. The gel maturation at 85°C was characterized by storage moduli that were close to one another for ovine and caprine WPC, but these were far from those of bovine WPC. The decrease in temperature from 85 to 20°C had a strong effect on the viscoelastic moduli of WPC gels, but the initial structure could be recovered by reheating the gels from 20 to 85°C.

Foaming and Emulsifying Properties of Whey Protein Concentrates As Affected by Lipid Composition

ABSTRACTFreeze‐dried WPC, containing 35 and 75% protein were manufactured by pretreating whey with calcium chloride and heat. These and commercial WPC were subjected to proximate analysis and lipid classes, phospholipid classes, free fatty acids (FFA), and monoacylglycerols (MAG) composition were determined. Solubility, thermal, foaming, and emulsifying properties of the WPC were studied. Pretreatment increased calcium and phosphorus contents and decreased the contents of all other minerals. The pretreatment had no effect on solubility, denaturation enthalpy, and onset temperature of denaturation of WPC. These values were comparable to those of commercial WPC. Foaming capacity and emulsion stability were unaffected, but foam stability increased and emulsifying capacity decreased due to pretreatment. Overall, total lipids and lipid class contents of experimental WPC were too low to affect surface properties of WPC.

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response.

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.