Thermal Denaturation Process Research Articles

Thermal Inactivation, Denaturation and Aggregation Processes of Papain-Like Proteases.

The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

May DNA analyses be biased by hidden oxidative damage? Voltammetric study of temperature and oxidation stress effect.

The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.

Enhancement of phycocyanin productivity and thermostability from Arthrospira platensis using organic acids

Intracellular hyperaccumulation of phycocyanin (PC) and its high susceptibility to degradation at higher temperatures are major challenging problems associated with its production from cyanobacteria. The present study evaluated different concentrations of organic acids (1, 2, and 3 mM) (citric acid, acetic acid, succinic acid, fumaric acid, and oxalic acid) under fed-batch mode on the biomass and phycobiliproteins’ production from Arthrospira platensis. Besides they were evaluated at 2.5–7.5 mM as preservative to stabilize PC at high temperatures. The incorporation of 3 mM of succinic acid into the cultivation medium enhanced the biomass and PC productivity to 164.05 and 26.70 mg L−1 day−1, which was ~ 2- and threefold higher than control, respectively. The produced PC in this treatment was food-grade with a 2.2 purity ratio. The use of organic acids also enhanced the thermal stability of PC. Citric acid (7.5 mM) markedly promoted the half-life values of PC to 189.44 min compared to 71.84 min in the control. The thermodynamic analysis confirmed higher thermostability of PC in the presence of organic acids and indicated the endothermic and non-spontaneity of the thermal denaturation process. The findings of the present study confirmed that organic acids could be utilized as cost effective and sustainable compounds for promoting not only phycobiliproteins’ production but also the thermostability of PC for potential application in food industry.

Study on the mechanism of soy protein isolate to improve quality of reduced-salt Hypophthalmichthys molitrix surimi gel: Focus on gel quality, protein structure, and in vitro digestibility

Excessive intake of sodium chloride may bring a series of diseases; as a result, reduced-salt surimi gels have gained growing popularity for sodium reduction. This paper studied soy protein isolate (SPI, 2.0%, 4.0%, and 6.0%, w/w) as a gel enhancer for reduced-salt silver carp surimi. Compared with the control (2.0% NaCl), the addition of SPI significantly increased (P < 0.05) the total SH content, hydrophobic interaction force, disulfide bond, hardness, gel strength, and water-holding capacity of the gels. During the thermal denaturation process, SPI and myofibrillar protein jointly participated in the formation of the gel network, resulting in a G′ value increase at 90 °C, forming a denser/more stable gel network structure. In vitro pepsin digestion results showed the digestibility of the reduced-salt gel with SPI was higher than that of the control. Therefore, appropriate SPI addition can improve the gel performance of reduced-salt surimi gel without affecting digestion and absorption.

Preparation of Gelatin from Broiler Chicken Stomach Collagen.

With the increasing consumption of poultry meat around the world, the use of chicken stomachs as a source of collagen is being offered. The objective of this study was to extract gelatin from the stomachs of broiler chickens and to estimate their gel strength, ash content, viscosity, gelling point, melting point, clarity and digestibility. An innovative biotechnological method based on the conditioning of collagen with a microbial endoproteinase (Protamex®) and hot-water extraction was used to control the chemical and thermal denaturation process of collagen to prepare gelatin. The experiments were planned using a Taguchi design, 2 factors at 3 levels; factor A for the amount of proteolytic enzyme (0.10, 0.15 and 0.20%) and factor B for the extraction temperature (55.0, 62.5 and 70.0 °C). Data were statistically processed and analyzed at a significance level of 95%. The gelatin yield averaged 65 ± 8%; the gel strength ranged from 25 ± 1 to 439 ± 6 Bloom, the viscosity from 1.0 ± 0.4 to 3.40 ± 0.03 mPa·s, gelling point from 14.0 ± 2.0 to 22.0 ± 2.0 °C, melting point from 28.0 ± 1.0 to 37.0 ± 1.0 °C. The digestibility of gelatin was 100.0% in all samples; the ash content was very low (0.44 ± 0.02-0.81 ± 0.02%). The optimal conditions for the enzymatic treatment of collagen from chicken stomachs were achieved at a higher temperature (70.0 °C) and a lower amount of enzyme (0.10-0.15%). Conditioning chicken collagen with a microbial endoproteinase is an economically and environmentally friendly processing method, an alternative to the usual acid- or alkaline-based treatment that is used industrially. The extracted products can be used for food and pharmaceutical applications.

Characterization of Secondary Structure and Thermal Stability by Biophysical Methods of the D-alanyl,D-alanine Ligase B Protein from Escherichia coli.

Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔHUcal and ΔHUvH ), as well as characterization of elements of secondary structure at three different pHs. DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔHUvH U of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of β-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in β-structures that can contribute to the formation of protein aggregates. Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.

Focusing on intramuscular connective tissue: Effect of cooking time and temperature on physical, textual, and structural properties of yak meat

This study aimed to evaluate the effects of different cooking time (2, 4, and 6 h) and temperature (50, 60, 70, 80, and 90 °C) on physical, textual, and structural properties of longissimus lumborum muscle of yak, and to explore the thermal denaturation process of intramuscular collagen by using a new tool (collagen hybridizing peptide staining, CHP staining). The results showed that tenderness was affected by the interaction of cooking time and temperature and the changes in moisture and collagen composition. In comparison with cooking time, temperature had more obvious effects on cooking loss, moisture content and redness. Scanning electron microscopy showed that as the temperature increased, intramuscular connective tissue gradually degraded, and muscle fibers became more compact. CHP staining showed that the collagen in the perimysium first denatured at 50 °C, and more and more collagen denatured and degraded as the temperature increased.

Molecular imprinting of doxorubicin by refolding thermally denatured bovine serum albumin and cross-linking with hydrogel network

In this work, we demonstrate a novel molecular imprinting method by unfolding and refolding an auxiliary protein in the presence of a small-molecular template, followed by fixation of the resultant protein/template assembly via polymerization of additional functional monomers. Employing doxorubicin (DOX) as a model template and bovine serum albumin (BSA) as an auxiliary binding protein, we optimized the conformation of BSA through thermal denaturation and renaturation process in the presence of template DOX. Then, in-situ polymerization of isopropylacrylamide, N-[3-(dimethylamino)propyl]methacrylamide and acrylamide was carried out to form imprinted hydrogels of the dBSA&DOX complex. The conformation of BSA in the resultant polymer was further fixed via formaldehyde cross-linking with the hydrogel network. The obtained imprinted hydrogel showed much higher affinity and selectivity to DOX than both native BSA and the control hydrogel prepared by using native BSA instead of unfolded/refolded BSA. The method may be further extended to prepare water compatible imprinted hydrogel against other small molecules by using easily accessible proteins as auxiliary ligands.

Determining the temperatures to which the bone was heated in archaeological contexts. Distinguishing between boiled and grilled bones

A study carried out in fresh cow (Bos taurus) cortical-femur bones which allows determining the maximum annealing temperature to which a bone was subjected is shown. It is focused on temperatures near the ones used in culinary activities. Mechanical spectroscopy, optical microscopy, differential thermal analysis and thermogravimetry studies are used in the present work. The study takes into account the behaviour of the thermal denaturation processes from the remnant collagen as a function of temperature. The survey of the relaxation process related to the triple helix to random coil transition from the residual collagen which occurs at 510 K/237 °C, is proposed as a potentially useful tool for determining that the bone was not previously heated above this temperature. In addition, by using mechanical spectroscopy a procedure for discriminating between grilled- and boiled-bones is shown. Likewise, a reference chart of the thermodynamic states of denaturation of collagen in the bone as a function both of temperature and cooking mode is reported. The relationships between the denaturation stages and the responses from mechanical spectroscopy and differential thermal analysis are also shown.

Biophysical characterization of the structural stability of Helix lucorum hemocyanin

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L −1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (T m) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L −1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Е a of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein.

Proteinen desnaturalizazio termalaren bidezko analisia konposatu bioeraginkor berrien aurkikuntzarako

Konposatu bioeraginkorrak bizidunetan jarduera zelularraren edota fisiologikoaren gain eragin dezaketen substantzia kimiko naturalak zein sintetikoak dira. Horien artean daude, adibidez, farmakoak, prozesu biologikoen modulazioaren bitartez osasunaren gain efektu onuragarriak bideratu ditzaketenak. Konposatu bioeraginkor berrien eta horien itu biologikoak bilatzeko estrategia berrien garapena inpaktu handiko erronka da. Itu proteinak edota ekintza mekanismoak aurkitzeko metodologia ezberdinak garatu dira. Itu proteinak aurkitzeko metodoen artean, lan honetan jorratzen dena daukagu: konposatu bioeraginkorrek proteinen desnaturalizazio termalean eragindako aldaketen azterketan oinarritutako metodoa. Lan honetan, konposatu bioeraginkorrak bilatzeko metodologia horren oinarriak eta haren inplementaziorako irizpideak azaltzen dira.

Evidence for Many Unique Solution Structures for Chymotrypsin Inhibitor 2: A Thermodynamic Perspective Derived from vT-ESI-IMS-MS Measurements.

Chymotrypsin inhibitor 2 (CI-2) is a classic model for two-state cooperative protein folding and is one of the most extensively studied systems. Alan Fersht, a pioneer in the field of structural biology, has studied the wild-type (wt) and over 100 mutant forms of CI-2 with traditional analytical and biochemical techniques. Here, we examine wt CI-2 and three mutant forms (A16G, K11A, L32A) to demonstrate the utility of variable-temperature (vT) electrospray ionization (ESI) paired with ion mobility spectrometry (IMS) and mass spectrometry (MS) to map the free energy folding landscape. As the solution temperature is increased, the abundance of each of the six ESI charge states for wt CI-2 and each mutant is found to vary independently. These results require that at least six unique types of CI-2 solution conformers are present. Ion mobility analysis reveals that within each charge state there are additional conformers having distinct solution temperature profiles. A model of the data at ∼30 different temperatures for all four systems suggests the presence of 41 unique CI-2 solution conformations. A thermodynamic analysis of this system yields values of ΔCp as well as ΔG, ΔH, and ΔS for each state at every temperature studied. Detailed energy landscapes derived from these data provide a rare glimpse into Anfinsen's thermodynamic hypothesis and the process of thermal denaturation, normally thought of as a cooperative two-state transition involving the native state and unstructured denatured species. Specifically, as the temperature is varied, the entropies and enthalpies of different conformers undergo dramatic changes in magnitude and relative order to maintain the delicate balance associated with equilibrium.

Thermal and emulsifying properties of globulins from chan ( Hyptis suaveolens L. Poit) seeds

This research aims to study the thermal and emulsifying properties of the chan globulins (CG), which are the most abundant Osborne fraction of the seed of Hyptis suaveolens. In this sense, the calorimetric analysis indicated a cooperative behavior in thermal denaturation process with a denaturation temperature of 100.9°C and an enthalpy change of 124.6 kJ/mol for CG. Besides, strong gels (Gʹ ~7.5 × 105 Pa) were produced, with a gelation temperature of 80°C. Also, CG presented high surface activity with critical micelle concentration (CMC) of 0.3 mg/ml. The best emulsifying properties and colloidal stability were found at basic pH values and they were negatively influenced by the content of NaCl along a pH gradient. In conclusion, CG may be used as thermostable emulsifier at basic pH values (preferably without NaCl), and as a potential ingredient for protein-rich gel formulations. Practical applications This work describes a simple and practical procedure to obtain CG at high yield (96%) and acceptable purity, which could be easily implemented without further costs and tedious chromatographic techniques. In addition, the high gelation capacity and strong gel formation for CG, make them suitable for formulation of protein-based gels with potential application in the drug and food industry. CG also showed high emulsifying activity and emulsifying stability indexes, implying their promising use as a thermostable emulsifier at basic pH values. Finally, the incorporation of CG as food ingredient, is an alternative to other pseudocereals to expand the offer of gluten-free food products for celiac consumers.

Conformational structure variation of human serum albumin after binding interaction with black phosphorus quantum dots

Herein, binding interaction between black phosphorus quantum dots (BPQDs) and human serum albumin (HSA) was systematically characterized for deep illustration of conformational structure variation of HSA affected by BPQDs. The results confirmed that the intrinsic fluorescence of HSA was statically quenched by BPQDs mainly through van der Waals interaction and hydrogen bond. BPQDs bound strongly with the site I of HSA to form ground state complex with molar ratio of 1 to 1. The secondary structure of HSA was changed obviously after its binding interaction with BPQDs, and the α-helix structure of HSA was transformed to the β-sheet structure. The melting temperature of HSA was decreased after its binding interaction with BPQDs, suggesting that BPQDs promoted the thermal denaturation process of HSA. BPQDs could also reduce the molar enthalpy change and the thermal stability of HSA. These results explored the exact conformational structure variation of HSA after its binding interaction with BPQDs, which provides vital information for possible biological influence of BPQDs on human beings.

C-mannosylation supports folding and enhances stability of thrombospondin repeats.

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.

THE STUDY OF THERMAL DENATURATION OF BEEF, PORK, CHICKEN AND TURKEY MUSCLE PROTEINS USING DIFFERENTIAL SCANNING CALORIMETRY

In the temperature range from 45 °C to 90 °C the process of thermal denaturation of a whole complex of muscle proteins in meat takes place. An effective mode to register the thermal denaturation process is the method of differential scanning calorimetry (DSC). As a result of studies the differences during the process of thermal denaturation of muscle proteins of pork, beef, chicken and turkey were defined by the appearance of endothermic peaks in DSC thermograms. The main variances are associated with the process of denaturation of myosin and sacroplasmic proteins and indicate indirectly their quantitative ratio in meat. The values of effective specific heat capacity in the temperature range from 20 °C to 90 °C are obtained as well as those of heat spent on the denaturation process.At reheating, the values of specific heat capacity increased by 0.1 J/(g*K) on the average, and peaks of thermal denaturation were not detected, that certifies the irreversibility of the denaturation process and the decrease in the bound moisture proportion in meat after thermal processing. Knowledge of the nature of protein thermal denaturation of each kind of meat product is one of the necessary tools for developing the technology of meat product thermal processing.

Whole-body cryostimulation impact on blood serum thermal denaturation profiles of cross-country skiers

Blood plasma and serum are important diagnostic materials as they clearly reflect an individual’s metabolism. The study has evaluated the effect of whole-body cryostimulation (WBC) on the blood serum of professional cross-country skiers. The experiment involved eight athletes (two women and six men) who underwent a series of ten WBC treatments. Aqueous solutions of human blood serum samples before WBC procedures, after one treatment and after a series of ten treatments were measured by means of differential scanning calorimetry (DSC), a relatively novel diagnostic tool. DSC results showed rather little impact of cryostimulation on heat capacity changes accompanying the process of thermal denaturation of blood serum proteins in elite athletes. However, the statistically significant reduction in the intensity of the serum denaturation transition in its low temperature range has been observed after ten WBC treatments. The results have been interpreted by changes in the serum proteome profile, notably in the ratio of ligated to unligated albumin molecules. As a side result, the relationships between the relative change in body fat mass after ten WBC treatments and the levels of alpha2-globulins and beta2-globulins fractions have been found.

Heterologous expression, purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris.

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (Tm ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.

Microscopic description of DNA thermal denaturation

We propose a microscopic model describing the process of DNA thermal denaturation. The process consists of the splitting of DNA base pairs, or nucleotides, resulting in the separation of two complementary DNA strands. In contrast to the previous modelling attempts we take into account the states of all base pairs of DNA which in fact imposes the microscopic nature of the approach. The model is a linear integro-differential non-autonomous equation describing the dynamics of probability density which characterizes the distances between the bases within individual base pairs. The non autonomous structure of the equation comes from the dependence on temperature that may be in the general case a (given) function of time. To our knowledge it is the first model taking into account not only the strength of double and triple hydrogen bonds between the complementary bases but also the stacking interactions between neighborhood base pairs. In the present paper the basic preliminary properties of the model including the existence, uniqueness and stability results in various cases are discussed. Moreover the symmetrized version of the model is considered and the “macroscopic limit” is studied. The performed numerical simulations reproduce the sigmoid shape of DNA melting curves and reveal the appearance of experimentally observed denaturation bubbles.

Molecular mechanism for thermal denaturation of thermophilic rhodopsin.

Understanding the factors affecting the stability and function of proteins at the molecular level is of fundamental importance. In spite of their use in bioelectronics and optogenetics, factors influencing thermal stability of microbial rhodopsins, a class of photoreceptor protein ubiquitous in nature are not yet well-understood. Here we report on the molecular mechanism for thermal denaturation of microbial retinal proteins, including, a highly thermostable protein, thermophilic rhodopsin (TR). External stimuli-dependent thermal denaturation of TR, the proton pumping rhodopsin of Thermus thermophilus bacterium, and other microbial rhodopsins are spectroscopically studied to decipher the common factors guiding their thermal stability. The thermal denaturation process of the studied proteins is light-catalyzed and the apo-protein is thermally less stable than the corresponding retinal-covalently bound opsin. In addition, changes in structure of the retinal chromophore affect the thermal stability of TR. Our results indicate that the hydrolysis of the retinal protonated Schiff base (PSB) is the rate-determining step for denaturation of the TR as well as other retinal proteins. Unusually high thermal stability of TR multilayers, in which PSB hydrolysis is restricted due to lack of bulk water, strongly supports this proposal. Our results also show that the protonation state of the PSB counter-ion does not affect the thermal stability of the studied proteins. Thermal photo-bleaching of an artificial TR pigment derived from non-isomerizable trans-locked retinal suggests, rather counterintuitively, that the photoinduced retinal trans-cis isomerization is not a pre-requisite for light catalyzed thermal denaturation of TR. Protein conformation alteration triggered by light-induced retinal excited state formation is likely to facilitate the PSB hydrolysis.