Kinetics Of Protein Denaturation Research Articles

Precooked state based on protein denaturation kinetics impacts moisture status, protein oxidation and texture of prepared chicken breast

The quality of meat in prepared dishes deteriorates due to excessive protein denaturation resulting from precooking, freezing, and recooking. This study aimed to link the precooked state with chicken breast's recooked quality. Cooked Value (CV), based on protein denaturation kinetics, was established to indicate the doneness of meat during pre-heating. The effects of CVs after pre-heating on recooked qualities were investigated compared to fully pre-heated samples (control). Mild pre-heating reduced water migration and loss. While full pre-heating inhibited protein oxidation during freezing, intense oxidation during pre-heating led to higher oxidation levels. Surface hydrophobicity analysis revealed that mild pre-heating suppressed aggregation during recooking. These factors contributed to a better texture and microstructure of prepared meat with mild pre-heating. Finally, a potential mechanism of how pre-heating affects final qualities was depicted. This study underlines the need for finely controlling the industrial precooking process to regulate the quality of prepared meat.

The Nature of the Enthalpy–Entropy Compensation and “Exotic” Arrhenius Parameters in the Denaturation Kinetics of Proteins

Protein unfolding is a ubiquitous process responsible for the loss of protein functionality (denaturation), which, in turn, can be accompanied by the death of cells and organisms. The nature of enthalpy–entropy compensation (EEC) in the kinetics of protein unfolding is a subject of debate. In order to investigate the nature of EEC, the “completely loose” transition state (TS) model has been applied to calculate the Arrhenius parameters for the unfolding of polyglycine dimers as a model process. The calculated Arrhenius parameters increase with increasing dimer length and demonstrate enthalpy–entropy compensation. It is shown that EEC results from the linear correlations of enthalpy and entropy of activation with dimer length, which are derived directly from the properties of the transition state. It is shown that EEC in solvated (hydrated, etc.) proteins is a direct consequence of EEC in proteins themselves. The suggested model allows us also to reproduce and explain “exotic” very high values of the pre-exponential factor measured for the proteins unfolding, which are drastically higher than those known for unimolecular reactions of organic molecules. A similar approach can be applied to analyzing the nature of EEC phenomena observed in other areas of chemistry.

Calcium Chelation by Phosphate Ions and Its Influence on Fouling Mechanisms of Whey Protein Solutions in a Plate Heat Exchanger.

Fouling of plate heat exchangers (PHEs) is a recurring problem when pasteurizing whey protein solutions. As Ca2+ is involved in denaturation/aggregation mechanisms of whey proteins, the use of calcium chelators seems to be a way to reduce the fouling of PHEs. Unfortunately, in depth studies investigating the changes of the whey protein fouling mechanism in the presence of calcium chelators are scarce. To improve our knowledge, reconstituted whey protein isolate (WPI) solutions were prepared with increasing amounts of phosphate, expressed in phosphorus (P). The fouling experiments were performed on a pilot-scale PHE, while monitoring the evolution of the pressure drop and heat transfer coefficient. The final deposit mass distribution and structure of the fouling layers were investigated, as well as the whey protein denaturation kinetics. Results suggest the existence of two different fouling mechanisms taking place, depending on the added P concentration in WPI solutions. For added P concentrations lower or equal to 20 mg/L, a spongy fouling layer consists of unfolded protein strands bound by available Ca2+. When the added P concentration is higher than 20 mg/L, a heterogeneously distributed fouling layer formed of calcium phosphate clusters covered by proteins in an arborescence structure is observed.

Use of Microwave Radiometry to Monitor Thermal Denaturation of Albumin.

This study monitored thermal denaturation of albumin using microwave radiometry. Brightness Temperature, derived from Microwave Emission (BTME) of an aqueous solution of bovine serum albumin (0.1 mM) was monitored in the microwave frequency range 3.8–4.2 GHz during denaturation of this protein at a temperature of 56°C in a conical polypropylene cuvette. This method does not require fluorescent or radioactive labels. A microwave emission change of 1.5–2°C in the BTME of aqueous albumin solution was found during its denaturation, without a corresponding change in the water temperature. Radio thermometry makes it possible to monitor protein denaturation kinetics, and the resulting rate constant for albumin denaturation was 0.2 ± 0.1 min−1, which corresponds well to rate constants obtained by other methods.

Kinetics of Protein Degradation and Physical Changes in Thermally Processed Atlantic Salmon (Salmo salar)

Protein denaturation is the main reason for physicochemical changes in muscle foods during thermal processing. The purpose of this study was to understand the kinetics of quality degradation and protein denaturation of Atlantic salmon (Salmo salar) during thermal pasteurization. Findings indicate that cook loss, area shrinkage, and protein denaturation parameters (both dielectric loss and differential scanning calorimetry (DSC) studies) were best fitted to a first-order reaction. In addition, color parameters followed zero-order kinetics. Fourier transform infrared spectroscopy (FTIR) results showed that increasing heating time and temperature reduced the α-helix peak, indicating protein denaturation. At the same time, the β-sheet peak intensity increased, which is related to protein aggregation. Correlation loading plots from FTIR spectra showed that protein denaturation was dominant below 75 °C. Above this transition temperature, protein denaturation occurred rapidly and protein aggregation became more obvious. The dielectric properties in cooked salmon at different times and temperature increased with increasing heating time and temperature, while penetration depth decreased significantly (P < 0.05). The kinetics of protein denaturation from DSC showed that the activation energy of 300.6 kJ/mol and k 0 was 1.1 × 1042 min−1. Our findings on kinetic data from protein denaturation and physical changes may be used to improve the processing schedule in the production of safe, high-quality pasteurized salmon.

Model for electrical conductivity of muscle meat during Ohmic heating

A model is presented for predicting the electrical conductivity of muscle meat, which can be used for the evaluation of Ohmic heating. The model computes the conductivity as a function of composition, temperature and microstructure. The muscle meat is thought to be composed of protein, water, salt. Concerning the microstructure, the model takes into account the muscle fiber orientation with respect to the electric field, and the development of drip channels due to protein denaturation. The model includes a description of the protein denaturation kinetics. The model has been validated for different types of meat, varying in composition and heating rate. The submodel for protein denaturation is validated using independent DSC measurements. For meats heated faster than 20° per minute, the conductivity is a linear function of temperature - due to the absence of protein denaturation, and thus drip channel formation. If meat is heated slower than 10° per minute the conductivity is showing non-linear behaviour, with a significant decrease at temperatures above 70 degrees Celsius. This decrease is explained by the action of the complete protein denaturation. Our study shows that if Ohmic heating of meat is performed at fast rates, there is a large potential to retain most of its moisture during heating.

Theoretical and practical aspects relating to the photothermal therapy of tumors of the retina and choroid: A review

Photothermal treatment of tumors of the retina and choroid such as retinoblastomas, malignant melanomas, benign tumors as well as of vascular malformations can be performed by using laser radiation. A number of basic physical laws have to be taken into account in this procedure. Of particular importance thereby are: Arrhenius' law to approximate the kinetics of protein denaturation and photocoagulation, furthermore the electromagnetic radiation field, the distribution of both radiant and thermal energy induced in tumors and vascular structures, the influence of the wavelength and laser pulse duration (exposure time), as well as of the optical properties of the tissue. Strict confinement of the extent of the photothermal damage is critical since such pathological entities are frequently located close to the macula or optic nerve head.The conditions for tumor destruction are best fulfilled when using radiation in the near-infrared range of the electromagnetic spectrum such as that emitted from the diode (810 nm) and the Nd: YAG (1064 nm) laser, because of the good optical penetration properties of these radiations in tissue. Short wavelength sources of radiation, such as the argon ion (488, 514 nm) or the freqeuency-doubled Nd: YAG (532 nm) laser are less well suited for the irradiation of large vascular structures due to their poor penetration depths. However, for vascular formations with a small thickness (1 mm or less), short wavelength sources appear to be the most appropriate choice. Optical coupling of radiant energy to the eye by means of indirect ophthalmoscopic systems or positive contact lenses is furthermore of importance. Strong positive lenses may lead to severe constrictions of the laser beam within the anterior segment, that leads to high irradiance increasing the probability for structures to be damaged; with negative contact lenses, such as the -64 D Goldmann type lens, this danger is largely absent.

Study of Ca2+-ATPase Activity and Solubility in the Whole Kuruma Prawn (Marsupenaeus japonicus) Meat During Heating: Based on the Kinetics Analysis of Myofibril Protein Thermal Denaturation

Protein denaturation is considered to be the main cause of physicochemical changes in prawns during heating. However, no studies have been analyzed the kinetics of protein denaturation and the relationship between the degree of denaturation and chemical changes. Therefore, we investigated the changes in Ca2+-ATPase activity, protein solubility, and total sulfhydryl content of whole prawn meat during heating by determining the thermal denaturation kinetics of the proteins. Activation energies (E a ) for the denaturation of myosin (183.2 kJ/mol) and actin (178.8 kJ/mol) were obtained by non-isothermal differential scanning calorimetry analysis. Using the kinetic parameters, the distribution of protein denaturation was predicted in whole prawns under arbitrary heating conditions. The results revealed an uneven distribution of the protein denaturation in prawns that was dependent on the heating conditions. Ca2+-ATPase activity decreased with increasing heating times at 51 or 85 °C and was strongly related to the average degree of protein denaturation. The results of protein solubility analysis suggested that hydrogen bonds, hydrophobic interactions, and ionic bonds changed with protein denaturation. The number of ionic bonds was reduced, while hydrogen content was enhanced at both temperatures. Hydrophobic interactions increased gradually at 51 °C (p 0.05). Our results indicate that the extent of protein solubility is significantly correlated with the average degree of protein denaturation during the heating process.

Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.

Optically Induced Thermal Gradients for Protein Characterization in Nanolitre-scale Samples in Microfluidic Devices

Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry.

Mathematical modeling of the heat transfer process and protein denaturation during the thermal treatment of Patagonian marine crabs

Southern Ocean swimming crab Ovalipes trimaculatus and the Patagonian stone crab Platyxanthus patagonicus are fishing resources with commercial value. Thermal treatment of crabs is necessary to denature muscle proteins, facilitating meat detachment from the crab shell (picking procedure). The proximal composition, protein patterns of crab muscle, thermophysical properties and heat transfer coefficients were determined. Heat transfer during thermal processing of body (i.e., cephalothorax) and claws of both crab species was simulated using a finite element computational code; the simulations were experimentally validated. Color changes in crab muscle during the heating process were measured. Thermal denaturation kinetics of myofibrillar proteins was determined using Differential Scanning Calorimetry (DSC) in small samples previously heated in water under controlled conditions. DSC thermograms of raw crab muscle showed two peaks at 49.0±0.4 and 77.5±0.6°C corresponding to myosin and actin respectively. Activation energies for the denaturation of myosin (145.70kJ/mol) and actin (156.42kJ/mol) were calculated from Arrhenius equation. The degree of denaturation achieved by the myofibrillar proteins at the coldest point of the muscle in body and claws during the heating process was established by considering the protein denaturation kinetics determined by DSC, the activation energies and the heat penetration curves. Adequate conditions for the detachment of meat from the crab exoskeleton were established. The obtained results may help in determining the optimal heating times during the industrialization of these crustaceans.

Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease

ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an N-terminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting single-molecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function.

Microstructural characterization of β-lactoglobulin–konjac glucomannan systems: Effect of NaCl concentration and heating conditions

In the initial part of this study, the high temperature (85 °C) microscopic phase behaviour of β-lactoglobulin (0.4–6%, w/w)–konjac (0.05–0.75%, w/w) mixtures containing 50 mM NaCl was established using confocal laser scanning microscopy (CLSM). Also, the effects of heating time (heating temperature: 78 °C) and NaCl concentration (0–75 mM) on protein denaturation kinetics and the phase behaviour in 2%, w/w, β-lactoglobulin–0.4%, w/w, konjac mixtures were investigated using turbidimetry, protein denaturation measurement, CLSM and image analysis techniques. Segregative phase separation occurred in heat-treated β-lactoglobulin–konjac mixtures containing biopolymer and NaCl concentrations exceeding certain critical levels, due to heat and NaCl induced β-lactoglobulin denaturation/aggregation. The microstructural properties of selected heated (to 85 °C for 30 min) and cooled (to 25 °C) β-lactoglobulin–konjac mixtures containing different NaCl levels were studied using CLSM and rheological measurements and the results showed that the microstructure can be distinguished as miscible, phase separated or phase separated containing stable protein inclusions dependent on NaCl concentration. Response surface methodology was used to determine the minimum NaCl concentrations required for phase separation and for formation of phase separated systems containing stable inclusions in a wide concentration range of heated and cooled β-lactoglobulin (0.8–2%, w/w)–konjac (0.2–0.75%, w/w) mixtures. The results show that the microstructural and rheological properties of β-lactoglobulin–konjac mixtures can be controlled by selecting appropriate mixture biopolymer and NaCl concentrations and heating conditions.

Effect of agitation on heat-induced deproteination process of buffalo milk whey

The heat induced deprotienation of buffalo milk whey was carried out in a bench top continuous process after optimization of process parameters for maximum protein removal at lab scale. The optimum conditions for maximum heat induced aggregation were found to be 92 ± 2 °C, 30 min at a pH 5.3 ± 0.1 and in the presence of 10 mM CaCl 2. The CSTR (continuous stirred tank reactor) was then used as a tool to study the kinetics of heat induced denaturation at different agitation rates. The kinetics was also studied with batch system under similar conditions. The values of deproteination rate constants were in close agreement from both batch and continuous studies. The shear rate was found to have great effect on the protein denaturation kinetics as well as protein aggregate size distribution. The rate of deproteination was found to increase as the shear rate increased. The denaturation kinetics also changed from second order to first order as the shear was increased from 360 s −1 to 1020 s −1, in both batch as well as continuous process.

Thermal Stability of Proteins

Protein stability is critical to the outcome of nearly all thermally mediated applications to biomaterials such as thermal therapies (including cryosurgery), burn injury, and biopreservation. As such, it is imperative to understand as much as possible about how a protein loses stability and to what extent we can control this through the thermal environment as well as through chemical or mechanical modification of the protein environment. This review presents an overview of protein stability in terms of denaturation due to temperature alteration (predominantly high and some low) and its modification by use of chemical additives, pH modification as well as modification of the mechanical environment (stress) of the proteins such as collagen. These modifiers are able to change the kinetics of protein denaturation during heating. While pH can affect the activation energy (or activation enthalpy) and the frequency factor (or activation entropy) of the denaturation kinetics, many other chemical and mechanical modifiers only affect the frequency factor (activation entropy). Often, the modification affecting activation entropy appears to be linked to the hydration of the protein. While the heat-induced denaturation of proteins is reasonably well understood, the heat denaturation of structural proteins (e.g., collagen) within whole tissues remains an area of active research. In addition, while some literature exists on protein denaturation during cold temperatures, relatively little is known about the kinetics of protein denaturation during both freezing and drying. Further understanding of this kinetics will have an important impact on applications ranging from preservation of biomaterials and pharmaceutics to cryosurgery. Interestingly, both freezing and drying involve drastic shifts in the hydration of the proteins. It is clear that understanding protein hydration at the molecular, cellular, and tissue level will be important to the future of this evolving area.

Microbial inactivation by new technologies of food preservation

The increasing consumer demand for 'fresh-like' foods has led to much research effort in the last 20 years to develop new mild methods for food preservation. Nonthermal methods allow micro-organisms to be inactivated at sublethal temperatures thus better preserving the sensory, nutritional and functional properties of foods. The aim of this review is to provide an overview of the microbiological aspects of the most relevant nonthermal technologies for microbial inactivation currently under study, including irradiation, high hydrostatic pressure, pulsed electric field and ultrasound under pressure. Topics covered are the mechanisms of inactivation, sensitivity of different microbial groups and factors affecting it and kinetics of inactivation.

A Modular Laser Apparatus for Polarimetry, Nephelometry, and Fluorimetry in General Chemistry

In most chemistry departments general chemistry students are the widest audience for engaging public understanding of the wonders and importance of the discipline of chemistry. Use of lasers for medical, commercial, and scientific purposes has increased, yet students often have to wait until upper-level courses to gain experience with lasers. We present an apparatus suitable for multiple uses in the general chemistry laboratory including polarimetry, fluorescence, and nephelometry. The main advantage of the instrument is its modular nature; the students assemble the instrument themselves for each laboratory exercise, thereby developing an understanding of the apparatus and the measurements made using the device. The open design of the instrument also decreases the chance that students will contract the "black-box syndrome" that seems to develop when using instruments that only provide the final data in an experiment without showing how the measurements are obtained. This apparatus is well suited for use in organic, biochemistry, and physical chemistry classes. In addition, we present suitable experiments adapted for the instrument: determination of the kinetics of acid-catalyzed hydrolysis of sucrose, determination of sulfate ion concentration in tap water using nephelometry, and fluorescence-probed kinetics of protein denaturation.

Transpupillar laser phototherapy for retinal and choroidal tumors: a rational approach.

The physical laws are considered that need to be taken into account for optimal photothermal treatment of solid and vascular tumors, as well as other vascular anomalies of various etiology of the retina and choroid. Optimal irradiation therapy should take into account the distribution of both radiant and thermal energy in tumors, such as retinoblastomas, malignant melanomas and vascular malformations. Strict confinement of the extent of photothermal damage is critical since such pathological entities are frequently located close to the macula or optic nerve head. A formal treatment of the optical quantities related to these requirements is presented. In this analysis we emphasize the following topics: Arrhenius' law, the kinetics of protein denaturation, the electromagnetic radiation field, wavelength, laser pulse duration (exposure time), optical properties of tissue, photocoagulation and thermotherapy. Generally, the conditions are best fulfilled when using radiation in the near-infrared range of the electromagnetic spectrum, such as that emitted from the diode (810 nm) and Nd:YAG (1064 nm) laser, because of the good optical penetration properties of this radiation in tissue. The xenon arc lamp was a very effective and particularly appropriate energy source for such purposes, and its withdrawal from the world market may have been untimely. Short wavelength sources of radiation, such as the argon ion (488, 514 nm) or the frequency-doubled Nd:YAG (532 nm) laser, are unsuitable for the irradiation of large vascular structures, as they have poor penetration depths. However, for vascular formations with a short path length (1 mm or less), short wavelength sources appear to be the most appropriate choice. Optical coupling of radiant energy to the eye by means of indirect ophthalmoscopic systems or contact lenses to the eye is crucial. Strong positive lenses may lead to severe constriction of the laser beam which leads to high irradiance within the anterior segment, increasing the chances of it being damaged; with negative contact lenses, such as the -64 D Goldmann type, this danger is reduced. Photothermotherapy is not without risk unless the temperature field can be well adapted to the tumorous structure, as temperature elevations outside a small therapeutic range that affect vital structures are considered to be a risk factor.

Thermo-mechanical laser ablation of soft biological tissue: modeling the micro-explosions

Characteristics of thermo-mechanical laser ablation process are investigated using an original numerical model. In contrast with previous models, it is based on a microscopic physical model of the micro-explosion process, which combines thermodynamic behavior of tissue water with elastic response of the solid tissue components. Diffusion of dissipated heat is treated in one dimension, and the amount of thermal damage is assessed using the Arrhenius model of the protein denaturation kinetics. Influence of the pulse fluence and duration on temperature profile development, ablation threshold, and depth of thermal damage is analyzed for the case of Er:YAG laser irradiation of human skin. Influence of mechanical properties on the ablation threshold of soft tissue is predicted theoretically for the first time. In addition, feasibility of deep tissue coagulation with a repetitively pulsed Er:YAG laser is indicated from the model.

Exploring the reaction kinetics of whey protein denaturation/aggregation by assuming the denaturation step is reversible

A theoretical study is presented here to illustrate the fundamental differences between laboratory established milk protein denaturation kinetics and the underlined reaction kinetics. Applying the elementary reaction kinetics in the context of reaction engineering, cases where the systems of β-lactoglobulin (β-lg) in water and β-lactoglobulin and α-lactalbumin (α-lac) mixture in water have been analysed. Assuming that denaturation is reversible to certain extent, for a mixture where many native whey proteins co-exist, the current study shows that the kinetics of β-lg denaturation can be far more complicated than that described by the simple rate equation commonly applied in previous literature. In order to illustrate that it is possible to use the existing data to obtain the underlined kinetic parameters, a computational procedure has been followed, as an example, to calculate the parameters for the one protein system (i.e., with β-lg only). Some sensible recommendations for future work have been made.