Denaturation Rates Research Articles

Ethnobotanical and comparative study of the antioxidant and anti-inflammatory potential of three organs of Zanthoxylum zanthoxyloides, a plant used in the traditional treatment of sickle-cell disease in Bamako

The literature reports that the sickle cell patients frequently use plants to manage their recurrent attacks. The aim of the present study was to contribute to a better understanding of the medicinal plants used in the management of that disease. An ethnobotanical survey was conducted in and around Bamako. The most frequently cited species was selected: Zanthoxylum zanthoxyloides Lam. Zepernick. & Timler, and the in vitro antioxidant and anti-inflammatory potential of their leaf, fruit and root extracts were assessed. A total of 39 people were surveyed, including 21 herbalists, 6 traditional health practitioners and 12 sickle-cell patients. Analysis of the survey forms revealed 19 plant species belonging to 16 families, including Z. zanthoxyloides, which had the highest frequency of citation (20.51%). The phytochemical screening of this species revealed the presence of alkaloids, phenolic compounds (flavonoids, tannins, coumarins), saponins and terpenes. The hydroethanolic extracts of Z. zanthoxyloides fruits showed the best DPPH free radical scavenging activity with an inhibitory concentration (IC50) of 20.80±0.11 µg/mL followed by hydroethanolic extracts of bark with IC50=22.9±0.14 µg/mL. In the phosphomolybdate test, the aqueous fruit extracts with a value of 537.70±0.05 mg AAE/100g exhibited the highest antioxidant potential. At a concentration of 1 mg/mL extracts, the aqueous fruit extracts demonstrated the most effective protein denaturation rates (92.66±0.66%) followed by the ethanolic root extracts (92.95±1.10%). This study showed that the fruits exhibit the highest promising anti-free radical and anti-inflammatory potential.

Smaller Gold Nanoparticles Release DNA More Efficiently During fs Laser Pulsed Optical Heating.

This work investigates the effect of plasmonic gold nanoparticle (AuNP) size on the rate of thermal release of single-stranded oligonucleotides under femtosecond (fs)-pulsed laser irradiation sources. Contrary to the theoretical predictions that larger AuNPs (50-60nm diameter) would produce the most solution heating and fastest DNA release, it is found that smaller AuNP diameters (25nm) lead to faster dsDNA denaturation rates. Controlling for the pulse energy fluence, AuNP concentration, DNA loading density, and the distance from the AuNP surface finds the same result. These results imply that the solution temperature increases around the AuNP during fs laser pulse optical heating may not be the only significant influence on dsDNA denaturation, suggesting that direct energy transfer from the AuNP to the DNA (phonon-phonon coupling), which is increased as AuNPs decrease in size, may play a significant role.

Kinetic modelling of the heat stability of bovine lactoferrin in raw whole milk

The thermal denaturation of the naturally occurring lactoferrin in raw bovine whole milk has been measured over a range of temperatures from 65 to 121 °C for holding times from 2 up to 300 s designed to mimic heating processes used in the food manufacturing industry. Data were generated by heat treatments of raw whole milk from two different sources, New Zealand (NZ) and China, and with lactoferrin at two different levels of naturally occurring iron saturation. Denaturation rates for lactoferrin in the Chinese-sourced milk samples were at least two-fold greater than lactoferrin in the NZ milk over the temperature range 65–80 °C. At 85 °C rates of denaturation were the same although more than 80-fold greater than at 65 °C whereas, at 95 °C, lactoferrin in the NZ milk denatured at twice the rate of that in the Chinese milk. The differences in denaturation rates up to 80 °C likely reflected the greater heat stability of the more iron-saturated NZ milk samples. From these data, a combined kinetic and Arrhenius-based model has been developed and validated that allows calculation of native lactoferrin retained after thermal processing at any temperature-time combination in the experimental range (65–95 °C). This model should have applicability in the evaluation of existing processes and for the design of processes to improve the retention of lactoferrin during food manufacture.

Improving the mesoscopic modeling of DNA denaturation dynamics

Although previously developed mesoscopic DNA models have successfully reproduced thermodynamic denaturation data, recent studies show that these overestimate the rate of denaturation by orders of magnitude. Using adapted Peyrard–Bishop–Dauxois (PBD) models, we have calculated the denaturation rates of several DNA hairpins and made comparison with experimental data. We show that the addition of a barrier at the onsite potential of the PBD model gives a more accurate description of the unzipping dynamics of short DNA sequences. The new models provide a refined theoretical insight on the dynamical mechanisms of unzipping which can have implications for the understanding of transcription and replication processes. Still, this class of adapted PBD models seems to have a fundamental limitation which implies that it is not possible to get agreement with available experimental results on the dynamics of long DNA sequences and at the same time maintain the good agreement regarding its thermodynamics. The reason for this is that the denaturation rate of long DNA chains is not dramatically lowered by the additional barrier—as the base-pairs that open are more likely to remain open, facilitating the opening of the full DNA molecule. Some care has to be taken, since experimental techniques suitable to the study of denaturation rates of long sequences seem not to agree with other experimental data on short DNA sequences. Further research, both theoretical and experimental, is therefore needed to resolve these inconsistencies—which will be a starting point for new minimalistic models that are able to describe both thermodynamics and dynamics at a predictive level.

Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates

Shelf-stable whey concentrates produced by membrane filtration represent an energy efficient alternative to whey powder. However, to obtain products with sufficient microbiological shelf-life, preservation of concentrates is necessary. The idea of this study was, therefore, to investigate the influence of ionic composition and dry matter (DM) of whey concentrates on thermal stability of the major whey proteins. Taking the thermal impact of heating up into account, denaturation kinetics of β-lactoglobulin and α-lactalbumin were determined in reverse osmosis (RO) and nanofiltration (NF) whey concentrates with DM contents of 12–30% at heating temperatures of 90 and 125 °C. Denaturation caused by heating up was strongly increased with increasing DM of both types of concentrates. During holding at 90 °C, whey proteins showed a higher thermal stability in RO concentrates as compared with NF concentrates. However, denaturation rates at 125 °C of whey proteins were lower in NF than in RO concentrates.

PH- and temperature-dependent denaturation profiles of tuna myoglobin

Attempts have been made to elucidate the denaturation profiles of tuna myoglobin (Mb) in comparison with horse Mb. Intensive absorbance characterization was carried out for derivatives (deoxy, oxy, met forms) of Mb. The wavelength of the maximum absorbance of tuna Mb was shorter only by 1 nm for deoxy and metMb, while it was 4 nm shorter for the second peak of metMb. Percentage Mb denaturation (PMD) was measured under a combination of pH (5.6, 6.5, 7.4) and temperature (70, 75, 80 °C). Tuna Mb was almost completely denatured even during the initial incubation at 75 and 80 °C at all pHs examined. During the incubation at 70 °C, the PMD values for tuna Mb were 88.5, 52.1, and 67.7% at pH 5.6, 6.5, and 7.4, respectively. The denaturation of tuna Mb proceeded even at 55 °C, but denaturation rates were very slow at pH 6.5. On the other hand, horse Mb was found to be very stable at pH 6.5 and 7.4 at all temperatures examined, except at pH 6.5 and 80 °C. At pH 5.6, the PMD values of horse Mb gradually increased, especially at 75 and 80 °C. These different Mbs showed quite different denaturation profiles.

Effect of heat, pH, ultrasonication and ethanol on the denaturation of whey protein isolate using a newly developed approach in the analysis of difference-UV spectra

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

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

β-Lactoglobulin heat denaturation: A critical assessment of kinetic modelling

Bovine whey protein β-lactoglobulin (β-lg) will denature above 70 °C. Denaturation may contribute positively or negatively to food functionality, so it is important to control the degree of denaturation during milk processing. This work critically examined the kinetic modelling of β-lg denaturation in water, buffer or milk at 70–150 °C using the general rate law for a single-species reaction and the Arrhenius equation. Several published datasets, originally analysed in linearised form, were re-analysed with nonlinear regression, which preserves the error structure of data and estimates the precision of parameters. This work discusses statistical best practice for modelling the heat denaturation of β-lg, explores the criteria for using 2- or 3-parameter regression equations, and proposes new approaches for modelling the concentration- and temperature-dependence of denaturation rates. These new approaches improved the accuracy and precision of kinetic parameter estimates, and revealed new details of how solution conditions influence β-lg denaturation rates.

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison.

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimental data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.

Forensic determination of blood sample age using a bioaffinity-based assay.

A bioaffinity-driven cascade assay was developed to determine the time elapsed from the point a blood sample was left at a crime scene to the point of discovery. Two blood markers, creatine kinase (CK) and alanine transaminase (ALT), were utilized to determine the age of the blood spot based on their natural denaturation processes. The analysis with the proposed bioassay was performed in human serum samples, which underwent the aging process under environmental conditions that could be expected at crime scenes. The concentration of the markers in the sample was based on physiological levels present in healthy adults. These two markers were concerted in a biocatalytic cascade composed of two parallel subsystems, with each of them following the activity of one marker. Both markers have very distinct denaturation rates which would not allow them to be used in a single marker setup while still providing satisfactory results. However, by parallel tunable monitoring of both markers, it is possible to provide information of the blood sample age with low temporal error for a prolonged period of time. To mimic a possible real crime scene situation – the reliability of the proposed assay was then successfully tested on dried/aged serum samples (up to 5 days old) in environments with different temperatures.

The lower limits for protein stability and foldability in primary hyperoxaluria type I

Mutational effects on protein stability and foldability are important to understand conformational diseases and protein evolution. In this work, we perform a comprehensive investigation on the energetic basis underlying mutational effects on the stability of human alanine:glyoxylate aminotransferase (AGT). We study twenty two variants whose kinetic stabilities span over eleven orders of magnitude and are classified into two groups: i) ten naturally-occurring variants, including the most common mutations causing primary hyperoxaluria type I (PH1); and ii) twelve consensus variants obtained by sequence-alignment statistics. We show that AGT dimer stability determines denaturation rates, and mutations modulate stability by changes in the effective thermodynamic stability, the aggregation propensity of partially/globally unfolded states and subtle energetic changes in the rate-limiting denaturation step. In combination with our previous expression analyses in eukaryotic cells, we propose the existence of two lower limits for AGT stability, one linked to optimal folding efficiency (close to the major allele stability) and the other setting a minimal efficiency compatible with glyoxylate detoxification in vivo (close to the minor allele stability). These lower limits could explain the high prevalence of misfolding as a disease mechanism in PH1 and support the use of pharmacological ligands aimed to increase AGT stability as therapies for this disease.

Cold-Adapted Digestive Aspartic Protease of the Clawed Lobsters Homarus americanus and Homarus gammarus: Biochemical Characterization

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca2+ ATPase of squid myofibrils was significantly suppressed in the presence of Ca2+. Monomeric myosin content decreased much faster than Ca2+ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca2+ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca2+ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca2+ generated the apparently different myosin denaturation patterns in the two media.

Thermal stability of carp actin in its polymerized form

We investigated the thermal denaturation of carp F-actin by measuring the loss of polymerization ability. The thermal denaturation rates of carp F-actin were at least 10-fold higher than those of chicken F-actin. Binding of tropomyosin thermally stabilized carp F-actin with no appreciable change in activation energy, suggesting the instability was caused by a high frequency of fragmentation of the actin filaments. A comparison of the critical concentration for polymerization suggested that the subunit–subunit interactions of carp F-actin were indeed weaker than those of chicken F-actin. Furthermore, using fluorescence quenching we showed that the nucleotide binding region of carp F-actin was in a more open conformation. Together, our results suggest that the instability of carp F-actin is a function of both the rate of fragmentation and the dissociation of bound nucleotides.

Different denaturation rates between methylated and non-methylated genomic DNA can result in allele-specific PCR amplification

We analysed a DNA sample from a father and child who were both heterozygous for a 7 base pair insertion in the MEST gene differentially-methylated promoter region, previously shown by PCR analysis of bisulphite-treated DNA to be on the methylated allele in the unaffected father and the unmethylated allele in the affected child. PCR from genomic DNA was then carried out using a commercial PCR kit with its recommended initial DNA denaturation step of 2 minutes. Subsequent sequence analysis showed that only the non-methylated allele had been amplified, the father appearing to be homozygous normal and the child appearing to have a homozygous 7 b.p. insertion. The PCR protocol was then modified in order to use a longer DNA denaturation stage prior to the addition of the polymerase enzyme. Upon doing so, both the methylated and non-methylated alleles were then identifiable by sequencing with the mutation appearing in its expected heterozygous form. These results highlight the fact that the methylation status of DNA can affect the denaturation rate prior to PCR and result in allele drop-out, showing that the standard protocols of commercial kits should be used with caution when working with methylated regions of DNA.

FA cellular automaton model of crystalline cellulose hydrolysis by cellulases

BackgroundCellulose from plant biomass is an abundant, renewable material which could be a major feedstock for low emissions transport fuels such as cellulosic ethanol. Cellulase enzymes that break down cellulose into fermentable sugars are composed of different types - cellobiohydrolases I and II, endoglucanase and β-glucosidase - with separate functions. They form a complex interacting network between themselves, soluble hydrolysis product molecules, solution and solid phase substrates and inhibitors. There have been many models proposed for enzymatic saccharification however none have yet employed a cellular automaton approach, which allows important phenomena, such as enzyme crowding on the surface of solid substrates, denaturation and substrate inhibition, to be considered in the model.ResultsThe Cellulase 4D model was developed de novo taking into account the size and composition of the substrate and surface-acting enzymes were ascribed behaviors based on their movements, catalytic activities and rates, affinity for, and potential for crowding of, the cellulose surface, substrates and inhibitors, and denaturation rates. A basic case modeled on literature-derived parameters obtained from Trichoderma reesei cellulases resulted in cellulose hydrolysis curves that closely matched curves obtained from published experimental data. Scenarios were tested in the model, which included variation of enzyme loadings, adsorption strengths of surface acting enzymes and reaction periods, and the effect on saccharide production over time was assessed. The model simulations indicated an optimal enzyme loading of between 0.5 and 2 of the base case concentrations where a balance was obtained between enzyme crowding on the cellulose crystal, and that the affinities of enzymes for the cellulose surface had a large effect on cellulose hydrolysis. In addition, improvements to the cellobiohydrolase I activity period substantially improved overall glucose production.ConclusionsCellulase 4D simulates the enzymatic hydrolysis of cellulose to glucose by surface and solution phase-acting enzymes and accounts for complex phenomena that have previously not been included in cellulose hydrolysis models. The model is intended as a tool for industry, researchers and educators alike to explore options for enzyme engineering and process development and to test hypotheses regarding cellulase mechanisms.

Effects of cholesterol on thermal stability of discoidal high density lipoproteins

Reverse cholesterol transport in plasma involves variations in HDL cholesterol concentration. To understand physicochemical and functional implications of such variations, we analyzed stability of reconstituted HDL containing human apolipoproteins (apoA-I, apoA-II, or apoC-I), phosphatidylcholines varying in chain length (12-18 carbons) and unsaturation (0 or 1), and 0-35 mol% cholesterol. Lipoprotein heat denaturation was monitored by circular dichroism for protein unfolding/dissociation and by light scattering for particle fusion. We found that cholesterol stabilizes relatively unstable complexes; for example, incorporation of 10-30 mol% cholesterol in apoC-I:dimyristoyl phosphatidylcholine complexes increased their kinetic stability by deltaDeltaG* congruent with 1 kcal/mol. In more stable complexes containing larger proteins and/or longer-chain lipids, incorporation of 10% cholesterol did not significantly alter the disk stability; however, 15% or more cholesterol destabilized the apoA-I-containing complexes and led to vesicle formation. Thus, cholesterol tends to stabilize less stable lipoproteins, apparently by enhancing favorable packing interactions, but in more stable lipoproteins, where such interactions are already highly optimized, the stabilizing effect of cholesterol decreases and, eventually, becomes destabilizing. These results help uncouple the functional roles of particle stability and chain fluidity and suggest that structural disorder in HDL surface, rather than chain fluidity, is an important physicochemical determinant of HDL function.

Immunochemical investigation of human seminal plasma acid phosphatase.

Acid phosphatase of human seminal plasma is inhibited in presence of normal rabbit serum involving non-competitive mechanism whereas this character is normalized and activity is stabilized in presence of its rabbit antiserum. Kinetic studies on stabilization of acid phosphatase by antiserum were combined by L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate inhibited the enzyme in presence and absence of normal and antiserum by competitive mechanism and percentage of inhibition of L-tartrate was involve in the binding with acid phosphatase antibodies. The kinetics of heat denaturation of acid phosphatase suggested that seminal acid phosphatase is more heat labile in presence and absence of normal serum than in presence of antiserum. The temperature of denaturation corresponding to half the initial activity(T 1/2) in presence of normal and antisera were 34 degrees and 66 degrees C respectively. NOn-linearity of Arrhenius plots for denaturation rates of the enzyme suggested various conformational states for denaturation yielding Ea varying between 20.08 and 5.03 Kcals at 25-65 degrees C.

Between-Species Variation in the Kinetic Stability of TIM Proteins Linked to Solvation-Barrier Free Energies

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.