Intermediate Thermal Stability Research Articles

Cellular substrates and pro-apoptotic function of the human HtrA4 protease

The human HtrA4 protein, belonging to the HtrA family of proteases/chaperones, participates in oncogenesis and placentation, and plays a role in preeclampsia. As the knowledge concerning the biochemical features of this protein and its role at the molecular level is limited, in this work we characterized the HtrA4 molecule and searched for its cellular function. We found that recombinant HtrA4 composed of the protease and PDZ domains is a trimeric protein of intermediate thermal stability whose activity is considerably lower compared to other human HtrA proteases. By pull-down combined with mass spectrometry we identified a large array of potential HtrA4 partners. Using other experimental approaches, including immunoprecipitation, enzyme-linked immunosorbent assay and fluorescence microscopy we confirmed that HtrA4 formed complexes in vitro and in cellulo with proteins such as XIAP (inhibitor of apoptosis protein), caspases 7 and 9, β-tubulin, actin, TCP1α and S100A6. The recombinant HtrA4 degraded XIAP, the caspases, β-tubulin and actin but not TCP1α or S100A6. Together, these results suggest that HtrA4 may influence various cellular functions, including apoptosis. Furthermore, the panel of potential HtrA4 partners may serve as a basis for future studies of HtrA4 function.

Contribution of Catalytic Transesterification Reactions to the Compatibilization of Poly(lactic acid)/Polycarbonate Blends: Thermal, Morphological and Viscoelastic Characterization

Samarium acetylacetonate (Sm-Acac) was added to catalyze interchange reactions between poly(lactic acid) (PLA) and polycarbonate (PC) in order to promote compatibilization and enhance the performances of the PLA/PC blend. The effects of the composition and catalyzed transesterification reactions were investigated using differential scanning calorimetry (DSC), thermogravimetry (TG), dynamic mechanical thermal analysis (DMTA) and scanning electron microscopy (SEM). DMTA and DSC analysis revealed the immiscibility of the uncatalyzed PLA/PC blends for the studied compositions because the glass transition temperatures of PC and PLA were unchanged after blending. In the PLA glassy region, PLA/PC blends exhibited lower storage moduli which increased upon heating due to the cold crystallization process. During melt mixing with Sm-Acac catalyst, PLA/PC blends were submitted to two competing processes. In one hand, Sm-Acac acted as a plasticizer and contributed in decreasing significantly the glass transition, crystallization and melting temperatures of PLA phase. In the other hand, Sm-Acac proved its efficiency in catalyzing the transesterification reactions that were evidenced by the decrease of the PLA aptitude to crystallization due to the hindering effect of the PC units inserted into the PLA chains. PLA/PC blends melt mixed with 0.25% of Sm-Acac showed a significant strengthening effect, corresponding to an increase in the storage modulus in the temperature range comprised between 70 and 90 °C. This indicated the formation of a copolymer at the interface and the promotion of adhesion as it is confirmed from the decrease in the height of the PLA Tan δ peak. At 0.5% of Sm-Acac, (90/10) PLA/PC blend revealed a new peak assigned to the glass transition of the PLA-PC copolymer, whereas the (50/50) PLA/PC blend was converted into a new random copolymer. TG analysis proved the presence of a copolymer structure presenting an intermediate thermal stability in both the catalyzed and uncatalyzed blends.

Replication Protein A Unfolds G-Quadruplex Structures with Varying Degrees of Efficiency

Replication protein A (RPA) is known to interact with guanine- (G-) rich sequences that adopt G-quadruplex (GQ) structures. Most studies reported in the literature were performed on GQ formed by homogeneous sequences, such as the human telomeric repeat, and RPA's ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ-forming DNA sequences (PQSs) to their stability against RPA-mediated unfolding using single-molecule fluorescence resonance energy transfer (FRET) and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQSs in the genome. The three GQ constructs have thermal stabilities that differ significantly. Our measurements showed that the most thermally stable structure (Tm = 86 °C) was also the most stable against RPA-mediated unfolding, although the least thermally stable structure (Tm = 69 °C) had at least an order-of-magnitude higher stability against RPA-mediated unfolding than the structure with intermediate thermal stability (Tm = 78 °C). The significance of this observation becomes more evident when considered within the context of the cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these results, we conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single-molecule stopped-flow method. All three GQ structures were unfolded within Δt ≈ 0.30 ± 0.10 s, a surprising result considering that the unfolding time does not correlate with thermal stability or stability against RPA-mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

Photo, thermal, and ultrasonic degradation of EGDMA‐crosslinked poly(acrylic acid‐co‐sodium acrylate‐co‐acrylamide) superabsorbents

AbstractSuperabsorbent polymers (SAPs) based on acrylic acid (AA), sodium acrylate (SA), and acrylamide (AM) were synthesized by inverse suspension polymerization using ethylene glycol dimethacrylate as the crosslinking agent. The equilibrium swelling capacities and the rates of swelling of SAPs varied with the AM content and followed first‐order kinetics. The photodegradation of SAPs in their equilibrium swollen state was carried out by monitoring their swelling capacity and the residual weight fraction. The SAPs degraded in two stages, wherein the swelling capacity increased to a maximum and then subsequently decreased. Thermogravimetric analysis of the SAPs indicated that the copolymeric superabsorbents had intermediate thermal stability between the homopolymeric superabsorbents. The activation energies of SAPs with 0, 20, and 100 mol % AM content were determined by Kissinger method and were found to be 299, 248, and 147 kJ mol−1, respectively. The ultrasonic degradation of the superabsorbents was carried out in their equilibrium swollen state, and the change in the viscosity with ultrasonication time was used to quantify the degradation. The ultrasonic degradation of AA/SA superabsorbent was also investigated at various ultrasound intensities. The degradation rate coefficients were found to increase with the intensity of ultrasound. The ultrasonic degradation of AA/SA/AM (20% AM) was also carried out, and degradation rate was found to be more than that of the AA/SA superabsorbent. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Synthesis and Thermal Study of Cured Epoxy-Acrylate-Maleate, Unsaturated Polyester and Their Interpenetrating Networks of Varying Compositions

Epoxy-acrylate-maleate of bisphenol-C (ECAM), unsaturated polyester resin of phthalic anhydride, maleic anhydride and propylene glycol (PMP) were synthesized. ECAM, PMP and their varying compositions (25–75 wt%) were cured using MEKP and cobalt naphthenate and characterized by IR, DSC and TGA techniques. Interpenetrating networks possess intermediate thermal stability of ECAM (300°C) and PMP (227°C) and followed single step degradation with fractional order kinetics (0.6–2.4) leaving 10–25% residue above 400°C. Low magnitudes of energy of activation and frequency factor of interpenetrating networks indicated improvement in flexibility due to toughening of PMP by ECAM of varying compositions.

CLONING AND SEQUENCING OF THE α-AMYLASE GENE FROMBACILLUS SUBTILISUS116 STRAIN ENCODING AN ENZYME CLOSELY IDENTICAL TO THAT FROMBACILLUS AMYLOLIQUEFACIENSBUT DISTINCT IN THERMAL STABILITY

The gene encoding for the α-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the α-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure–function relationships of these biocatalysts.

Thermal stability of LDPE, iPP and their blends

The thermal decomposition reaction of LDPE, iPP and LDPE/PP blends irradiated with accelerated electrons has been investigated with TGA and DSC. TGA showed only one degradation step at different temperatures according to the type and composition of the samples. LDPE exhibited the highest thermal stability compared with either iPP or LDPE/iPP blends; LDPE/iPP blends showed intermediate thermal stability. TMPTMA loading and irradiation enhanced the thermal stability of all samples. The obtained TGA data by the stimulation method illustrated that the compatibility between LDPE and iPP in their blends takes place during mixing and irradiation processes, where only one degradation step was observed in the experimental TGA data instead of two degradation stages that appeared in the arithmetic calculation curve of the blend. The melting temperature decreased for TMPTMA loaded LDPE, whereas irradiation and/or loading with TMPTMA did not change the melting temperature of iPP. LDPE/iPP (50/50 wt%) showed a drop in melting temperature compared with the components. The glass transition temperatures increased as a result of modification either by TMPTMA loading or irradiation.

Thermal Characterization of Upper Critical Solution Temperature m‐LLDPE/Poly(ethylene‐ran‐butene) Elastomer Blends

AbstractSummary: The phase and thermal characteristics of blends consisting of linear low‐density polyethylene (LLDPE) (0.7 mol‐% hexene copolymer) and poly(ethylene‐ran‐butene) (PEB) (26 mol‐% butene copolymer) have been investigated using optical microscopy (OM), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). An upper critical solution temperature of 162 °C was exhibited. The addition of PEB not only slowed the overall crystallization rate of LLDPE but also changed the distribution of lamellar thickness or perfection of LLDPE crystals. The equilibrium melting temperature of LLDPE in the blends was reduced and kept relatively constant in the bi‐phase state. The blends showed a single‐stage degradation and an intermediate thermal stability between those of the individual components. It could be attributed to their homogeneous states at degradation temperatures and the similar decomposing mechanisms of two components. The kinetic analysis of thermal degradation also confirmed the above results.Phase diagram of LLDPE/PEB blends.magnified imagePhase diagram of LLDPE/PEB blends.

Thermal characteristics of addition-cure phenolic resins

The thermal and pyrolysis characteristics of four different types of addition-cure phenolic resins were compared as a function of their structure. Whereas the propargyl ether resins and phenyl azo functional phenolics underwent easy curing, the phenyl ethynyl- and maleimide-functional ones required higher thermal activation to achieve cure. All addition-cure phenolics exhibited improved thermal stability and char-yielding property in comparison to conventional phenolic resole resin. The maleimide-functional resins exhibited lowest thermal stability and those crosslinked via ethynyl phenyl azo groups were the most thermally stable systems. Propargylated novolac and phenyl ethynyl functional phenolics showed intermediate thermal stability. The maximum char yield was also given by ethynyl phenyl azo system. Non-isothermal kinetic analysis of the degradation reaction implied that all the polymers undergo degradation in at least two steps, except in the case of ethynyl phenyl azo resin, which showed an apparent single step degradation. The very low pre-exponential factor common to all polymers implied the significance of volatilisation process in the kinetics of degradation. Isothermal pyrolysis studies led to the conclusion that in the case of nitrogen-containing polymer, the pyrolysis occurs via loss of nitrogenous products, which is conducive for enhancing the carbon-content of the resultant char. FTIR spectra of the pyrolysed samples confirmed the presence of C–O groups in the char. XRD analysis of the partially carbonised polymers did not give any indication of crystallites except in the case of ethynyl phenyl azo system.

Human Erythrocyte Proteinl-Isoaspartyl Methyltransferase: Heritability of Basal Activity and Genetic Polymorphism for Thermal Stability

Proteinl-isoaspartyl methyltransferase (PIMT) is believed to play an important role in the disposition of age-damaged proteins by catalyzing the repair of abnormal isoaspartyl linkages resulting from the spontaneous deamidation of asparaginyl residues or isomerization of aspartyl residues. As a step toward testing the hypothesis that human disease- or age-related pathology might be associated with a deficiency in PIMT, we investigated basal activity and thermal stability of PIMT in erythrocyte lysates from 299 U.S. family members. Thermal stability was measured because it is a sensitive measure of variation in amino acid sequence. Basal activity was normally distributed with a mean ± SD of 558 ± 43 units/ml erythrocytes. Statistical analysis of the data revealed that basal PIMT activity exhibited a high degree of heritability. Enzyme thermal stability showed a skewed bimodal frequency distribution, and segregation analysis of family member pedigrees was consistent with Mendelian inheritance of two major alleles. No DNA was available from the family samples, so we tested two additional population samples for a known Ile/Val polymorphism at codon 119 and for PIMT activity and thermal stability, using blood donated by 25 Norwegians and by 20 Koreans. Single-stranded conformational polymorphism analysis using polymerase chain reaction revealed a 100% correlation between thermal stability grouping and this polymorphism. The high thermal stability samples were all homozygous Ile, the low thermal stability samples were all homozygous Val, and the intermediate thermal stability samples were all heterozygous. Furthermore, this polymorphism was responsible, in part, for the variation observed in basal erythrocyte PIMT activity. These results will help provide a foundation for future studies aimed at correlating levels of PIMT activity, or other properties of this enzyme, with human disease.

Characterization of C1 Inhibitor-Ta: A DYSFUNCTIONAL C1INH WITH DELETION OF LYSINE 251

Dysfunctional C1 inhibitor (C1INH)-Ta is a naturally occurring mutant from a patient with type II hereditary angioedema. This mutant has a deletion of the codon for Lys-251, which is located in the connecting strand between helix F and strand 3A, overlying beta sheet A. Deletion of this Lys modifies the amino acid sequence at this position from Asn-Lys-Ile-Ser to Asn-Ile-Ser and creates a new glycosylation site. To further characterize the mechanism of dysfunction, we have analyzed the recombinant normal and Ta proteins expressed by COS cells in addition to the proteins in serum and isolated from serum. Recombinant C1INH-Ta revealed an intermediate thermal stability in comparison with the intact and reactive center cleaved normal proteins. Analysis of the reactivity of this recombinant protein with target proteases demonstrated no complex formation with C1s, C1r, or kallikrein. Inefficient complex formation was, however, clearly detectable with beta-factor XIIa. Each protease produced partial cleavage of the recombinant mutant inhibitor. Recombinant C1INH-Ta, on 7.5% SDS-polyacrylamide gel electrophoresis and by size fractionation on Superose 12, showed a higher molecular weight fraction that was compatible in size with dimer formation. However, no multimerization of C1INH-Ta isolated from serum or of C1INH-Ta in serum, was observed. The C1INH-Ta dimer expressed the epitopes that normally are expressed only on the protease complexed or the cleaved inhibitor. These epitopes were not expressed on the monomeric inhibitor. The data suggest that the mutation in C1INH-Ta results in a folding abnormality that behaves as if it consists of two populations of molecules, one of which is susceptible to multimerization and one of which is converted to a substrate, but which retains residual inhibitory activity.

Phenolic resin

The thermal and pyrolysis characteristics of four different types of addition-cure phenolic resins were compared as a function of their structure. Whereas the propargyl ether resins and phenyl azo functional phenolics underwent easy curing, the phenyl ethynyl- and maleimide-functional ones required higher thermal activation to achieve cure. All addition-cure phenolics exhibited improved thermal stability and char-yielding property in comparison to conventional phenolic resole resin. The maleimide-functional resins exhibited lowest thermal stability and those crosslinked via ethynyl phenyl azo groups were the most thermally stable systems. Propargylated novolac and phenyl ethynyl functional phenolics showed intermediate thermal stability. The maximum char yield was also given by ethynyl phenyl azo system. Non-isothermal kinetic analysis of the degradation reaction implied that all the polymers undergo degradation in at least two steps, except in the case of ethynyl phenyl azo resin, which showed an apparent single step degradation. The very low pre-exponential factor common to all polymers implied the significance of volatilisation process in the kinetics of degradation. Isothermal pyrolysis studies led to the conclusion that in the case of nitrogen-containing polymer, the pyrolysis occurs via loss of nitrogenous products, which is conducive for enhancing the carbon-content of the resultant char. FTIR spectra of the pyrolysed samples confirmed the presence of C–O groups in the char. XRD analysis of the partially carbonised polymers did not give any indication of crystallites except in the case of ethynyl phenyl azo system.

Purification and characterization of a new alpha-amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae.

A new alpha-amylase from the extracellular culture of the yeast Lipomyces kononenkoae CBS 5608 has been purified to homogeneity by ammonium sulphate treatment, affinity binding on cross-linked starch, and DEAE-Biogel A chromatography. The enzyme was monomeric, with an apparent M(r) of 76 kilodaltons, pI < 3.5, and optimum pH 4.5-5.0, and exhibited intermediate thermal stability. The temperature for optimal enzyme activity was 70 degrees C. It is a glycoprotein with both N- and O-linked sugars. Kinetic analyses indicate that the enzyme has an endoamylolytic mechanism. The kM for soluble starch was 0.80 g.L-1 and the kcat was 622.s-1.