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PubMed2005

The in situ observation of the temperature and pressure stability of recombinant Aspergillus aculeatus pectin methylesterase with Fourier transform IR spectroscopy reveals an unusual pressure stability of beta-helices.

The stability of recombinant Aspergillus aculeatus PME (pectin methylesterase), an enzyme with high beta-helix content, was studied as a function of pressure and temperature. The conformational stability was monitored using FTIR (Fourier transform IR) spectroscopy whereas the functional enzyme stability was monitored by inactivation studies. Protein unfolding followed by amorphous aggregation, which makes the process irreversible, was observed at temperatures above 50 degrees C. This could be correlated to the irreversible enzyme inactivation observed at that temperature. Hydrostatic pressure greater than 1 GPa was necessary to induce changes in the enzyme's secondary structure. No enzyme inactivation was observed at up to 700 MPa. Pressure increased PME stability towards thermal denaturation. At 200 MPa, temperatures above 60 degrees C were necessary to cause significant PME unfolding and loss of activity. These results may be relevant for an understanding of the extreme stability of amyloid fibrils for which beta-helices have been proposed as a structural element.

Aspergillus↗
PubMed1999

Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis.

A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions. To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22-->Lys and Met256-->Val, that separately endowed the protein with further stability. We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations. Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II beta-turn play important roles in enhancing the protein stability. Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid. The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Modeling protein stability: a theoretical analysis of the stability of T4 lysozyme mutants.

Free energy calculations were conducted to determine the relative stability of the unnatural amino acid mutants of T4 lysozyme norvaline (Nvl) and O-methyl-serine (Mse) and of alanine at residue 133, which is leucine in the native sequence. These calculations were performed both to assess the validity of the methodology and to gain a better understanding of the forces which contribute to protein stability. Peptides of different length were used to model the denatured state. Restraints were employed to force sampling of the side chain chi1 dihedral of the perturbed side chain, and the effect of protein repacking in response to mutation was studied through the use of different constraint sets. In addition, the convergence behavior and hysteresis of the simulations in the folded and unfolded states were determined. The calculated results agree well with experiment, + 1.84 versus + 1.56 kcal/mol for Mse-->Nvl and -3.48 versus -2.2 to -3.6 kcal/mol for Nvl-->Ala. We find that free energy calculations can provide useful insights to protein stability when conducted carefully on a well chosen system. Our results suggest that loss of packing interactions in the native state is a major source of destabilization for mutants which decrease the amount of buried nonpolar surface area and that subtle responses of the backbone affect the magnitude of the loss of stability. We show that the conformational freedom of the chi1 dihedral has a noticeable effect on protein stability and that the solvation of amino acid side chains is strongly influenced by interactions with the peptide backbone.

Amino Acid Sequence↗
PubMed2002

Novel design architecture for genetic stability of recombinant poliovirus: the manipulation of G/C contents and their distribution patterns increases the genetic stability of inserts in a poliovirus-based RPS-Vax vector system.

Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.

Amino Acid Sequence↗
PubMed2001

Evaluation of the stability of (99m)Tc-ECD and stabilized (99m)Tc-HMPAO stored in syringes.

OBJECTIVE: To determine and compare the stability of (99m)Tc-ECD and stabilized (99m)Tc-HMPAO when stored in syringes over an 8-h period. METHODS: (99m)Tc-ECD and stabilized (99m)Tc-HMPAO were prepared according to the manufacturers' protocols, with the following exception: eluate less than 60 min old was used to prepare (99m)Tc-HMPAO rather than the recommended 30 min. Once prepared, 185 MBq (5 mCi) of both products were drawn into 5-mL syringes and allowed to sit at room temperature. At 2, 4, 6, and 8 h after preparation, the radiochemical purity (RCP) of the contents of the syringes was determined and compared to the RCP of the products in vials. Retention of activity of each product in syringes was also evaluated by measuring activity remaining in each syringe (and filter, in the case of (99m)Tc-HMPAO) after expressing its contents. RESULTS: The RCP of stabilized (99m)Tc-HMPAO stored in syringes decreased from a mean of 87.7% at 2 h to 74.0% at 8 h after preparation. In contrast, (99m)Tc-ECD retained an RCP of greater than 94% throughout the time tested. The impurity that appeared to increase over time with (99m)Tc-HMPAO was found to be sodium pertechnetate. Total retention of activity remaining in the syringe and filter ranged from 11.6% at 2 h to 9.5% at 8 h for (99m)Tc-HMPAO; the syringe itself retained less than 5% of the total activity at all time periods. (99m)Tc-ECD exhibited 6.2% to 11.3% retention of activity in the syringe. The sorption of sodium pertechnetate to the syringe for the same time period was less than 1%. CONCLUSIONS: (99m)Tc-ECD is a more stable product than stabilized (99m)Tc-HMPAO in a syringe. Both products demonstrate retention of radioactivity in the syringe. Some of this retention may denote sorption of the products to plastic.

Cysteine↗
PubMed1992

Effect of terminal heat sterilization on the stability of phospholipid-stabilized submicron emulsions.

Parenteral emulsions similar in composition to the commercially available fat emulsions were prepared using a Microfluidizer. The relationship of pH and terminal heat sterilization by autoclaving on the stability of phospholipid-stabilized emulsions was evaluated. The stability of these formulations was determined by using droplet size and zeta potential measurements. It was found that the zeta potentials of emulsions stabilized by more purified phospholipid were lower in magnitude compared to those stabilized by less purified phospholipid. Furthermore, acidic emulsions (between pH 5.0 and 7.0) showed an increased droplet size upon autoclaving, whereas the emulsions adjusted to an alkaline pH (pH 8.0 to 10.0) did not show a significant change in their droplet sizes upon autoclaving. The zeta potentials of all the emulsions "increased" (in magnitude) upon autoclaving and achieved values between -40 and -50 mV. The results of our studies could be explained on the basis of hydrolysis of phosphatidylcholine at different pH values, leading to a breakdown of the film surrounding the oil droplet, which results in coalescence and/or cracking of the emulsions.

Drug Stability↗
PubMed2006

Chemical stability of lipid excipients in SLN-production of test formulations, characterisation and short-term stability.

The study investigates the chemical stability of lipids used as excipients in the production of solid lipid nanoparticles (SLN). A total of 17 SLN formulations was produced using different lipids. Most of the formulations were produced using identical binary surfactant mixtures and concentrations to study the effect of the chemical nature of the lipid on its stability in SLN. In some formulations surfactants were exchanged to study the contribution of the surfactant. The particles were characterised by photon correlation spectroscopy, laser diffractometry, zeta potential determination and differential scanning calorimetry, the latter to assess potential effects of lipid crystallinity and modifications on lipid stability. Lipid analysis was performed by gas chromatography using a sampling preparation and analysis procedure especially developed for SLN. This short-term study provides primarily information about the stability of the lipid under production conditions, that means high pressure homogenisation (cavitation) at high temperature. No degradation products couldbe detected for all lipids, the production process itself did not impair excipient stability.

Calorimetry, Differential Scanning↗
PubMed1983

[Efficacy testing of stabilizing agents in epinephrine model solutions. 19: Stability of drugs and preparations].

The efficiency of 54 stabilizers was tested on epinephrine solutions using a selection-combination method. The addition of only one antioxidant produces but slight improvements in stability. Maximal additive activity is achieved by the combination of two antioxidants. Superadditive effects (which are necessary for considerable prolongation of the time of applicability of aqueous epinephrine solutions) are obtained by the combination of antioxidants of different modes of action and a discolouration-protective agent or a synergist. On this basis, the authors developed an efficient stabilizing procedure for pharmaceutical preparations containing active principles sensitive to oxidation. The relationship between the concentration of the stabilizer and the stabilizing effect is demonstrated by examples.

Antioxidants↗
PubMed1997

Stability and stabilization of recombinant peroxidase in reversed micelles.

Stability of recombinant peroxidase lacking carbohydrate residues on the surface of the protein molecule has been characterized in reversed micelles of Aerosol OT in octane. The enzyme stability was found to depend on the surfactant hydration degree (w0 = [H2O]/[AOT]). Residual activity after 1 h incubation dropped to zero at w0 = 7 but was 54% at w0 = 25. However, the residual activity levels at all values of hydration degree were definitely low compared to that of glycosylated wild-type horseradish peroxidase. The stability of the enzyme apparently depends on the presence of carbohydrate residues. Stabilization of recombinant peroxidase in reversed micellar system involved sugar-containing co-surfactants such as Tweens and Spans is proposed. As an example, addition of 1 mM Span 80 (1% relative to AOT concentration) increased the recombinant peroxidase stability up to that of wild-type peroxidase.

Detergents↗
PubMed1989

Comparison of alpha-helix stability in peptides having a negatively or positively charged residue block attached either to the N- or C-terminus of an alpha-helix: the electrostatic contribution and anisotropic stability of the alpha-helix.

An estimation of the thermodynamic effects of a charged random coil, which is attached either to the N- or C-terminus of polyalanine, upon alpha-helix stability is attempted. A temperature-induced helix-coil transition of Ala20Lys20Phe and Lys20Ala20Phe was studied under various conditions of salt concentration and pH. By combining the results with previous ones for Ala20Glu20Phe and Glu20Ala20Phe, which have opposite electric charges to the present system [S. Ihara et al. (1982) Biopolymers 21, 131-145], the free energy of the coil to helix transition of the polyalanine block could be separated into two terms--one term for the electrostatic interaction of electric charges in the random-coil block with the alpha-helix dipole, and a second term for the intrinsic stability of the helix. The first term indicates the significance of the helix dipole-charge interactions, which affects the helix stability depending on the attaching side of the charged block and on the sign of the charges. This clearly shows the anisotropic stability of the alpha-helix. Furthermore, analysis of the dependence of these thermodynamic quantities on salt concentrations showed, assuming that the effect of the attached electric charges was symmetric (in other words, the absolute values of the electrostatic interaction terms were independent of the sign of electric charges), that the intrinsic stability of the alpha-helix was dependent on which side of the helix was attached to the random coil: a random coil attached to the N-terminus of the alpha-helix had little effect while that attached to a C-terminal significantly destabilized the helix.

Circular Dichroism↗
PubMed1997

The Influence of Tetraalkylammonium Counterions on the Drainage and Stability of Thin Films and Foams Stabilized by Dilute Aqueous Solutions of Sodium Dodecyl Sulfate

It was demonstrated that the presence of tetraalkylammonium counterions in aqueous foams and thin film lamella stabilized by sodium dodecyl sulfate (at concentrations below the CMC) can act either to promote or to prevent foam stability. The increase in stability was explained by the congenial incorporation of the smaller and more hydrophilic counterions (tetraethylammonium cations and, to some extent, tetrabutylammonium cations) into the interfacial anionic layers. It was suggested that this increased the interfacial cohesive interaction and resiliency within the thin film lamella. This causes regular thin film drainage rates (within reasonable agreement with Reynold's theory) and was shown to promote black films at low surfactant concentrations in model horizontal thin film lamella. Also, high bulk foam lifetimes were observed, as determined by handshaking. The decrease in stability was associated with rapid drainage rates and was explained by the penetration of larger more hydrophobic tetrapentylammonium cations in the layers. This could disrupt and weaken the molecular interactions (less cohesion). This result is quite different from previously reported studies describing the action of tetraalkylammonium counterions on sodium dodecyl sulfate (at concentrations above the CMC). At these higher surfactant concentrations, a defoaming action was generally observed and could be explained in terms of a reduction in the dynamic stability (Gibbs surface elasticity).

Journal Article↗
PubMed1996

Stability and stabilization of Fusobacterium necrophorum hemolysin.

The stability and stabilization of the hemolytic activity of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme were monitored over a period of four weeks using culture supernatants. The hemolytic activity was completely lost after one week at room temperature and 37 degrees C. After a two-week storage at 4 degrees C and -80 degrees C only trace activity was detected with -80 degrees C being the better of the two conditions. The addition of cysteine monohydrochloride, bovine serum albumin or Tween 80 as stabilizers, however, led to the detection of a considerable amount of the hemolytic activity in the sample stored at 4 degrees C and - 80 degrees C throughout the period investigated. The hemolytic activity appeared to be more stable in the presence of Tween 80 at -80 degrees C. Cysteine monohydrochloride was found to crystallize at - 80 degrees C and was therefore ineffective as a stabilizer at this temperature. Hemoglobin was also ineffective as a stabilizer.

Albumins↗
PubMed2005

Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by beta-lactoglobulin-iota-carrageenan membranes.

An oil-in-water emulsion (5 wt% corn oil, 0.5 wt% beta-lactoglobulin (beta-Lg), 0.1 wt% iota-carrageenan, 5 mM phosphate buffer, pH 6.0) containing anionic droplets stabilized by interfacial membranes comprising of beta-lactoglobulin and iota-carrageenan was produced using a two-stage process. A primary emulsion containing anionic beta-Lg coated droplets was prepared by homogenizing oil and emulsifier solution together using a high-pressure valve homogenizer. A secondary emulsion containing beta-Lg-iota-carrageenan coated droplets was formed by mixing the primary emulsion with an aqueous iota-carrageenan solution. The stability of primary and secondary emulsions to sodium chloride (0-500 mM), calcium chloride (0-12 mM), and thermal processing (30-90 degrees C) were analyzed using zeta-potential, particle size and creaming stability measurements. The secondary emulsion had better stability to droplet aggregation than the primary emulsion at NaCl </=500 mM, CaCl(2) </= 2 mM, and holding temperatures </= 60 degrees C for 20 min. The interfacial engineering technology used in the study could therefore lead to the creation of food emulsions with improved stability to environmental stresses.

Calcium Chloride↗
PubMed2004

Protein stability in nanocages: a novel approach for influencing protein stability by molecular confinement.

Confinement of a protein in a small inert space and microviscosity are known to increase its thermodynamic stability in a way similar to the mechanisms that stabilize protein fold in the cell. Here, to examine the influence of confinement on protein stability we choose four test cases of single domain proteins characterized by a wide range of melting temperatures, from approximately 73 degrees C of titin I27 to approximately 36 degrees C of yeast frataxin. All proteins are stabilized when confined in the gel, the most dramatic stabilization being that of yeast frataxin, whose melting temperature increased by almost 5 degrees C in the gel. In addition to being simple to use, this approach allows us to change the viscosity of the solvent without changing its composition or altering the structure of the proteins. The dimensions of the pores of the gels fall in the nanometer range, hence they are similar to those of the chaperone cavity. This method could therefore be used as a novel and powerful approach for protein folding studies.

Acrylic Resins↗
PubMed2004

Structural stability of oligomeric chaperonin 10: the role of two beta-strands at the N and C termini in structural stabilization.

Chaperonin 10 (cpn10) is a well-conserved subgroup of the molecular chaperone family. GroES, the cpn10 from Escherichia coli, is composed of seven 10kDa subunits, which form a dome-like oligomeric ring structure. From our previous studies, it was found that GroES unfolded completely through a three-state unfolding mechanism involving a partly folded monomer and that this reaction was reversible. In order to study whether these unfolding-refolding characteristics were conserved in other cpn10 proteins, we have examined the structural stabilities of cpn10s from rat mitochondria (RatES) and from hyperthermophilic eubacteria Thermotoga maritima (TmaES), and compared the values to those of GroES. From size-exclusion chromatography experiments in the presence of various concentrations of Gdn-HCl at 25 degrees C, both cpn10s showed unfolding-refolding characteristics similar to those of GroES, i.e. two-stage unfolding reactions that include formation of a partially folded monomer. Although the partially folded monomer of TmaES was considerably more stable compared to GroES and RatES, it was found that the overall stabilities of all three cpn10s were achieved significantly by inter-subunit interactions. We studied this contribution of inter-subunit interactions to overall stability in the GroES heptamer by introducing a mutation that perturbed subunit association, specifically the interaction between the two anti-parallel beta-strands at the N and C termini of this protein. From analyses of the mutants' stabilities, it was revealed that the anti-parallel beta-strands at the subunit interface are crucial for subunit association and stabilization of the heptameric GroES protein.

Amino Acid Sequence↗
PubMed2000

Stability and stabilization of globular proteins in solution.

Proteins are multifunctional: their amino acid sequences simultaneously determine folding, function and turnover. Correspondingly, evolution selected for compromises between rigidity (stability) and flexibility (folding/function/degradation), to the result that generally the free energy of stabilization of globular proteins in solution is the equivalent to only a few weak intermolecular interactions. Additional increments may come from extrinsic factors such as ligands or specific compatible solutes. Apart from the enthalpic effects, entropy may play a role by reducing the flexibility (cystine bridges, increased proline content), or by water release from residues buried upon folding and association. Additional quaternary interactions and closer packing are typical characteristics of proteins from thermophiles. In halophiles, protein stability and function are maintained by increased ion binding and glutamic acid content, both allowing the protein inventory to compete for water at high salt. Acidophiles and alkalophiles show neutral intracellular pH; proteins facing the outside extremes of pH possess anomalously high contents in ionizable amino acids. Global comparisons of the amino acid compositions and sequences of proteins from mesophiles and extremophiles did not result in general rules of protein stabilization, even after including complete genome sequences into the search. Obviously, proteins are individuals that optimize internal packing and external solvent interactions by very different mechanisms, each protein in its own way. Strategies deduced from specific ultrastable proteins allow stabilizing point mutations to be predicted.

Hydrogen Bonding↗
PubMed1982

Heat stability of milk: influence of modification of lysine and arginine on the heat stability-pH profile.

Several dicarbonyl compounds (glyoxal, substituted glyoxals, diacetyl and 1, 2-cyclohexanedione) had a marked stabilizing effect on the heat stability of milk, especially in the presence of urea. These reagents are believed to modify arginine more or less specifically suggesting an important role for arginine residues in heat stability. In contrast, modification of lysine residues with dansyl chloride, acetic anhydride or cyanoborohydride had little effect on maximum heat stability although it did alter the HCT-pH profile. Since diacetyl is a natural constituent of fermented milks and cheese, it may be acceptable as an additive to increase the heat stability of milk.

Acetic Anhydrides↗
PubMed2005

Influence of protein concentration and order of addition on thermal stability of beta-lactoglobulin stabilized n-hexadecane oil-in-water emulsions at neutral pH.

The influence of protein concentration and order of addition relative to homogenization (before or after) on the extent of droplet flocculation in heat-treated oil-in-water emulsions stabilized by a globular protein were examined using laser diffraction. n-Hexadecane (10 wt%) oil-in-water emulsions (pH 7, 150 mM NaCl) stabilized by beta-lactoglobulin (beta-Lg) were prepared by three methods: (1) 4 mg/mL beta-Lg added before homogenization; (2) 4 mg/mL beta-Lg added before homogenization and 6 mg/mL beta-Lg added after homogenization; (3) 10 mg/mL beta-Lg added before homogenization. The emulsions were then subjected to various isothermal heat treatments (30-95 degrees C for 20 min), with the 150 mM NaCl being added either before or after heating. Emulsion 1 contained little nonadsorbed protein and exhibited extensive droplet aggregation at all temperatures, which was attributed to the fact that the droplets had a high surface hydrophobicity, e.g., due to exposed oil or extensive protein surface denaturation. Emulsions 2 and 3 contained a significant fraction of nonadsorbed beta-Lg. When the NaCl was added before heating, these emulsions were relatively stable to droplet flocculation below a critical holding temperature (75 and 60 degrees C, respectively) but showed extensive flocculation above this temperature. The stability at low temperatures was attributed to the droplets having a relatively low surface hydrophobicity, e.g., due to complete saturation of the droplet surface with protein or due to more limited surface denaturation. The instability at high temperatures was attributed to thermal denaturation of the adsorbed and nonadsorbed proteins leading to increased hydrophobic interactions between droplets. When the salt was added to Emulsions 2 and 3 after heating, little droplet flocculation was observed at high temperatures, which was attributed to the dominance of intra-membrane over inter-membrane protein-protein interactions. Our data suggests that protein concentration and order of addition have a strong influence on the flocculation stability of protein-stabilized emulsions, which has important implications for the formulation and production of many emulsion-based products.

Alkanes↗