Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris

Chondroitinase ABC I (cABC I) from Proteus vulgaris cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The clinical utilization of this enzyme is mainly limited by its thermal instability. This study has been undertaken to determine the effects of glycerol, sorbitol and trehalose on cABC I activity and thermal stability. The results indicated that the enzyme catalytic activity and intrinsic fluorescence intensity increased in the presence of these cosolvents whereas no considerable conformational changes observed in far-UV CD spectra. Thermal CD experiment revealed an increase in T(m) of cABC I in the presence of cosolvents which was significant for trehalose. Our results support the idea that cABC I has stabilized in the presence of glycerol, sorbitol and trehalose. Therefore, the use of these cosolvents seems to be promising for improvement in shelf-life and clinical applications of this drug enzyme.     

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (k_inact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of k_inact(0.7×10⁻⁴s⁻¹) in comparison with their absence (2.5×10⁻⁴s⁻¹). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.     

Stabilization of firefly luciferase against thermal stress by osmolytes

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.     

Improving the stability of uricase from Aspergillus flavus by osmolytes: Use of response surface methodology for optimization of the enzyme stability

Urate oxidase (UOX) is used for the treatment of hyperuricemia and gout. The low stability of protein drugs such as UOX is a significant drawback in their liquid solutions. In this study, UOX from Aspergillus flavus was overexpressed and purified. Then, the effect of osmolytes (glycerol, sorbitol, and sucrose) on UOX thermal stability was studied. The enzyme stability was optimized by the aid of osmolytes using response surface methodology (RSM). The thermal stability results and Pareto analysis showed that sucrose has a stronger stabilization effect on UOX relative to the other osmolytes and increases the enzyme stability up to 2.4-fold, solely. RSM results indicated that the stability of UOX increased up to 4.9-fold in the presence of 12 % of glycerol and sucrose. The combinatorial effect results indicated that the osmolytes affect each other stabilization effects on UOX, probably through influencing the hydrogen bonds and mixing the osmolytes at different concentrations does not always lead to increased stability of UOX. The results revealed that the effect of sorbitol on UOX stability was negligible in the presence of glycerol and sucrose. Finally, this study results showed that RSM can be used to select a proper set of osmolytes for enzyme stabilization.     

Local dielectric properties around polar region of lipid bilayer membranes

Summary Local dielectric constant was evaluated from the Stokes shifts of fluorescence spectra ofl--dansylphosphatidylethanolamine (DPE) incorporated into liposomes made of synthetic phosphatidylcholine (dipalmitoyl or distearoyl) or bovine brain phosphatidylserine. The evaluation was established as follows. First, the Stokes shift of DPE was assured to follow Mataga-Lippert's equation and was a function of the dielectric constant and the refractive index in some standard organic solvents. Second, the change of the refractive index did not contribute much to the change in the Stokes shift. Third, the time resolved fluorescence depolarization of DPE in liposomes showed that the cone wobbling diffusion was rapid relative to the fluorescence lifetime and therefore that the dielectric relaxation did not affect the evaluation of the constant in the polar region of membranes. We then investigated the characteristics of the local dielectric constant in the polar region of the lipid bilayer and found that the dielectric constant varies between 4 and 34 depending upon calcium binding and also gel/liquid-crystal phase transition. Such large changes of the local dielectric constant were further correlated with the dynamic structure of lipid bilayer membranes measured by conventional fluorescence depolarization techniques. The large changes of dielectric constant around the polar region suggest that electrostatic interactions at this region can be altered 10-fold by such ionic or thermotropic factors and therefore that local dielectric properties can play crucial roles in membrane functions.     

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.     

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.5 that does not show any cooperative transition upon heating. Does the presence of some of these osmolytes at pH 2.5 help in the retention of structure that is typical of native state? To answer this question, the effect of ethylene glycol (EG), glycerol, xylitol, sorbitol, trehalose and glucose at pH 2.5 on the structure and stability of yeast hexokinase A was investigated using spectroscopy and calorimetry. In presence of the above osmolytes, except EG, yeast hexokinase at pH 2.5 retains native secondary structure and hydrophobic core and unfolds with excessive heat absorption upon thermal denaturation. However, the cooperative structure binds to ANS suggesting that it is an intermediate between MG and the native state. Further, we show that at high concentration of polyols at pH 2.5, except EG, which populates a non-native ensemble, ΔH_cal/ΔH_van approaches unity indicative of two-state unfolding. The results suggest that osmolytes stabilize cooperative protein structure relative to non-cooperative ensemble. These findings have implications toward the structure formation, folding and stability of proteins produced under stress in cellular systems.     

Roles of trehalose and magnesium sulfate on structural and functional stability of firefly luciferase

Firefly luciferase is widely used in many analytical techniques. However, the enzyme is unstable, so that its relative inactivation results in low sensitivity of those techniques. In this study, we have investigated the effects of MgSO₄ and trehalose on the structural stability and function of luciferase from Photinus pyralis using circular dichroism (CD), conventional and stopped-flow fluorescence spectroscopy and bioluminescence assay. The secondary structural content, compactness and its melting temperature are also studied, which showed that the stability of luciferase increased in the presence of additives. Measurements of refolding rate constants under conditions that favor folding, show that MgSO₄ accelerates the folding of enzyme, on the contrary, refolding rate constant decreases in the presence of trehalose which can be attributed to its high viscosity. Finally, combined with remaining activity assay we concluded that magnesium sulfate and trehalose can be used for short- and long-term stabilization, respectively.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

Counteraction of trehalose on urea-induced protein unfolding: Thermodynamic and kinetic studies

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.     

Trehalose and glycerol stabilize and renature yeast inorganic pyrophosphatase inactivated by very high temperatures

A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins, and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. Similar to molecular chaperones, most of these compounds have no substrate specificity, but some specifically stabilize certain proteins. In the present work, the capacity of trehalose and glycerol, two well-known osmolytes, to stabilize and renature inorganic pyrophosphatase is demonstrated. Both trehalose and glycerol significantly protect pyrophosphatase against thermoinactivation achieved by incubating the enzyme at temperatures up to 95 degrees C, and allow the enzyme already inactivated in the presence of these osmolytes to renature upon incubation at low temperatures. To the best of our knowledge, there are no data on the effects of these compounds on renaturation of thermoinactivated proteins. The correlation between the recovery of enzyme activity and structural changes indicated by fluorescence spectroscopy contribute to better understanding of the protein stabilization mechanism.     

Evidence for a novel cryoprotective protein from freeze-tolerant larvae of the goldenrod gall fly Eurosta solidaginis

Third-instar larvae of the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae) from populations in northern North America transition from freeze-susceptible to freeze-tolerant just prior to the onset of winter. While studies have documented the accumulation of carbohydrate cryoprotectants during this transition, protein cryoprotectants common to other freeze-tolerant species have not been reported in the gall fly. Using larvae collected from a population in Madison County, NY, which changes from freeze-susceptible to freeze-tolerant in early October, we assayed for the presence of factors that could preserve the catalytic activity of the cold-labile enzyme, rabbit muscle lactate dehydrogenase. Freezing this enzyme with a heat-stable, hydrophilic fraction derived from homogenates of both freeze-tolerant larvae and those in the process of becoming freeze-tolerant preserved between 70% and 80% of this enzyme's activity. Neither a comparable solution of bovine serum albumin nor the naturally-occurring carbohydrates (glycerol, sorbitol, or trehalose) conferred this level of cryoprotection. The putative cryoprotective protein from gall fly larvae did not bind to a weak anion exchanger, implying that its character may be cationic.     

LysK,the enzyme lysing Staphylococcus aureus cells: Specific kinetic features and approaches towards stabilization

LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspension of autoclaved Staphylococcus aureus cells) has been found to be adequately described by the two-stage Michaelis–Menten kinetic scheme. Ionization of the enzyme and enzyme–substrate complex is important for revealing catalytic activity, which is controlled by two ionogenic groups with pK 6.0 and 9.6. Ionization energy of the group with pK 6.0 is of 30 kJ/mol, thus, pointing out on His residue; pK 9.6 might be attributed to metal ion or metal-bound water molecule. At temperatures lower than 40 °C, LysK stability depends on its concentration, pH and presence of low molecular weight additives. Results of electrophoresis under native and denaturing conditions as well as sedimentation analysis strongly suggest that aggregation is behind LysK inactivation. Decrease in the enzyme concentration, as well as addition of low molecular mass polyols (glycerol, sorbitol, sucrose, trehalose) and Ca²⁺ cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40–42 °C) was accompanied by changes in LysK secondary structure as confirmed by CD spectroscopy studies. According to computer modeling data, Cys and His residues and metal cation might play a crucial role for LysK catalytic activity. Our data on the enzyme activity in the presence of ethylenediaminetetraacetic acid and different metal cations confirmed the importance of metal cation in LysK catalysis.     

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.     

Sex-specific differences in desiccation resistance and the use of energy metabolites as osmolytes in Drosophila melanogaster flies acclimated to dehydration stress

In the Indian subcontinent, there are significant between-population variations in desiccation resistance in Drosophila melanogaster, but the physiological basis of adult acclimation responses to ecologically relevant humidity conditions is largely unknown. We tested the hypothesis that increased desiccation resistance in acclimated flies is associated with changes in cuticular permeability and/or content of energy metabolites that act as osmolytes. Under an ecologically relevant humidity regime (~50 % relative humidity), both sexes showed desiccation acclimation which persisted for 2–3 days. However, only females responded to acclimation at ~5 % relative humidity (RH). Acclimated flies exhibited no changes in the rate of water loss, which is consistent with a lack of plastic changes in cuticular traits (body melanization, epicuticular lipid). Therefore, changes in cuticular permeability are unlikely in drought-acclimated adult flies of D. melanogaster. In acclimated flies, we found sex differences in changes in the content of osmolytes (trehalose in females versus glycogen in males). These sex-specific changes in osmolytes are rapid and reversible and match to corresponding changes in the increased desiccation resistance levels of acclimated flies. Further, the increased content of trehalose in females and glycogen in males support the bound-water hypothesis for water retention in acclimated flies. Thus, drought acclimation in adult flies of D. melanogaster involves inducible changes in osmolytes (trehalose and glycogen), while there is little support for changes in cuticular permeability.     

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T ^*, temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.     

Counteraction of Trehalose on N,N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation

Candida rugosa lipase (CRL) has been widely used as a biocatalyst for non-aqueous synthesis in biotechnological applications, which, however, often suffers significant loss of activity in organic solvent. Experimental results show that trehalose could actively counteract the organic-solvent-induced protein denaturation, while the molecular mechanisms still don’t unclear. Herein, CRL was used as a model enzyme to explore the effects of trehalose on the retention of enzymatic activity upon incubation in N,N-dimethylformamide (DMF). Results showed that both catalytic activity and conformation changes of CRL influenced by DMF solvent were inhibited by trehalose in a dose-dependent fashion. The simulations further indicated that the CRL protein unfolded in binary DMF solution, but retained the native state in the ternary DMF/trehalose system. Trehalose as the second osmolyte added into binary DMF solution decreased DMF-CRL hydrogen bonds efficiently, whereas increased the intermolecular hydrogen bondings between DMF and trehalose. Thus, the origin of its denaturing effects of DMF on protein is thought to be due to the preferential exclusion of trehalose as well as the intermolecular hydrogen bondings between trehalose and DMF. These findings suggest that trehalose protect the CRL protein from DMF-induced unfolding via both indirect and direct interactions.