Compatibility of organic solvents with the Salmonella/microsome test

We have screened 14 solvents for compatibility with the Salmonella mutagenicity test and have found 12 to be satisfactory under the conditions specified. These 12 solvents are: dimethyl sulfoxide, glycerol formal, dimethyl formamide, formamide, acetonitrile, 95% ethanol, acetone, ethylene glycol dimethyl ether, 1-methyl-2-pyrrolidinone, p-dioxane, tetrahydrofurfuryl alcohol and tetrahydrofuran.The influence of the nutrient broth culture medium on the spontaneous mutation rate of the tester strains is also discussed.     

Synthesis of a highly fluorescent N,N‐dimethyl benzylamine–palladium(II) curcuminate complex and its application for determination of trace amounts of water in organic solvents

In this work a new highly fluorescent N,N‐dimethyl benzylamine–palladium(II) yu complex was synthesized by the reaction of [Pd₂{(C,N–C₆H₄CH₂N(CH₃)₂}₂(μ‐OAc)]₂] with curcumin. The structure of the synthesized complex was characterized using Fourier transform infra‐red (FT‐IR) spectroscopy, ¹H nuclear magnetic resonance spectroscopy, and elemental analysis. Fluorescence quantum yield (Φ_F) values of the synthesized complex in dimethyl sulfoxide (DMSO), acetonitrile, ethanol, and methanol were 0.160, 0.104, 0.068, and 0.061, respectively. The fluorescence signal of the complex in the organic solvents was very sensitive to the water content of the organic solvent. The quenching effect of water was used to determine trace amounts of water in the heteroatom‐containing organic solvents (ethanol, methanol, acetonitrile) and redox‐active solvents (DMSO). The linear ranges for determination of water (v/v %) in ethanol, DMSO and acetonitrile were found to be 0.03–14.5, 0.08–13.8, and 0.07–18.8, respectively. Two linear ranges were found for determination of water (v/v %) in methanol (0.1–1.2 and 4.7–25.0). Detection limit (DL) values were calculated to be 0.001, 0.05, 0.004, and 0.01 (v/v %) in ethanol, methanol, acetonitrile, and DMSO, respectively. The proposed method overcomes the problems of the standard Karl Fischer method for determination of water in DMSO. In addition, it gave the best DL value for determination of water in ethanol compared with all published papers to date.     

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.     

Preparation and physicochemical characterization of 5 niclosamide solvates and 1 hemisolvate

The purpose of the study was to characterize the physicochemical, structural, and spectral properties of the 1∶1 niclosamide and methanol, diethyl ether, dimethyl sulfoxide, N,N' dimethylformamide, and tetrahydrofuran solvates and the 2∶1 niclosamide and tetraethylene glycol hemisolvate prepared by recrystallization from these organic solvents. Structural, spectral, and thermal analysis results confirmed the presence of the solvents and differences in the structural properties of these solvates. In addition, differences in the activation energy of desolvation, batch solution calorimetry, and the aqueous solubility at 25°C, 24 hours, showed the stability of the solvates to be in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > methanol solvate > dimethyl sulfoxide solvate > N,N' dimethylformamide solvate. The intrinsic and powder dissolution rates of the solvates were in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > N,N' dimethylformamide solvate > methanol solvate > dimethyl sulfoxide solvate. Although these nonaqueous solvates had higher solubility and dissolution rates than the monohydrous forms, they were unstable in aqueous media and rapidly transformed to one of the monohydrous forms.     

Toxicity assessment of common organic solvents using a biosensor based on algal photosynthetic activity measurement

The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: ${\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}The acute toxicities of common organic solvents (e.g., methanol, ethanol, isopropanol, acetone, acetonitrile, and dimethylformamide) were evaluated using a biosensor based on microalgal photosynthesis measurement. The biosensor was air-tight, with no headspace, preventing volatile organic toxicants from escaping into the environment as well as partitioning from the aqueous phase into the headspace until equilibrium was reached. Both the incubating and exposure times were set at 10 min. It was observed that only 2 h was needed to obtain complete dose-related inhibition of photosynthetic activity. The results showed that all the tested organic solvents inhibited algal photosynthesis with EC₅₀ ranging between 589 and 2,570 mM. The inhibition of these solvents was in the order: isopropanol > acetone > acetonitrile > ethanol > dimethylformamide > methanol. The quantitative structure-activity relationship (QSAR) between toxicity data and partition coefficient of the examined compounds could be modeled as follows: \textlog₁₀ \textEC₅₀   ( m\textM ) = - 0.6428  \textlog  P + 5.76  ( \textR² ? 0.88 ){\text{log}}_{{10}} {\text{EC}}_{{50}} \;{\left( {\mu {\text{M}}} \right)} = - 0.6428\;{\text{log}}\;P + 5.76\;{\left( {{\text{R}}^{2} \approx 0.88} \right)}. This indicates that the photosynthetic activity of the microalga Pseudokirchneriella subcapitata is highly dependent on the hydrophobicity of these commonly used organic solvents.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

Purification,characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258

Ten strains of Bjerkandera adusta from the University of Alberta Microfungus Collection and Herbarium (UAMH) were compared for manganese peroxidase production. The enzyme from B. adusta UAMH 8258 was chosen for further study. After purification the enzyme showed a molecular weight of 43 kDa on 15% SDS-PAGE, 36.6 kDa on matrix-assisted laser desorption ionization-time of flight mass spectrometry, and an isoelectric point of 3.55. The N-terminal amino acid sequence was determined to be VAXPDGVNTATNAAXXALFA, and the amino acid composition showed no tyrosine residues in the enzyme. Manganese peroxidase exhibited both Mn(II)-dependent (optimum pH 5) and Mn(II)-independent activity (optimum pH 3). The purified enzyme was chemically modified with cyanuric chloride-activated methoxypolyethylene glycol to enhance its surface hydrophobicity. The modified and native enzymes showed similar catalytic properties in the oxidation of Mn(II) and other substrates such as 2,6-dimethoxylphenol, veratryl alcohol, guaiacol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate). However, the modified enzyme showed greater resistance to denaturation by hydrogen peroxide and stability to organic solvents such as acetonitrile, N,N-dimethylformamide, tetrahydrofuran, methanol, and ethanol. The PEG-modified enzyme also showed greater stability to higher temperatures and lower pH than the native enzyme. Thus, chemical modification of manganese peroxidase from B. adusta increases its potential usefulness for applied studies. Received: 12 October 2001 / Accepted: 14 November 2001     

Preparation of chitin butyrate by using phosphoryl mixed anhydride system

Acylation of chitin with butyric acid was performed in the presence of trifluoroacetic anhydride/phosphoric acid mediated system. The products were characterized by ¹H NMR and FT-IR spectroscopy and their solubility was tested in different organic solvents. Inclusion of butyric acid moieties into the parent molecule was confirmed from the ¹H NMR and FT-IR spectra. FT-IR analysis revealed that the degree of acid substitution (DS) of the products was in a range of 1.9–2.38, which increased with increasing the amounts of butyric acid added to the reaction system. Degree of N-deacetylation (DD) of the products, as determined by ¹H NMR was between 54.2% and 65.6%. The products with DS >2.0 were soluble in dimethyl sulfoxide, N,N-dimethylformamide, tetrahydrofuran, methanol, acetone, chloroform, and acetic acid.     

N-acetyl- -arginine ethyl ester synthesis catalysed by bovine trypsin in organic media

The bovine trypsin-catalysed synthesis of N-acetyl- -arginine ethyl ester from N-acetyl- -arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl- -arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.     

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

PREPARATION OF GLUTATHIONE IMPRINTED POLYMER

Glutathione imprinted polymer was prepared using 1-vinyl imidazole and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, in dimethyl sulfoxide. The adsorption selectivity of glutathione-imprinted polymer was tested by reduced glutathione, oxidized glutathione, and L-Gly-Leu-Tyr in 30% phosphate buffer (0.01 M, pH 5.0)–70% acetonitrile and binding affinity values were compared. Reusability of molecularly imprinted polymer particles was also investigated. Molecularly imprinted polymer particles were found to be stable and to maintain glutathione adsorption capacity at 95% when washed with methanol–acetic acid (10%) after seven usages. Functional monomer 1-vinyl imidazole and cross linker ethylene glycol dimethacrylate-based glutathione imprinted polymer could be used as solid phase extraction material for recognition of glutathione in biological samples.     

Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

Bimolecular fluorescence quenching reactions of the biologically active coumarin composite 2‐acetyl‐3H‐benzo[f]chromen‐3‐one in different solvents

A study on fluorescence quenching was carried out for the coumarin derivative 2‐acetyl‐3H‐benzo[f]chromen‐3‐one (2AHBC) with aniline at room temperature. Efficient fluorescence quenching was observed and Stern–Volmer (S–V) plots showed upward curves from linearity in all solvents of different polarities. For the solute 2AHBC, ground state complex formation does not hold in our study. The kinetic distance (r) value was found to be greater than the encounter distance (R) and indicated that the quenching reaction was held within the sphere of action. Diffusion‐limited reactions were found to be more prominent in high polarity solvents, namely dimethyl sulfoxide (DMSO), DMF, ACN, methanol, ethanol, propanol and DCM. The relationships between quenching constant (K_SV) and dielectric constants (ε) of the different solvents were studied.     

Characterization of a novel cold-active esterase isolated from swamp sediment metagenome

A functional screen of a metagenomic library from “Upo” swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser₁₀₃–Asp₂₄₈–His₂₆₈ that is typical of esterases. The Ser₁₀₃ residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca²⁺, retaining more than 60% of its original activity after 12 h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, K_m and k_cat, for p-nitrophenyl-acetate were 185.8 μM and 186.6 s^?1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications.

Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol     

Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films

Synopsis The influence of different histochemical conditions on some metachromatic staining reactions has been studied using polyacrylamide films containing pure glycosaminoglycans. The films were incubated in fixatives without staining, and in glycerol, diethylene glycol and other glycols, formamide,N,N-dimethylformamide, dimethyl sulphoxide and ethanol (of several concentrations) after staining and their absorption (metachromatic) spectra recorded. In the case of heparin and heparan sulphate the metachromasy was disturbed when the films were immersed before staining in some fixative solutions containing formaldehyde and acid. After equilibration of stained films in organic solvents, changes in the absorption peaks were found to depend on the type and concentration of solvent, the type of glycosaminoglycan and the type of dye.Films containing glycosaminoglycan plus protein were used to investigate the blocking of the metachromatic reaction as the result of ionic interactions with proteins. The parameters that influence this phenomenon (e.g type of protein, glycosaminoglycan and dye, pH of staining) are discussed and a three-dimensional picture is introduced which can explain some of the results obtained in these experiments.     

Functional stability and structural transitions of Kallikrein: spectroscopic and molecular dynamics studies

Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96 h, showed moderate tolerance in acetonitrile (45% activity after 72 h) and much lower stability in methanol (40% activity after 24 h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 °C as indicated by secondary structure monitoring, it was functionally stable only up to 45 °C, implicating thermolabile active site geometry. In GdnHCl [1.0 M], 75% of the activity of KLKp was retained after incubation for 4 h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.