alpha-chymotrypsin and tetrahydrofuran

Carnosine (beta-Ala-L-His) has high antioxidant activity, and it is widely used in biology, chemical engineering, medicine and other fields. Its analogue syntheised in non-aqueous solvent and catalyzed by enzymes is high-effective but low-price, so it has great prospect. Here, we synthesized a carnosine analogue imidazole 4(5)-alanylamide-5(4)-carboxylic acid with imidazole-4,5-dicarboxylic acid and L-Alanine as substrates, alpha-chymotrypsin as catalyst in tetrahydrofuran (THF) solvent. Based on the orthogonal experiments, the optimized synthetic conditions are 4,5-dicarboxylic acid: L-alanine = 1:3 (m/m), alpha-chymotrypsin: substrates (4,5-dicarboxyl acid and L-alanine) = 1:200 (m/m), pH 8 phosphate buffer:THF = 1.6:10 (V/V), reaction temperature 35 degrees C, time 1.5 h. We separated the product with silica gel G60 thin-layer chromatography (TLC), and a new spot appeared at Rf (ratio to front) = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram (HPLC) and 13C NMR (13C nuclear magnetic resonance). The UV spectra shows a new absorption peak at 310 nm, and the peak in 253 nm is largely strengthened; HPLC reserve times are all 4.5 min at 253 nm, 310 nm, 330 nm; 13C NMR shows 8 carbons. Combing with the catalytic mechanism of alpha-chymotrypsin, structure of the analogue is confirmed, i.e., imidazole 4(5)-alanylamide-5(4)-carboxylic acid.

Topics: Carnosine; Catalysis; Chromatography, High Pressure Liquid; Chymotrypsin; Furans; Solvents

Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.

Topics: Acetonitriles; Adenosine Triphosphate; Alcohol Dehydrogenase; Animals; Buffers; Chromatography, High Pressure Liquid; Chymotrypsin; Circular Dichroism; Dimethyl Sulfoxide; Dimethylformamide; Ethanol; Ethylene Glycols; Fireflies; Formamides; Furans; Hydrophobic and Hydrophilic Interactions; Kinetics; Luciferases, Firefly; Pyrrolidinones; Solvents; Temperature; tert-Butyl Alcohol; Water

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.

Topics: Acetone; Alcohol Dehydrogenase; Catalysis; Chymotrypsin; Dose-Response Relationship, Drug; Enzyme Activation; Freeze Drying; Furans; Hexanes; Horseradish Peroxidase; Protein Denaturation; Protein Folding; Solvents; Substrate Specificity; Subtilisins; Urea