alpha-chymotrypsin and acetonitrile

LC-HRMS-based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent-related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid-phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP-2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (H

Topics: Acetonitriles; Chromatography, High Pressure Liquid; Chymotrypsin; Ethanol; Humans; Hydrophobic and Hydrophilic Interactions; Peptides; Plasma; Proteolysis; Solid Phase Extraction; Tandem Mass Spectrometry; Trypsin

The aim of our study is to monitor the preferential hydration/solvation of the protein macromolecules at low and high water content in water-organic mixtures. Our approach is based on the analysis of the absolute values of the water/organic solvent sorption. We applied this approach to estimate the protein stabilization/destabilization due to the preferential interactions of α-chymotrypsin with water-acetonitrile mixtures. At high water content, α-chymotrypsin is preferentially hydrated. At the intermediate water content, the preferential interaction changed from preferential hydration to preferential binding of acetonitrile. From infrared spectra, changes in the structure of α-chymotrypsin were determined through an analysis of the structure of the amide I band. Acetonitrile augments the intensity of the 1626 cm

Topics: Acetonitriles; Chymotrypsin; Solubility; Spectroscopy, Fourier Transform Infrared; Thermodynamics; Water

The use of enzymes in nonaqueous solvent has been one of the most exciting facets of enzymology in recent times; however, the mechanism of how organic solvent and essential water influence on structure and function of enzyme has been not satisfactorily explained in experiments, which limit its further application. Herein, we used molecular dynamics (MD) simulation to study γ-chymotrypsin in two types of media (viz., acetonitrile media with inclusion of 151 crystal water molecules and aqueous solution). On the basis of the MD result, the truncated active site modes containing two specific solvent molecules are furthered studied at the B3LYP/6-31+G(d,p) level of theory within the framework of PCM model. The results show that the acetontrile solvent gives rise to an extent deviation of enzyme structure from the native one, a drop in the flexibility and the total SASA of enzyme. The QM study further reveals that the structure variation of the active pocket caused by acetonitrile would lead to a weakened strength in the catalytic H-bond network, a drop in the pK(a) value of His57, and an increase in the proton transfer barriers from the Ser195 to the His57 residue, which may contribute to the drop in the enzymatic activity in acetontrile media. In addition, the crystal waters play an importance role in retaining the catalytic H-bond network and weakening the acetonitrile-induced variations above, which may be associated with the fact that the enzyme could retain catalytic activity in microhydration acetonitrile media.

Topics: Acetonitriles; Catalytic Domain; Chymotrypsin; Hydrogen Bonding; Molecular Dynamics Simulation; Quantum Theory; Solvents; Thermodynamics; Water

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Topics: 2-Propanol; Acetone; Acetonitriles; Acetylcholinesterase; Animals; Chickens; Chitinases; Chymotrypsin; Enzyme Assays; Ethanol; Humans; Methanol; Muramidase; Neutrophils; Pancreas; Pancreatic Elastase; Solvents; Spectrometry, Mass, Electrospray Ionization; Swine; Time Factors

This work focused on the conformational changes of α-chymotrypsin from bovine pancreas for various concentrations as well as the effects of chemical denaturation and organic solvents using both of viscosity and dynamic light scattering techniques. A wide range of concentrations varying between 0.1 and 30 mg/ml is tested to determine the critical concentration c** that separates the extremely dilute regime to the dilute regime and the overlapping concentration c* that separates the dilute regime to the semi-dilute regime. The knowledge of α-chymotrypsin folding process is followed for a range of chemical denaturant concentrations varying from 1 to 6 M. We showed that the α-chymotrypsin folds through a cooperative two-state transition without detectable kinetic intermediates and the formation of 50% unfolded happens for 5.5 M of urea or guanidinium chloride (GdmCl) concentrations. The action modes of the acetonitrile on the conformational changes of α-chymotrypsin are also achieved for concentrations varying between 10 and 50%.

Topics: Acetonitriles; Animals; Cattle; Chemistry Techniques, Analytical; Chymotrypsin; Guanidine; Hydrodynamics; Light; Protein Conformation; Protein Denaturation; Scattering, Radiation; Solvents; Temperature; Urea; Viscosity

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 alpha-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 degrees C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.

Topics: Acetonitriles; Chymotrypsin; Dimethylformamide; Enzymes, Immobilized; Ethanol; Kinetics; Polymers; 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

The effects of different concentrations (20-95%) of organic solvents (ethanol, 1,4-dioxane and acetonitrile) were studied on alpha-chymotrypsin and trypsin from bovine pancreas. The changes in secondary structure were followed by CD measurements, and the apparent Michaelis constants (KMapp) and the stabilities of the enzymes were determined. Significant alterations in the CD spectra were found for both enzymes at the different organic solvent concentrations. The apparent KM values of trypsin and alpha-chymotrypsin decreased as the low solvent concentrations were elevated, but then increased in the presence of higher organic solvent concentrations. The stabilities of the enzymes changed on increase of the organic solvent concentration; trypsin exhibited a higher stability than that of alpha-chymotrypsin in all organic solvents. These results show that at an organic solvent content of 95% the manifestation of an enzyme activity similar to that measured in water can be attributed to the similar compositions of the secondary structural elements.

Topics: Acetonitriles; Animals; Cattle; Chymotrypsin; Circular Dichroism; Dioxanes; Dose-Response Relationship, Drug; Enzyme Stability; Ethanol; Hydrogen-Ion Concentration; Organic Chemicals; Protein Structure, Secondary; Solvents; Structure-Activity Relationship; Trypsin

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.

Topics: Acetonitriles; Acylation; Animals; Biochemistry; Cattle; Chymotrypsin; Diatomaceous Earth; Diffusion; Dipeptides; Enzyme Activation; Enzymes, Immobilized; Kinetics; Structure-Activity Relationship; Substrate Specificity

Topics: Acetonitriles; Buffers; Chymotrypsin; Enzymes, Immobilized; Kinetics; Substrate Specificity; Water

The characterization of the S' subsite specificity of native and ethylated alpha-chymotrypsin has been studied via acyl transfer reaction in acetonitrile containing 10 vol% of water. Using Ac-Tyr-OEt as acyl donor, we investigated the partitioning of acyl-chymotrypsins between water and amino acid and peptide-derived nucleophiles. For the investigation of S'2 subsite specificity, a series of 19 dipeptides of the general structure Ala-Xaa (Xaa represents all natural amino acids except cysteine) were used. From the values of the apparent partition constants rho app, the order of preference for the P'2 position is estimated to be: positively charged > hydrophilic > or = hydrophobic > aromatic > Pro > negatively charged side chain. Concerning the S'1 specificity, the same preference is deduced based on the study with the series of amino acid amides and Xaa-Ala dipeptides. In contrast to the nucleophilic specificity of alpha-chymotrypsin in aqueous solutions, free dipeptides and hydrophilic amino acid derivatives as nucleophiles exhibit much higher reactivities for acyl transfer in acetonitrile. We have not observed a significant difference in nucleophilic specificity between native and ethylated chymotrypsin.

Topics: Acetonitriles; Acylation; Amino Acid Sequence; Animals; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin; Molecular Sequence Data; Oxidation-Reduction; Peptides; Solvents; Substrate Specificity; Water

The influence of eight different N-terminal protecting groups (For, Ac, Boc, Fmoc, Mal, Pheac, Aloc and Z) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH2 in organic media was studied. Groups such as Ac, For, Boc, Z, Mal, Pheac and Alloc always rendered good peptide yields (92% to 99%) either in acetonitrile or in ethyl acetate. Good correlations were found between molecular and physico-chemical characteristics of the N-alpha moiety such as the hydrophobicity (log P), ovality and dipole moment and the global reaction rate parameter k'. High k' values were obtained with the less hydrophobic groups, Ac, For and Mal, that have ovality values close to one and the highest dipole moments. Furthermore, it was found that the relative rate of hydrolysis and aminolysis of the acyl-enzyme intermediate expressed as the partition parameter p is affected by the N-alpha moiety of the acyl donor. Correlations between this parameter and the dipole moment of the protecting group were observed.

Topics: Acetates; Acetonitriles; Amides; Chymotrypsin; Enzymes, Immobilized; Kinetics; Peptide Biosynthesis; Peptides

11 amino acid derivatives were tested as alpha-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. alpha-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determined. All the amino acid derivatives tested were esterified, and the highest values of kcat/KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the alpha-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interaction of this part of the molecule with the enzyme to a large extent.

Topics: Acetates; Acetonitriles; Amino Acids; Animals; Cattle; Chymotrypsin; Esters; In Vitro Techniques; Kinetics; Methanol; Solvents; Structure-Activity Relationship; Substrate Specificity; Water

Peptide synthesis with chymotrypsin in organic solvents was investigated and the apparent partition constants have been measured. We find that the Papp values of the most amino acids and peptide derivatives are drastically changed and the stereo- and regiospecificity in acetonitrile/water mixture is reduced.

Topics: Acetonitriles; Amino Acids; Animals; Catalysis; Cattle; Chymotrypsin; Kinetics; Peptides; Substrate Specificity; Water

Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.

Topics: Acetonitriles; Adsorption; Catalysis; Chymotrypsin; Dipeptides; Drug Stability; Hydrolysis; Indicators and Reagents; Kinetics; Peptide Hydrolases; Peptides; Solubility; Solutions; Thermolysin; Water

alpha-Chymotrypsin was adsorbed on solid support materials and the catalytic activity of the preparations in organic solvents was studied. The activity was highly dependent on the nature of the support material and on the amount of water present in the reaction mixture. There appears to be competition for the water in the system between the enzyme, the support material and the solvent. The support materials were characterized by measuring their ability to absorb water from water-saturated diisopropyl ether. For the quotient: (amount of water on the support)/(amount of water in the solvent) in the model system the term aquaphilicity was proposed. The activity of adsorbed chymotrypsin in diisopropyl ether decreased with increasing aquaphilicity of the support material. The same trend was observed when the activity of horse liver alcohol dehydrogenase adsorbed on different supports was measured in diisopropyl ether.

Topics: Acetonitriles; Adsorption; Alcohol Dehydrogenase; Animals; Catalysis; Cattle; Chromatography, Gas; Chymotrypsin; Enzymes; Ethers; Horses; Liver; Pancreas; Solubility; Solvents; Water