alpha-chymotrypsin and benzoyltyrosine-ethyl-ester

An immobilised enzyme reactor (IMER) in the form of capillary monolith was developed for a micro-liquid chromatography system. The plain monolith was obtained by in situ thermal copolymerisation of glycidyl methacrylate and ethylene dimethacrylate in a fused silica capillary (200 × 0.53 mm ID) by using n-propanol/1,4-butanediol as porogen. The enzyme, α-chymotrypsin (CT), was covalently attached onto the monolith via triazole ring formation by click-chemistry. For this purpose, the monolithic support was treated with sodium azide and reacted with the alkyne carrying enzyme derivative. CT was covalently linked to the monolith by triazole-ring formation. The activity behaviour of monolithic IMER was investigated in a micro-liquid chromatography system by using benzoyl-L-tyrosine ethyl ester (BTEE) as synthetic substrate. The effects of mobile-phase flow rate and substrate feed concentration on the final BTEE conversion were investigated under steady-state conditions. In the case of monolithic IMER, the final substrate conversion increased with increasing feed flow rate and increasing substrate feed concentration. Unusual behaviour was explained by the presence of convective diffusion in the macropores of monolith. The results indicated that the monolithic-capillary IMER proposed for micro-liquid chromatography had significant advantages with respect to particle-based conventional high-performance liquid chromatography-IMERs.

Topics: Butylene Glycols; Chromatography, Liquid; Chymotrypsin; Click Chemistry; Enzymes, Immobilized; Epoxy Compounds; Hydrolysis; Methacrylates; Polymerization; Triazoles; Tyrosine

A convenient and rapid method for the photo-regulation of the proteolytic enzyme alpha-chymotrypsin is described. When alpha-chymotrypsin is coated with photolytic 1-(2-nitrophenyl)ethanol residues this not only markedly reduces the capability of the enzyme to digest both of the small substrates N-benzoyl-L-tyrosine ethyl ester and N-succinyl-L-phenylalanine p-nitroanilide, but also completely inhibits the enzyme's proteolytic activity. The inactivated alpha-chymotrypsin can then be reactivated under physiological conditions, when and where it is required, by exposure to UV-A light. These results further demonstrate that 1-(2-nitrophenyl)ethanol coated proteins can often be used as light sensitive biological switches as a simple alternative to site directed procedures.

Topics: Animals; Cattle; Chymotrypsin; Enzyme Activation; Ethanol; Molecular Structure; Nitrobenzenes; Phenylalanine; Photochemistry; Serum Albumin, Bovine; Tyrosine; Ultraviolet Rays

Chemical glycosylation of bovine alpha-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of alpha-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of alpha-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.

Topics: Animals; Caseins; Cattle; Chromatography, Affinity; Chymotrypsin; Electrophoresis, Capillary; Glucosamine; Glycosylation; Histidine; Hydrogen Bonding; Imino Acids; Mass Spectrometry; Metals; Structure-Activity Relationship; Tyrosine

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.

Topics: Acrylic Resins; Animals; Cattle; Chymotrypsin; Diatomaceous Earth; Dimethylformamide; Dipeptides; Endorphins; Enzyme Stability; Enzymes, Immobilized; Kinetics; Solvents; Tyrosine

Diisopropyl fluorophosphate (DFP), a volatile highly toxic enzyme inhibitor, in buffer (pH 3, pH 5, pH 7, pH 9, pH 11, Hank's, Dulbecco's, PBS, TBE, and HEPES) or water (10 mM), in DMF solution (200 mM), and bulk quantities can be degraded by adding 1M NaOH. The DFP was completely degraded, as determined by enzymatic assay, and the final reaction mixtures were not mutagenic.

Topics: Buffers; Chymotrypsin; Hydrolysis; Isoflurophate; Mutagenicity Tests; Salmonella typhimurium; Sodium Hydroxide; Solvents; Spectrophotometry, Ultraviolet; Tyrosine; Waste Disposal, Fluid

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p'-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intracellular Ca2+ concentration [( Ca2+]i) and pH (pHi) were measured with fura-2 and 2',7'-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca2+]i, which was depressed by BTEE. Egg jelly also caused a transient rise of pHi, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca2+]i. The rise of pHi was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca2+ channels, which take part in the acrosome reaction.

Topics: Acrosome; Animals; Calcium; Calcium Channels; Chymotrypsin; Female; Hydrogen-Ion Concentration; Ionophores; Male; Protease Inhibitors; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa; Tosylphenylalanyl Chloromethyl Ketone; Tyrosine

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.

Topics: Animals; Chymotrypsin; Fluorometry; Microchemistry; NAD; Rats; Spectrophotometry, Ultraviolet; Tyrosine