alpha-chymotrypsin and 4-nitroaniline

A molecular probe with l-phenylalanine

Topics: Aniline Compounds; Chymotrypsin; Fluorescent Dyes; Humans; Molecular Probes; Papain; Trypsin

The entrapment of α-chymotrypsin (α-CT) within 70-140 nm liposomes formed from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) leads to an unexpected and remarkable increase in the thermal stability of the enzyme. This finding is based on the observation that heating aqueous suspensions of α-CT-containing POPC liposomes to 80 °C for 30 minutes resulted in partial enzyme inactivation, whereas the same treatment of aqueous solutions of free α-CT inactivated the enzyme completely. The stabilizing effect of enzyme confinement in the attoliter volumes of the liposomes was found to increase with decreasing numbers of α-CT molecules per liposome. Single-enzyme confinement was particularly effective, as intermolecular interactions between heat-denatured α-CT molecules (causing irreversible inactivation) are not possible.

Topics: Aniline Compounds; Animals; Ascomycota; Cattle; Chymotrypsin; Endopeptidase K; Heating; Oligopeptides; Particle Size; Phosphatidylcholines; Protein Stability; Unilamellar Liposomes

The synthesis of the chromogenic substrates for trypsin-like proteinases catalyzed by alpha-chymotrypsin and subtilisin from B. subtilis strain 72 were carried out in the organic media at a low water content using the enzymes adsorbed on different porous materials. The method proposed allows us to vary the structure of the compounds to be synthesized and is a suitable technique for their scaling-up.

Topics: Adsorption; Amino Acid Sequence; Aniline Compounds; Arginine; Chromogenic Compounds; Chymotrypsin; Endopeptidases; Enzymes, Immobilized; Lysine; Molecular Sequence Data; Oligopeptides; Substrate Specificity; Subtilisins

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.

Topics: Alanine; Amino Acid Sequence; Amino Acids; Aniline Compounds; Aprotinin; Binding Sites; Chromatography, High Pressure Liquid; Chymotrypsin; Coumarins; Fluorometry; Humans; Indicators and Reagents; Kallikreins; Kinetics; Leukocytes; Lysine; Molecular Sequence Data; Pancreatic Elastase; Serine Endopeptidases; Spectrophotometry; Trypsin

A method--enzymoblotting--was developed for localizing various enzymes after electrophoretic separation, transfer to nitrocellulose, and incubation with specific substrates. As an application, the proteinases porcine trypsin (EC 3.4.21.4), bovine chymotrypsin (EC 3.4.21.1), porcine elastase (EC 3.4.22.11), and their zymogen forms from porcine pancreas homogenate were analyzed utilizing specific p-nitroanilide substrates. After agarose gel electrophoresis, transfer of the separated proteinases to a nitrocellulose membrane was performed by capillary diffusion for 30 min. After air-drying of the nitrocellulose membrane, it was incubated in the appropriate substrate solution for 60 min. N-alpha-Benzoyl-DL-arginine-para-nitroanilide HCl was used as a substrate for trypsin, N-benzoyl-L-tyrosine-para-nitroanilide and succinyl-L-phenylalanine-para-nitroanilide for chymotrypsin, and N-succinyl-L-alanyl-L-alanyl-L-alanine-para-nitroanilide for elastase. p-Nitroaniline, the product thus obtained, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye, visible at the site of the proteinases on the nitrocellulose membrane. The results could be preserved at -18 degrees C. Zymogen forms of the pancreas proteinases were detected in a similar manner. They were converted to active proteinases in situ on the nitrocellulose membrane after preincubating the nitrocellulose membrane in the activation enzymes enteropeptidase or trypsin.

Topics: Aniline Compounds; Animals; Cattle; Chymotrypsin; Collodion; Electrophoresis, Agar Gel; Endopeptidases; Methods; Pancreas; Pancreatic Elastase; Swine; Trypsin

A number of peptide-4-nitroanilide substrates containing proline within the peptide chain have been synthesized and subjected to chymotryptic hydrolysis. Values of kcat and Km have been obtained from measurements at pH 7.8 and 25.0 degrees C. Kinetic studies at high enzyme concentrations up to 6.0 X 10(-4) mol X 1(-1) have allowed the evaluation of the conformational specificity of chymotrypsin due to the observation of various kinetic phases during the time-course of the reaction. When proline occupies the P2 position within the peptide chain, it is shown that the enzyme cleaves only the trans isomer of the substrate. The conformational specificity has also been studied for proline in P4 and P5 positions of the substrate. In some cases, an enzyme-catalyzed hydrolysis of the cis isomer was detected. From the amplitude ratios and the rate constants of the kinetic phases, information about the structural dependency of the cis/trans interconversion could be obtained. Charged residues N-terminal to the isomeric bond are of little influence on either cis/trans ratio or the rate of cis to trans interconversion. Extending the peptide chain N-terminal to the isomeric bond by alanine decreases to a low extent the cis content and increases the rate constant of the trans isomer formation.

Topics: Aniline Compounds; Chymotrypsin; Kinetics; Magnetic Resonance Spectroscopy; Mathematics; Proline; Protein Conformation; Structure-Activity Relationship; Temperature