alpha-chymotrypsin and Autolysis

Trypsin is a protease, which is commonly used for the digestion of protein samples in proteomic experiments. The process of trypsin autolysis is known to produce autolytic peptides as well as active enzyme forms with one or more intra-chain splits. In consequence, their variable presence can influence the digestion of a protein substrate in the reaction mixture. Besides two major and well-studied forms named β-trypsin and α-trypsin, there are also other active trypsin forms known such as γ-trypsin and pseudotrypsin (ψ-trypsin). In this work, the cleavage specificity of ψ-trypsin was evaluated using in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the resulting peptides. The numbers of produced and matching peptides were similar to those obtained using α-/β-trypsin. The same experience was obtained with a real complex protein sample from rat urine. In previous reports, ψ-trypsin was supposed to generate non-specific cleavages, which has now been reevaluated. Purified ψ-trypsin cleaved all analyzed proteins preferentially on the C-terminal side of Lys and Arg residues in accordance with the canonical tryptic cleavage. However, a minor nonspecific cleavage performance was also registered (particularly after Tyr and Phe), which was considerably higher than in the case of trypsin itself.

Topics: Animals; Autolysis; Chymotrypsin; Proteins; Proteinuria; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin

The molecular mechanism of the autolysis of rat alpha-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site-directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114-->Ile and Tyr146-->His/Asn147-->Ser, that had the same enzymatic activity and molecular stability as the wild-type enzyme, displayed altered routes of autolytic degradation. The Phe114-->Ile mutant also exhibited a significantly slower autolytic inactivation (its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146-Asn147 --> Phe114 --> other sites; (b) the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other; and (c) the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity.

Topics: Amino Acid Sequence; Animals; Autolysis; Binding Sites; Chymotrypsin; Enzyme Stability; Half-Life; In Vitro Techniques; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Tertiary; Rats; Recombinant Proteins; Sequence Homology, Amino Acid

Amino acid sequencing of a subtilisin-type bacterial protease and a bio-engineered variant was carried out by investigating various enzymatic digests using HPLC-frit fast atom bombardment MS methods. The fast atom bombardment mass spectral data allowed rapid identification of the enzymatically generated peptides and differentiation between both proteins. The feasibility of determining the positions and nature of mutations in the amino acid sequence depends mainly on the size of the peptides containing the modifications.

Topics: Amino Acid Sequence; Autolysis; Bacteria; Chromatography, High Pressure Liquid; Chymotrypsin; Cyanogen Bromide; Hydrolysis; Molecular Sequence Data; Pepsin A; Peptide Fragments; Recombinant Proteins; Spectrometry, Mass, Fast Atom Bombardment; Subtilisins; Trypsin

Topics: Amino Acids; Autolysis; Bacteriolysis; Calcium; Cations, Divalent; Cell Division; Cell Wall; Chymotrypsin; Edetic Acid; Freezing; Hydrogen-Ion Concentration; Klebsiella pneumoniae; Lipoproteins; Magnesium; Microscopy, Electron; Molecular Conformation; Mucoproteins; Muramidase; Mutation; Phosphorus; Pronase; Sodium Dodecyl Sulfate; Trypsin

Topics: Animals; Autolysis; Chickens; Chromatography, Paper; Chymotrypsin; Copper; Dithiothreitol; Female; Hydrolysis; Muramidase; Ovum; Oxidation-Reduction; Pepsin A; Protein Conformation; Sulfhydryl Compounds; Urea

Topics: Animals; Autolysis; Bromelains; Cartilage; Cattle; Chloroquine; Chymotrypsin; Colorimetry; Densitometry; Glycosaminoglycans; Hyaluronoglucosaminidase; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Microbial Collagenase; Papain; Pepsin A; Peptide Hydrolases; Prednisolone; Pyridinium Compounds; Sternum; Stimulation, Chemical; Testis; Trypsin

Topics: Amino Acid Sequence; Amino Acids; Animals; Autolysis; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Chymotrypsin; Enzyme Activation; Molecular Weight; Pancreas; Swine

Topics: Animals; Antibody Specificity; Antigens; Antigens, Neoplasm; Autolysis; Chemistry Techniques, Analytical; Chymotrypsin; Edetic Acid; Epitopes; Histocompatibility; Hydrogen-Ion Concentration; Immune Sera; Methods; Mice; Papain; Peptide Hydrolases; Sarcoma; Solubility; Spleen; Thermolysin; Transplantation Immunology; Trypsin

Topics: Animals; Autolysis; Bacteriolysis; Carbon Isotopes; Chymotrypsin; Escherichia coli; Hydrogen-Ion Concentration; Pepsin A; Rumen; Sheep; Tissue Extracts; Trypsin

Topics: Amino Acids; Animals; Autolysis; Calcium; Carboxypeptidases; Cattle; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Echinodermata; Electrophoresis, Disc; Endopeptidases; Enzyme Activation; Enzyme Precursors; Hydrogen-Ion Concentration; Molecular Weight; Trypsin

Topics: Acetylcholinesterase; Animals; Autolysis; Benzoates; Centrifugation, Density Gradient; Cesium; Chemical Phenomena; Chemistry; Chymotrypsin; Eels; Electric Organ; Freezing; Hydrogen-Ion Concentration; Kinetics; Membranes; Mercury; Osmolar Concentration; Pepsin A; Sharks; Sucrose; Toluene; Trypsin

Evans blue has been demonstrated to promote the autolysis of alpha-chymotrypsin at low dye-to-protein ratios in alkaline solution. This effect has been attributed to the stabilization of less tightly folded conformers of the protein by the dye. The effect is specific. Of 20 other strongly acidic dyes tested, only trypan red showed activity comparable to that of Evans blue. A general discussion of the influence of ligand binding on the stability of proteins is presented.

Topics: Autolysis; Chromatography, Gel; Chymotrypsin; Coloring Agents; Hydrogen-Ion Concentration; Models, Chemical; Protein Binding; Spectrophotometry

Topics: Amines; Animals; Autolysis; Chromatography, Thin Layer; Chymotrypsin; Culture Techniques; Guinea Pigs; Humans; Peptide Hydrolases; Protein Hydrolysates; Skin

Topics: Autolysis; Chromatography, Gel; Chymotrypsin; Temperature

Topics: Autolysis; Blood; Chymotrypsin; Dipeptidases; Hematologic Tests; Kidney; Rats; Research; Serum

Topics: Autolysis; Blood; Carboxypeptidases; Chymotrypsin; Dipeptidases; Liver; Rats; Research; Serine Endopeptidases; Serum