alpha-chymotrypsin and hydrazine

Synthesis of colloidal functional graphene is challenging because graphene is water-insoluble and its relatively inert surface made the functionalization a difficult task. Here we report interdigited bilayer type coating that provide both colloidal stability and functionalization option for graphene. Colloidal graphene oxide is first converted into interdigited bilayer coated graphene oxide and next they are transformed into colloidal graphene by hydrazine reduction. These coated graphenes can be further transformed into colloidal functional graphene using covalent conjugation chemistry. Functional graphene has been synthesized for optical detection of enzyme where a fluorescent dye is covalently linked through a peptide so that the dye fluorescence is quenched by graphene but switches on once enzymes cleave the peptide bond. The interdigited bilayer coating reported here is unique as it provides coating thickness <3 nm, offering optically responsive graphene-fluorophore substrate with high colloidal stability.

Topics: Animals; Cell Line; Chymotrypsin; Colloids; Enzymes; Fluorescent Dyes; Graphite; Hydrazines; Oxidation-Reduction; Oxides; Peptides; Rats; Spectrometry, Fluorescence; Water

Surface morphology changes of hydrazine-RF-plasma-exposed cellophane surfaces were monitored under 40 kHz and 13.56 MHz CW and pulsed discharge environments and the immobilization of alpha-chymotrypsin onto plasma-modified substrates was studied. It has been shown, using SEM and AFM techniques, that significantly different cellophane topographies are generated under different frequency and pulsing parameter conditions. ESCA and ATR-FTIR analyses of plasma-modified surfaces indicated the presence of primary amide and primary amine functionalities. It was found that the relative ratios of crystalline vs amorphous zones of the nascent surface layers can also be controlled by properly selected plasma parameters, including the duty cycles of pulsed plasma environments. Enzyme immobilization reactions with alpha-chymotrypsin were accomplished both from oxygen-plasma-generated carbonyl and hydrazine-plasma-created primary amine functionalities by anchoring the biomolecules either directly to the cellophane surface or by involving spacer molecules. It was found with the cellulose substrates that fairly good enzyme activity was retained without the necessity of intercalated spacer chains. It appears that the ability of the cellulose substrate to swell in the aqueous environment allows sufficient freedom of mobility for the immobilized enzyme to retain a significant part of its activity on the cellulose. However, the activities both of the free enzyme in the presence of cellophane, and that of the immobilized enzyme molecules are significantly diminished in comparison to the activity of the free enzyme, as a result of the incorporation of these molecules into the swollen network. Potential applications of immobilized enzymes from cold-plasma-functionalized surfaces are discussed.

Topics: Argon; Biocompatible Materials; Cellophane; Chymotrypsin; Hydrazines; Hydrogen-Ion Concentration; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Models, Chemical; Models, Theoretical; Spectrometry, Fluorescence; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Time Factors; Ultraviolet Rays

We introduce a new potent inhibitor, N-[2, 2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine alpha-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme-inhibitor dissociation constant (Ki) against alpha-chymotrypsin were 0.0028 s-1 and 0.0045 microM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful alpha-chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, beta-substituent at the beta-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the beta-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.

Topics: Animals; Binding, Competitive; Cattle; Chymotrypsin; Enzyme Reactivators; Humans; Hydrazines; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phenylalanine; Structure-Activity Relationship; Trypsin Inhibitors

Pro-Val pseudo dipeptides incorporating protio and halo enol lactones were tested for inhibitory activity against the serine proteases human leukocyte elastase (HLE), porcine pancreatic elastase, alpha-chymotrypsin, trypsin, thrombin, and urokinase. The protio enol lactones 1a-c were found to be HLE substrates but were poor alternate substrate inhibitors. The bromo enol lactone trans isomer 2a was found to be a very effective inhibitor of HLE and chymotrypsin, as shown by the binding constants (KI), acylation rates (ka), inactivation rates, and partition ratios determined for each enzyme. This inhibitor shows better specificity toward its target enzyme HLE than monosubstituted halo enol lactones; we attribute this to a pseudo dipeptide acyl enzyme whose structure is similar to that adopted by good peptide substrates of HLE. Inactivation of chymotrypsin by the bromo enol lactone 2a is permanent, but inactivation of HLE is partially recoverable upon treatment with the nucleophile hydrazine, indicating that lactone 2a produces two species of inactivated HLE. The more stable of these species could be the result of alkylation of His-57 by the electrophilic bromomethyl ketone revealed in the acyl enzyme, and the less stable, hydrazine-reactivatable species could be the result of alkylation of Asp-102 or the hydrolysis of the bromomethyl ketone group in the initially formed acyl enzyme to form a new, more stable acyl enzyme.

Topics: Amino Acid Sequence; Animals; Binding Sites; Chymotrypsin; Dipeptides; Humans; Hydrazines; Kinetics; Lactones; Leukocyte Elastase; Models, Molecular; Molecular Sequence Data; Molecular Structure; Pancreatic Elastase; Protease Inhibitors; Protein Conformation; Substrate Specificity; Swine; Thrombin

We have found that alpha-aryl-substituted halo enol lactones (I and II) are effective mechanism-based inactivators for chymotrypsin. In this study, we have investigated, for comparative purposes, halo enol lactones with aryl functions situated beta and gamma to the lactone carbonyl group. We synthesized 4-phenyl-5(E)-(iodomethylidene)tetrahydro-2-furanone (1), 4-phenyl-5(E)-(iodomethylidene)dihydro-2-furanone (2), 4-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (3), and 5-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (4), using a halolactonization reaction to convert the appropriate phenyl-substituted acetylenic acid precursor into the corresponding 5(E)-(halomethylidene)furanone and 6(E)-(halomethylidene)pyranone system. The 4-phenylfuranone (1 and 2) and the 5-phenylpyranone (4) proved to be only reversible, competitive inhibitors. By contrast, the 4-phenyltetrahydropyranone (3) inactivated alpha-chymotrypsin in a time-dependent manner. This inactivation was very rapid but reversible, with regeneration of enzyme activity being spontaneous and hydrazine-accelerated, suggestive of the intermediacy of a stable acyl enzyme. Kinetic comparison of the iodomethylene lactone 3 with the corresponding protio lactone 25 indicates that the iodine accelerates the rate of chymotrypsin acylation but produces an acyl enzyme that is more hydrolytically labile than that formed from lactone 25. From the results of this study, we conclude that a phenyl group situated at C-3 (alpha to the lactone carbonyl group) in both the 5(E)-(iodomethylidene)tetrahydro-2-furanone (I) and 6(E)-(iodomethylidene)tetrahydro-2-pyranone (II) series is essential for their activity as mechanism-based irreversible inactivators of chymotrypsin. The corresponding beta-aryl-substituted lactones, by contrast, are potent acylating agents that lead to acyl enzymes of high stability.

Topics: Acylation; Binding, Competitive; Chromatography, Gel; Chymotrypsin; Hydrazines; Lactones; Protease Inhibitors; Serine Endopeptidases; Structure-Activity Relationship