A conjugate of trypsin and chymotrypsin

A heteroenzyme conjugate retaining activities of two component enzymes from trypsin and chymotrypsin was prepared using N-succinimidyl pyridyl dithiopropionate as crosslinking reagent. The conjugate bound to both trypsin and chymotrypsin affinity columns. Trypsin and chymotrypsin were linked in the ratio of 1:1 on mol basis. The conjugate, when treated with dimethyladipimidate, showed decreased autolysis of its trypsin component.     

Behavior of serine proteases thrombin,trypsin, and chymotrypsin under conditions of MALDI-TOF-Mass spectrometry

Features of ion formation under the influence of laser emission under the conditions of MALDI-TOF mass spectrometry in the glycosylated protein thrombin and the related proteases trypsin and chymotrypsin were studied for the first time. It was shown that trypsin and chymotrypsin are ionized in the form of molecular ions with masses 23800 and 25660 Da respectively. The molecular ion of thrombin was not detected; three main fragment ions with masses 9951, 10230, and 11982 Da were detected in the mass spectrum of thrombin. It was suggested that these fragments relate to peptide and glycopeptides fragment ions formed as a result of bond cleavage in the region of Asn60G during exposure to laser emission under the conditions of MALDI-TOF mass spectrometry. __________ Translated from Teoreticheskaya i éksperimental’naya Khimiya, Vol. 44, No. 2, pp. 74–78, March–April, 2008.     

An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

In biomedical research and clinical diagnostics, it is a major challenge to measure disease‐related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time‐consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge‐changing fluorescent peptide substrates. Charge‐changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic α‐chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both α‐chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross‐reactivity with the trypsin‐like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin‐specific), a detection limit of about 10–20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point‐of‐care diagnostics.     

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.     

High concentrations of sodium chloride facilitate the use of immobilized chymotrypsin for separating virgin forms of specific trypsin inhibitors.

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.     

Immobilization of chymotrypsin on silver nanoparticles

Russian Chemical Bulletin - The method of immobilization of chymotrypsin based on the reduction reaction of silver nitrate with sodium borohydride in an aqueous medium in the presence of the enzyme...     

Complexes of cellulose and trypsin

Adsorption of trypsin to microcrystalline cellulose has been determined as functions of protein concentration and pH of the aqueous medium. The study of adsorption at several pH values indicates that interaction of trypsin to the microcrystalline cellulose interface is controlled by the electrostatic effect. The FTIR, desorption, and SEM data reveal that a part of trypsin is strongly bound to the microcrystalline cellulose matrix. Resulting complexes consist of microcrystalline cellulose, trypsin, and water.     

A fluorescence study of DNS-labelled chymotrypsin and related enzymes

Various dansyl (DNS)-conjugates of chymotrypsin, chymotrypsinogen and elastase have been prepared. Excitation and emission spectra, and fluorescence depolarization measurements have been obtained in a spectrophotofluorometer, and relevant fluorescent lifetimes have been derived from fluorescence decay measurements.Free DNS-OH gave a lifetime of 13 nsec in fair agreement with other estimates, but combined DNS gave significantly lower values. In particular, ?-DNS lysine in water gave a value of 4 nsec, with higher values (up to 10 nsec) at increasing glycerol concentration. Of the various protein conjugates, specifically labelled chymotrypsin gave the lowest lifetime (6 nsec); for the others, higher values were observed, the values increasing with pH of labelling. Of the protein conjugates, only in the case of specifically labelled elastase, did 'glycerol concentration cause an increase in the measured τ value from 8 to 12 nsec.Active-site labelled chymotrypsin was unusual in that its fluorescent spectrum was dependent upon excitation wavelength and its excitation spectrum upon the selected emission wavelength. The addition of further non-specifically attached DNS-groups caused a shift towards the spectra of such DNS-groups, and saturation with indole gave spectra of such a type as well as erasing wavelength dependence. It seemed that specifically attached DNS groups were probably within the “tosyl hole” but could be dislodged by indole, a competitive inhibitor.Depolarization properties were in all cases markedly dependent upon excitation wavelength. Experiments performed with varying temperature gave relaxation times which were smaller than expected for a rigid molecule with the molecular weight of chymotrypsin but, also, considerable increases were observed with increasing excitation wavelength. It seemed that the susceptibility to temperature-activated rotation of the DNS group increased with the energy of the incident radiation. At constant temperature, apart from specifically-labelled chymotrypsin, spuriously high relaxation times were observed, for which an explanation is required. Until this is provided, depolarization measurements cannot be regarded as providing a sound method of measuring relaxation times of protein molecules in solution.     

Immobilized chymotrypsin on reversibly precipitable polymerized liposome

A polymerized liposome (PLS) was prepared using a synthesized phosphatidylethanolamine with a diacetylene moiety that showed a reversibly precipitable property on addition and removal of salt. To prepare a soluble-insoluble immobilized enzyme, chymotrypsin was covalently immobilized on the outer surface of the PLS. The carbodiimide method was employed for the enzyme immobilization. Coupling was rapid and nearly complete at a weight ratio of enzyme to the PLS of < 0.12. The immobilized enzyme showed favorable activity yields for both low-and high-mol-wt substrates, i.e., 90 ±9% forN-benzoyl-L-tyrosine ethyl ester and 59 ±5% for casein up to an enzyme coupling density of 0.38 g/g-PLS. The immobilized enzyme was reusable and more stable at high temperature and long-term incubation than the native enzyme.     

Sunflower trypsin inhibitor-1

SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.     

Conformer selection and intensified dynamics during catalytic turnover in chymotrypsin

Intensified searching: In enzymes, conformational dynamics are linked to the catalytic reaction coordinate. A novel analytical approach was used to monitor catalysis-linked dynamics in chymotrypsin, revealing that in some enzymes, catalysis is promoted by intensified, but undirected conformational sampling after substrate binding.     

Non-specific binding in the affinity chromatography of chymotrypsin

The strong and specific binding of chymotrypsin on chromatographic columns containing agarose substituted with N--amino Caproyl- -tryptophan methyl ester is abolished when the -amino groups on the surface of the enzyme are reacted with acetic anhydride. Because the catalytic properties of the acetylated chymotrypsin are identical to those of the underivatized enzyme, it is concluded that the high affinity of chymotrypsin for this column is not due solely to biospecific inhibitor binding, which is by itself very weak, but requires reinforcement through weak non-specific interactions with the column support. It is postulated that these non-specific interactions include electrostatic interactions between agarose matrix and positively charged lysyl residues on the enzyme. The results demonstrate for the first time that residues on the surface of an enzyme not associated with its active site can play an important role in some chromatographic systems previously thought to be based on purely biospecific interactions.     

Resolution of α-methyl amino esters by chymotrypsin

The resolution of DL-α-MeTrp-OMe HCl, Dl-α-MePhe-OMe HCl and DL-α-Me-pF-Phe-OMe HCl have been achieved using α-chymotrypsin at pH 5.0.     

Photochemical inactivation of trypsin

Abstract —The quantum yield for inactivation of aqueous trypsin fits the expression φ_f=Σ_rf_rφ‘_r, where f_r, is the fraction of incident light absorbed by residues of type r and the φ’_r are constants. The values φ‘_trp= 0.012, φ_tyr= 0.005 and φ’_eys= 0.10, obtained at pH 3 in the wavelength range 240–290 nm, are attributed to independent events by comparing with quantum yields of the initial photochemical products and permanent residue destruction. The proposed inactivating processes are photoionization of one essential tryptophyl residue, photolysis of one essential cystyl residue, and splitting of an essential cystyl residue induced by light absorption in a nearby tyrosyl residue. The initial photochemical process from pH 3–7 identified by flash photolysis is the ejection of electrons from approximately two tryptophyl residues, leading to the formation of the disulfide bridge electron adduct and the hydrated electron. It is proposed that one photoionized tryptophyl residue is permanently disrupted and the other is restored through a back reaction that leads to a damaged, active enzyme form. An enhanced inactivation quantum yield at flash photolysis light intensities is attributed to a biphotonic process. A model based on one-photon photoionization of tryptophan from a short-lived precursor of the fluorescent state and the biphotonic photoionization of tryptophan via the triplet state is consistent with the experimental results.