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 new specific fullerene-based fluorescent probe for trypsin

A novel fluorescent probe (C(60)-FL) was designed and synthesized for the direct determination of trypsin, based on photo-induced electron transfer (PET). The probe consists of two functional moieties: fluorescein which performs as a fluorophore and an electron donor, and fullerene (C(60)) which acts as an electron acceptor and trypsin substrate analogue. In the presence of trypsin, the probe exhibited fluorescence increase due to the inhibition of electron transfer by the combination of C(60)-FL with trypsin. The response of the probe to trypsin was direct and rapid. Experimental results showed that the increase in fluorescence intensity is proportional to the concentration of trypsin within the range of 4.40×10(-7) to 7.04×10(-5) g mL(-1) under the optimized experimental conditions. The detection limit of the proposed method was 40 ng mL(-1). The method had high selectivity for trypsin over other enzymes and proteins, such as lipase, α-amylase, bovine serum albumin, zinc metallothionein, glutathione reductase, thioredoxin and α-chymotrypsin etc. The remarkable properties of C(60)-FL help to extend the development of fluorescent probes for investigating enzymes in a biological context.     

Free radical inactivation of trypsin

Reactivities of free radical oxidants, ^.OH, Br^-·₂ and Cl₃COO^. and a reductant, CO^-·₂, with trypsin and reactive protein components were determined by pulse radiolysis of aqueous solutions at pH 7, 20°C. Highly reactive free radicals, ^.OH, Br^-·₂ and CO^-·₂, react with trypsin at diffusion controlled rates, k(^.OH + trypsin) = 8.2 × 10¹⁰ M^-1 s^-1, k(Br^-·₂ + trypsin) = 2.55 × 10⁹ M^-1 s^-1 and k(CO^-·₂ + trypsin) = 2.6 × 10⁹ M^-1 s^-1. Moderately reactive trichloroperoxy radical, k(Cl₃COO^. + trypsin) = 3 × 10⁸ M^-1 s^-1, preferentially oxidizes histidine residues. The efficiency of inactivation of trypsin by free radicals is inversely proportional to their reactivity. The yields of inactivation of trypsin by ^.OH, Br^-·₂ and CO^-·₂ are low, G(inactivation) = 0.6-0.8, which corresponds to ∾ 10% of the initially produced radicals. In contrast, Cl₃COO^. inactivates trypsin with ∾ 50% efficiency, i.e. G(inactivation) = 3.2.     

Immobilization of trypsin in polycation-polyanion complexes

Trypsin was immobilized in quasi-soluble polyanion-polycation complexes with retention of the enzyme activity. The activity of the immobilized enzyme strongly depends on pH of the prepared solution, composition, relative concentrations and molar masses of the polyelectrolytes. The highest activity of trypsin (about 93%) was found in the case of the complexes Na-poly(styrenesulfonate)-poly(diallyldimethylammonium chloride): trypsin = 10:1, prepared at pH 3.0, with high molar mass of the polyanion (about one million). This method can be proposed for immobilization of various serine proteases.     

Immobilization of trypsin on sub-micron skeletal polymer monolith

A new kind of immobilized trypsin reactor based on sub-micron skeletal polymer monolith has been developed. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multi-step reaction. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N-α-benzoyl-L-arginine (BA) which is the digestion product of a substrate N-α-benzoyl-L-arginine ethyl ester (BAEE). Results showed that the digestion speed was about 300 times faster than that performed in free solution. The performance of such an enzyme reactor was further demonstrated by digesting protein myoglobin. It has been found that the protein digestion could be achieved in 88 s at 30°C, which is comparable to 24 h digestion in solution at 37°C. Furthermore, the immobilized trypsin exhibits increased stability even after continuous use compared to that in free solution. The present monolithic enzyme-reactor provides a promising platform for the proteomic research.     

Comparative evaluation of bioactivity of crystalline trypsin for drying by Fourier-transformed infrared spectroscopy

The purpose of this study was to evaluate the enzymatic stability of colloidal trypsin powder during heating in a solid-state by using Fourier transform infrared (FT-IR) spectra with chemoinformatics and generalized two-dimensional (2D) correlation spectroscopy. Colloidal crystalline trypsin powders were heated using differential scanning calorimetry. The enzymatic activity of trypsin was assayed by the kinetic degradation method. Spectra of 10 calibration sample sets were recorded three times with a FT-IR spectrometer. The maximum intensity at 1634 cm⁻¹ of FT-IR spectra and enzymatic activity of trypsin decreased as the temperature increased. The FT-IR spectra of trypsin samples were analyzed by a principal component regression analysis (PCR). A plot of the calibration data obtained was made between the actual and predicted trypsin activity based on a two-component model with γ² = 0.962. On the other hand, a 2D method was applied to FT-IR spectra of heat-treated trypsin. The result was consistent with that of the chemoinformetrical method. The results for deactivation of colloidal trypsin powder by heat-treatment indicated that nano-structure of crystalline trypsin changed by heating reflecting that the β-sheet was mainly transformed, since the peak at 1634 cm⁻¹ decreased with dehydration. The FT-IR chemoinformetrical method allows for a solid-state quantitative analysis of the bioactivity of the bulk powder of trypsin during drying.     

Surface imprinting strategies for the detection of trypsin

Self-organized receptor layers are synthesized by molecular imprinting methods directly on pre-coated 10 MHz quartz-crystal microbalances (QCMs). The surface-imprinting is performed by three methods using amorphous, crystalline and solubilized trypsin, respectively, as templates. These attempts allowed us to compare imprinting results obtained with templating proteins in the dry state as well as in aqueous solution. All methods are generally applicable for surface imprinting of thin films. The biomimetic sensor layers allow selective enzyme enrichment on the imprinted electrode with detection limits as low as 100 ng ml(-1) and response times of a few minutes. Solution-based polymer imprinting with native trypsin as template resulted in the highest specific enzyme recognition, which even allowed us to distinguish denatured trypsin from the native form.     

Adsorption of trypsin on hydrophilic and hydrophobic surfaces

The adsorption of trypsin onto polystyrene and silica surfaces was investigated by reflectometry, spectroscopic methods, and atomic force microscopy (AFM). The affinity of trypsin for the hydrophobic polystyrene surface was higher than that for the hydrophilic silica surface, but steady-state adsorbed amounts were about the same at both surfaces. The conformational characteristics of trypsin immobilized on silica and polystyrene nanospheres were analyzed in situ by circular dichroism and fluorescence spectroscopy. Upon adsorption the trypsin molecules underwent structural changes at the secondary and tertiary level, although the nature of the structural alterations was different for silica and polystyrene surfaces. AFM imaging of trypsin adsorbed on silica showed clustering of enzyme molecules. Rinsing the silica surface resulted in 20% desorption of the originally adsorbed enzyme molecules. Adsorption of trypsin on the surface of polystyrene was almost irreversible with respect to dilution. After adsorption on silica the enzymatic activity of trypsin was 10 times lower, and adsorbed on polystyrene the activity was completely suppressed. The trypsin molecules that were desorbed from the sorbent surfaces by dilution with buffer regained full enzymatic activity.     

Clinical usefulness of a trypsin radioimmunoassay kit

From the clinical use of RIA-gnost trypsin kit, the following results were obtained. 1. Standard curve showed a steep and good curve was shown. 2. Incubation: The condition for the first incubation was set at the room temperature for 10-24 hours and that for the second incubation at the room temperature for 3-5 hours. With these settings, satisfactory results were obtained. 3. Reproducibility and recovery: The C.V. of the reproducibility and the recovery were considered superior, and the values were below 10% and +/- 3%, respectively. 4. Correlation between trypsin and serum elestase-1: An excellent positive correlation (coefficient of correlation r = 0.889) was shown. 5. Serum trypsin concentration of normal and pancreatic diseases: The normal range was from 100 to 500 ng/ml. Acute pancreatitis rose obviously. Diabetes mellitus and chronic pancreatitis was below 500 ng/ml and the pancreatic cancer showed a tendency to scatter in the range of 50-1,250 ng/ml. The above results indicated that serum trypsin can be easily measured with high precision by using this method. Thus the method is considered useful for the diagnosis of pancreatic diseases.     

Membrane affinity chromatography used for the separation of trypsin inhibitor.

Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability.     

Studies on natural and modified peptide Trichosanthes trypsin inhibitors

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C18 column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3-4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.     

Fluorimetric study of interaction of merbromin with trypsin

Interaction of merbromin with trypsin is of bovine origin has been studied by monitoring the absorption steady-state and time-resolved fluorescence spectral properties of the dye. Studies have been done in media of varying pH at different trypsin concentrations. It has been observed that trypsin brings about a quenching of fluorescence of the dye. The quenching is static in nature and the equilibrium constant of dye-trypsin interaction in the ground-state has been determined from quenching studies. Steady-state anisotropy of the dye increases in presence of trypsin in the medium. Values of micro-viscosity in the vicinity of the fluorophore in media containing trypsin have been determined from measurements of fluorescence anisotropy. Time-resolved fluorescence studies indicate the existence of two decaying states for the dye. The fractional contribution to the time-resolved decay changes with pH. The average lifetime, however, does not depend on the concentration of trypsin.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

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.     

The properties of covalently immobilized trypsin on soap-free P(MMA-EA-AA) latex particles

The covalent immobilization of trypsin onto poly[(methyl methacrylate)-co-(ethyl acrylate)-co-(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55 degrees C and 8.5, both of which were higher than that of the free form. It was found that K(m) (Michaelis constant) was 45.7 mg . ml(-1) and V(max) (maximal reaction rate) was 793.0 microg . min(-1) for immobilized trypsin, compared to a K(m) of 30.0 mg . ml(-1) and a V(max) of 5 467.5 microg . min(-1) for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times.     

Interaction of sodium benzoate with trypsin by spectroscopic techniques

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software.     

Covalent attachment of trypsin on plasma polymerized allylamine

This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV–vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.     

Label-free electrical determination of trypsin activity by a silicon-on-insulator based thin film resistor.

A silicon-on-insulator (SOI) based thin film resistor is employed for the label-free determination of enzymatic activity. We demonstrate that enzymes, which cleave biological polyelectrolyte substrates, can be detected by the sensor. As an application, we consider the serine endopeptidase trypsin, which cleaves poly-L-lysine (PLL). We show that PLL adsorbs quasi-irreversibly to the sensor and is digested by trypsin directly at the sensor surface. The created PLL fragments are released into the bulk solution due to kinetic reasons. This results in a measurable change of the surface potential allowing for the determination of trypsin concentrations down to 50 ng mL(-1). Chymotrypsin is a similar endopeptidase with a different specificity, which cleaves PLL with a lower efficiency as compared to trypsin. The activity of trypsin is analyzed quantitatively employing a kinetic model for enzyme-catalyzed surface reactions. Moreover, we have demonstrated the specific inactivation of trypsin by a serine protease inhibitor, which covalently binds to the active site of the enzyme.     

Kinetic study on the mechanism of inhibition of trypsin and trypsin-like enzymes by p-guanidinobenzoate ester

It was found that [4-(2-succinimidoethylthio)phenyl 4-guanidinobenzoate]methanesulfonate (E-3123) inhibits trypsin, thrombin and kallikrein, and its inhibitory activity is most potent toward trypsin. The interactions of these enzymes with E-3123 were studied mainly by using stopped-flow spectrophotometry. E-3123 behaved as a quasi-substrate of the enzymes and the inhibitory property was due to the efficient production of the stable acyl-enzyme. The acylation process with trypsin was exceedingly effective, and the resulting acyl-enzyme was the most stable among the three enzymes tested. This observation affords a rational basis for explaining the action of E-3123, which is a transient inhibitor most active toward trypsin.