Variety characteristics of soybean seeds in relation to protein and oil contents and activities of proteinase inhibitors

The protein and oil contents and the activity of proteinase inhibitors in six varieties of soybean have been studied. It has been found that the specific amidase activity of trypsin inhibitors ranges from 170 to 320 nominal units. Electrophoretic results indicates the presence in the water-soluble fraction of seven or eight components possessing inhibitor activity in relation to trypsin and chymotrypsin.Institute of Plant Physiology and Biophysics, Academy of Sciences of the Tadzhik SSR, Dushanbe. Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 761–765, November–December, 1984.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

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.     

Suppressive activity of protease inhibitors from buckwheat seeds against human T-acute lymphoblastic leukemia cell lines

The buckwheat protease inhibitor designated BWI-1, a member of the potato inhibitor I family, inhibits trypsin, chymotrypsin, and subtilisin, whereas the buckwheat protease inhibitor designated BWI-2a, a novel protease inhibitor homologous to the vicilin family, inhibits only trypsin. We examined the suppressive activity of BWI-1 and BWI-2a against T-acute lymphoblastic leukemia (T-ALL) cells, such as JURKAT and CCRF-CEM, and human normal blood lymphocytes. Both inhibitors significantly suppressed the growth of T-ALL cells as judged by the soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (tetrazolium/formazan assay). JURKAT cells showed slightly higher susceptibility to buckwheat inhibitors than CCRF-CEM cells. Modification of Arg residue(s) in inhibitors by 1,2-cyclohexandione inactivated their trypsin inhibitory activity, considerably abolishing their suppressive activity. This suggests that the trypsin inhibitory activity is involved in the suppression of growth of human T-ALL cell lines. It was further found that both inhibitors triggered programmed cell death (apoptosis) of these cell strains with DNA fragmentation.     

Isolation and properties of trypsin inhibitors from the seeds ofLupinus luteus andL. polyphyllus

From dormant seeds of European yellow lupin (Lupinus luteus) and Washington lupin (Lupinus polyphyllus) inhibitors have been isolated that effectively suppress the activity of trypsin and interact nonstochiometrically with chymotrypsin. From their physicochemical properties and aminoacid compositions, the inhibitors from the species of lupin investigated belong to different types.     

Inhibitory property characterization and reactive site exploration of the arrowhead proteinase inhibitor

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymotrypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymotrypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the structural characteristics of inhibitors A and B, it was predicated that the two reactive sites should be located in the positions Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), respectively. In inhibitor A, there exists another hydrophobic residue involved in inhibiting chymotrypsin. This residue might be situated in the reactive region composed of the Arg reactive site.     

Separation and quantitation of urinary trypsin inhibitors by reversed-phase high-performance liquid chromatography

Summary Human urines contain a family of trypsin inhibitors (UTIs) in small quantities, which seem to be involved in important biological processes. A procedure for separation and quantitative determination of such endogenous inhibitors in human urine has been developed. The urine sample is adjusted to pH 8.3 and percolated through a trypsin-Sepharose 4B column: the inhibitors are eluted with acid solution. The eluate (1000 l) is analysed by RP-HPLC with programmed elution and ultraviolet detection (200 nm). Three principal peaks have been evidenced: they are due to the elution of urinary trypsin inhibitors (UTIs) having apparent m.w. of ca. 6000, 72000, 18000 daltons, respectively. Characteristics of the procedure are: limited sample volume (ca. 200 ml) and recovery of the global inhibition activity (95%). For each UTI determination reproducibility and linearity ranges are reported.

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Trennung und Bestimmung von Trypsin-Inhibitoren im Harn durch Umkehrphasen-HPLC

     

Improvement of electrophoretic detection of antitrypsin activity in fish blood and seminal plasma

The original method of Uriel and Berges for detection of trypsin inhibitors lacks specificity due to masking effects of nonspecific esterases. We report a modification of this method based on inhibition of esterases in samples by phenylmethylsulfonyl fluoride (PMSF). This method can be particularly useful for characterization profiles of antitrypsin activity in seminal plasma of salmonid fish where esterases and inhibitors migrate at the same mobility.     

INHIBITORY PROPERTY CHARACTERIZATION AND REACTIVE SITE EXPLORATION OF THE ARROWHEAD PROTEINASE INHIBITOR

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymo-trypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymo-trypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the st     

胰酶的小肽类抑制剂的研究

利用噬菌体表面展示15肽库技术对胰酶抑制剂进行了3轮特异性的筛选.从中得到18个不同的肽序列,与胰酶天然抑制剂活性部位比较,对抑制剂的活性序列进行了分析.根据分析结果合成了1个9肽,其抑制常数为89±10μmol/L.     

Selectivity of neutral/weakly basic P1 group inhibitors of thrombin and trypsin by a molecular dynamics study

Molecular dynamics (MD) simulations followed by molecular mechanics generalized Born surface area (MM-GBSA) analyses have been carried out to study the selectivity of two neutral and weakly basic P1 group inhibitors (177 and CDA) to thrombin and trypsin. Detailed binding free energies between these inhibitors and individual protein residues are calculated by using a per-residue basis decomposition method. The analysis of the detailed interaction energies provides insight on the protein-inhibitor-binding mechanism and helps to elucidate the basis for achieving selectivity through interpretation of the structural and energetic results from the simulations. The study shows that the dominant factor of selectivity for both inhibitors is van der Waals energy, which suggests better shape complementarity and packing with thrombin. Nonpolar solvation free energy and total entropy contribution are also in favor of selectivity, but the contributions are much smaller. Binding mode and structural analysis show that 177 binds to thrombin and trypsin in a similar binding mode. In contrast, the CDA binds to thrombin and trypsin in very different modes.     

Synthesis and evaluation of guanidine-containing Schiff base copper(II), zinc(II), and iron(III) chelates as trypsin inhibitors

3-Formyl-4-hydroxyphenylguanidine hydrochloride and its Schiff base copper(II), zinc(II), and iron(III) chelates were synthesized and their inhibitory activity against bovine beta-trypsin were determined. Syntheses of Schiff base metal chelates were carried out from 3-formyl-4-hydroxyphenylguanidine, various L-amino acids, and divalent metal acetate. Their structures were established on the basis of spectroscopic evidence and elemental analysis. The inhibitory activity of these chelates against bovine beta-trypsin was determined. The guanidine-containing copper(II) and zinc(II) chelates behaved as potent competitive inhibitors of trypsin. However, similar inhibitory activity was not observed for guanidine-containing iron(III) chelates. The inhibition constants (K(i) values, ca. 10(-5) M) of guanidine-containing Schiff base copper(II) and zinc(II) chelates were slightly lower than those (ca. 10(-6) M) of the corresponding amidine-containing Schiff base chelates with regard to bovine trypsin.     

A sensitive colorimetric label-free assay for trypsin and inhibitor screening with gold nanoparticles

Gold nanoparticles (Au-NPs) with negative charges aggregate in the presence of Arg(6) due to electrostatic interactions resulting in the red-shift of the plasmon absorption. But, after incubation of Arg(6) with trypsin the aggregation of Au-NPs can be prohibited. Accordingly, a newly designed-Au-NPs based colorimetric assay method for trypsin activity is established. Trypsin with a concentration as low as 1.6 ng mL(-1) can be assayed with this new colorimetric assay. This colorimetric label-free assay for trypsin can be performed in aqueous solution and both Au-NPs and Arg(6) are easily accessible. Thus, this assay method is useful for screening inhibitors of trypsin.     

Isolation and properties of trypsin inhibitors from the seeds ofLupinus luteus andL. polyphyllus

From dormant seeds of European yellow lupin (Lupinus luteus) and Washington lupin (Lupinus polyphyllus) inhibitors have been isolated that effectively suppress the activity of trypsin and interact nonstochiometrically with chymotrypsin. From their physicochemical properties and aminoacid compositions, the inhibitors from the species of lupin investigated belong to different types.V. F. Kuprevich Institute of Experimental Botany, Belarusian Academy of Sciences, Minsk. A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 879–884, November–December, 1993.     

Protein content,activity, and electrophoretic composition of the inhibitor-containing fraction of pea seeds

The amounts of protein and the activities of the proteinase (trypsin) inhibitors of 16 varieties of pea have been studied. The amount of protein ranged from 19.3 to 25.2% and the amidase activities of the trypsin inhibitors from 18 to 40.8 IU. It was found that the electrophoretic spectrum of the inhibitor-containing fraction of the pea seed protein consisted of 12–16 components with molecular masses of 14–89 kDa. Features of the electrophoretic spectrum of some varieties of pea have been established.V. F. Kuprevich Institute of Experimental Botany, Belorussian SSR Academy of Sciences, Minsk. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 243–247, March–April, 1989.     

Synthesis of novel bridged dinitrogen heterocycles and their evaluation as potential fragments for the design of biologically active compounds

A library of saturated bridged heterocycles based on 3,6-diazabicyclo[3.2.1]octane-2,4-dione and bispidine scaffolds (mean compound molecular weight is approximately 300 Da) with up to three stereocenters and four diversity points has been synthesized. Synthetic scaffold modifications leading to an increase in molecular complexity were studied. Well-defined stereochemical structures of both compound sets was confirmed by X-ray studies and halogenoaryl substituents were inserted appropriately for the design of novel non-basic serine protease inhibitors. Comprehensive molecular modeling has been performed for all synthesized compounds giving rationales of ligand–enzyme interactions with thrombin and trypsin. Biological testing confirmed moderate inhibitory activity of halogen-substituted saturated diazabicyclic small molecules towards thrombin.     

Determination of dissociation constants of complexes of trypsin and its low molecular weight inhibitors by affinity chromatography in zonal and frontal analysis arrangement

The inhibitory constants of complexes of trypsin and its soluble or immobilized inhibitors were determined from volumes in which trypsin emerged from the column of its immobilized inhibitor (p-aminobenzamidine coupled through hexamethylenediamine to hydroxyalkyl methacrylate gel, Spheron) eluted by solutions of soluble trypsin inhibitors (benzylamine, benzoyl-L-arginine, N-butylamine, benzamidine, and p-aminobenzamidine). The values of constants obtained by affinity chromatography in the zonal and frontal analysis arrangement were in good agreement and in accordance with data obtained kinetically. The plot of l/(V_i-V₀) versus l/K_I (determined by zonal analysis) or of V_i versus K_I(V-V_i) (determined by frontal analysis) for identical concentrations of various inhibitors was linear. The fact that the dissociation constant of the complex of trypsin and immobilized p-aminobenzamidine (1.6-3.7 x 10⁶ M) is lower than the dissociation constant of the complex of trypsin and free p-aminobenzamidine (1.9 x 10^-5 M) seems to indicate possibilities of nonspecific adsorption in the binding of trypsin to p-aminobenzamidine-NH₂-Spheron. Submitted as RNDr.-Thesis at the Faculty of Natural Sciences, Charles University, Prague, May 1977.     

Affinity chromatography of proteases of hydroxyalkyl methacrylate gels with covalently attached inhibitors

The efficient isolation of trypsin and chymotrypsin from a crude pancreatic extract was achieved by affinity chromatography on specific adsorbents prepared by coupling of both naturally occurring protease inhibitors and also synthetic low-molecular-weight protease inhibitors to hydroxyalkyl methacrylate gels. Specific sorbents prepared with synthetic inhibitors are stable and are suitable for the isolation of chymotrypsin and trypsin even on a large scale.     

Brown Kidney Bean Bowman–Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity

A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. ‘brown kidney bean’. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman–Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0–14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC₅₀ of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.