PROTEINS AND TRYPSIN INHIBITORS IN SEEDS OF VARIOUS PLANTS

Process Biochemistry, 2012

A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20 kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (K i) calculated was 1.75 nmol/L, displaying a high affinity between enzyme and inhibitor. Both Native PAGE and RP-HPLC revealed that EATI is composed of four isoinhibitors that share the amino acid composition and the amino-terminal sequence homolog to Kunitz-type inhibitors. EATI is stable to denaturation by heat (up to 70 • C), pH (2-10), urea (8 mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100 mmol/L). CD analysis revealed that EATI in reduced form underwent structural modifications associated with a decrease in thermal and pH stabilities, suggesting that their disulfide bonds are not involved in the structuring of its reactive site, but are important for maintenance of its conformational stability. This behavior makes EATI one of the few inhibitors described in the literature with high DTT resistance.

Phytochemistry, 2008

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS–PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies

Journal of Food Science, 2010

ABSTRACT: The trypsin inhibitor from navy beans (Phaseoulus vulgaris), red kidney beans (Phaseoulus vulgaris L.), and adzuki beans (Vigna angularis) provided by the Royal Project Foundation in Thailand was isolated by heat and ammonium sulfate (AS) precipitation. Incubation at 70 °C for 10 min produced the highest trypsin inhibitor recovery for all legumes. The AS precipitation with 60% to 80% saturation (precipitate IV) resulted in 41-, 88-, and 34-fold of the purity and (−)26%, 126%, and (−)47% of percentage of activity increase for navy beans, red kidney beans, and adzuki beans, respectively. The trypsin inhibitors had a molecular weight of 132 kDa for navy beans, 118 kDa for red kidney beans, and 13 kDa for adzuki beans under nonreducing conditions. The obtained precipitate IV fraction from each legume effectively prevented the degradation of the tilapia muscle with concentration dependent. The myosin heavy chain increased as the concentration of the inhibitor fraction increased, especially at the highest level of addition. The result indicated that the precipitate IV from these legumes have potential for use as a protease inhibitor in fishery related products.

Process Biochemistry, 2009

Three-phase partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert-butanol (t-butanol) in order to obtain the three phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions (p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.

Braz. J. Plant …, 2010

Inga cylindrica Trypsin Inhibitor (ICTI) was purified as a single polypeptide chain by one step anion-exchange chromatography from a crude extract of Inga cylindrica (Vell.) Mart. seeds. ICTI is a 19.5 kDa protein presenting a remarkable inhibitory activity against bovine trypsin (EC 3.4.21.4) (K i = 4.3 nM). Circular dichroism analysis revealed that this inhibitor is a β type protein (40.4% of β-strand; 24.6% of β-turn and 6.7% of α-helix) in accordance with properties displayed in Kunitz type inhibitors. ICTI is a thermal stable protein within a wide range of pH (1.6 to 10.0) exhibiting highest stability at pH 7.0 as indicated by T m of 70.0 o C and ∆G 25 of 48.5 ± 0.7 kJ.mol -1 . The values of ∆G 25 at pH 1.6 (22.5 ± 1.2 kJ.mol -1 ) and pH 10.0 (31.5 ± 1.0 kJ.mol -1 ) indicate a reduced structural stability of the protein under these conditions. This is likely to result from pK a differences of the acid and basic side chains reflecting the changes in the non-covalent interactions in the folded state.

Journal of Biological Chemistry, 1967

Inhibitors of trypsin and chymotrypsin from several highly inbred genetic types of lima beans were fractionated by ion exchange chromatography. Six chromatographically distinct inhibitors were found in all cases. However, the relative distribution of these components fell into two types, dependent upon the variety of bean used. One milligram of one of the chromatographically homogeneous fractions inhibited 2.6 mg of bovine trypsin and 1.2 mg of a-chymotrypsin. The presence of a "double-headed" inhibitor was indicated, in which both inhibitory sites were present on the same molecule. The molecular weights of this fraction calculated from the amino acid composition and from the stoichiometry of trypsin inhibition were 9,670 and 9,050 g per mole, respectively. However, a molecular weight of 16,200 g per mole was found by the Ehrenberg method, and an szo,,,, value of 1.81 S was obtained. This indicates a possible aggregation or a molecular weight twice that indicated by the minimum one from amino acid analyses. Chemical modification of the ammo groups of this fraction with trinitrobenzenesulfonic acid completely destroyed the trypsin-inhibitory activity, with no effect on the chymotrypsin-inhibitory activity. Amidination of the amino groups, which results in no change in charge, also greatly lowered the trypsin-inhibitory activity. The results of the chemical modifications indicate that a "fast reacting" lysine residue is critical for the trypsin-inhibitory activity. The first major investigation of trypsin-inhibitory activity in different species of legumes was made by Borchem, Ackerson,

Food Science and Technology International, 2015

A novel trypsin inhibitor of protease (CqTI) was purified from Chenopodium quinoa seeds. The optimal extracting solvent was 0.1M NaCl pH 6.8 (p < 0.05). The extraction time of 5h and 90 °C was optimum for the recovery of the trypsin inhibitor from C. quinoa seeds. The purification occurred in gel-filtration and reverse phase chromatography. CqTI presented active against commercial bovine trypsin and chymotrypsin and had a specific activity of 5,033.00 (TIU/mg), which was purified to 333.5-fold. The extent of purification was determined by SDS-PAGE. CqTI had an apparent molecular weight of approximately 12KDa and two bands in reduced conditions as determined by Tricine-SDS-PAGE. MALDI-TOF showed two peaks in 4,246.5 and 7,908.18m/z. CqTI presented high levels of essential amino acids. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. Its activity was stable over a pH range (2-12), temperatures range (20-100 °C) and reducing age...

Food and health, 2022

Trypsin, an enzyme from the serine protease class, is known to be involved in the degradation of proteins. Excessive activity of trypsin is strongly implicated in triggering many diseases, such as acute pancreatitis, inflammation, and tumour. Therefore, this enzyme's regular and balanced activity is necessary for normal physiological functions. Thus, there is a need to develop new trypsin inhibitors from natural sources and chemical compounds. In this study, the inhibitory effects of aqueous extracts prepared from 29 different plants and 10 different chemical compounds were investigated on the activity of trypsin due to its importance in the health sector. The present study's plant extracts and chemical compounds showed trypsin-inhibitory effects. The inhibitory activities of the extracts and chemical compounds increased in a dose-dependent manner. Several plant extracts and chemical compounds that showed high trypsin inhibitory activities may be appropriate for use as trypsin inhibitors to provide additional support to drug treatment in the health field.

Phytochemistry, 2007

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o’clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5 M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP–HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (Ka) with bovine β-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I–III) were found to be about 107 M−1. Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.Five trypsin inhibitors from the ripe seeds of the garden four-o’clock and spinach were isolated. The primary structures and disulfide bridge pattern were established for natural and chemical synthesized inhibitors. There are probably representatives of a family of serine proteinase inhibitors.

Natural Product Research, 2012