Purification and characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gel-filtration chromatography followed by preparative polyacrylamide gel electro-phoresis. One of the inhibitors, Cajanus trypsin-chymotrypsin inhibitor (CTCI), inhibits both bovine trypsin and chymotrypsin while the other, Cajanus trypsin inhibitor(CTI), inhibits only bovine trypsin. The two inhibitors contained no carbohydrate and had an isoelectric point of 6. CTCI and CTI had average molecular weights of 15000 and 10500, respectively. The purified inhibitors in solution were stable to heat at 80°C for 15 min and pH 7–10. In the pH range 3–5, 80% of the activity was retained. Autoclaving totally destroyed the inhibitor activity. CTCI had two sites for trypsin binding and one site for chymotrypsin binding while CTI had only one site for trypsin binding. The inhibitors were very specific towards mammalian serine proteases and did not inhibit other proteases or serine proteases of bacterial origin.     

DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10^?7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.     

REACTION OF LENTIL PROTEINASE INHIBITORS WITH HUMAN AND BOVINE TRYPSIN AND CHYMOTRYPSIN1

The two main trypsin-chymotrypsin isoinhibitors previously purified from lentils (Lens culinaris Medik.), LCI-1 and LCI-4, inhibited one mol of human trypsin (1.05 and 1.00), more than one mol of bovine trypsin (1.53 and 1.38) and human chymotrypsin (1.70 and 1.43) as well as less than one mol of bovine chymotrypsin (0.62 and 0.54, respectively) per mol of inhibitor. Complex formation, together with chemical and enzymatic modification studies, showed that they were Bowman-Birk inhibitors with two independent reactive sites. One of these sites, mainly reacting with trypsin, contained arginine and bound tightly to bovine trypsin, less tightly to human trypsin and loosely to human chymotrypsin. The other reactive site, preferring chymotrypsin, contained tyrosine and bound tightly to human chymotrypsin, less tightly to bovine chymotrypsin and loosely to bovine trypsin. The amounts of bound enzyme exceeding one mol per mol of inhibitor reacted with the “wrong” sites: bovine trypsin with the chymotrypsin-reactive and human chymotrypsin with the trypsin-reactive one. The much higher inhibition of human chymotrypsin compared to that of bovine chymotrypsin resulted from a combination of two effects: the additional binding of human chymotrypsin at the “wrong” reactive site and the weak binding of the bovine chymotrypsin.     

Natural plant enzyme inhibitors. Isolation of a trypsin/α-amylase inhibitor and a chymotrypsin/trypsin inhibitor from ragi (Eleusine coracana) grains by affinity chromatography and study of their properties

A trypsin/α-amylase inhibitor (TAI) and a chymotrypsin/trypsin inhibitor (CTI) were isolated in homogeneous forms from ragi grains by affinity chromatography using immobilised chymotrypsin and immobilised trypsin. Both the inhibitors were capable of inhibiting the caseinolytic and amidolytic activities of bovine trypsin whereas the esterolytic activity of the enzyme was only weakly affected. While TAI had no action on chymotrypsin, the CTI exerted an inhibitory effect on the caseinolytic activities of bovine α-, β-, γ- and ?-chymotrypsins. Both the inhibitors could inactivate the proteolytic actions of bovine as well as human crude pancreatic preparations, TAI showed inhibitory activity against human pancreatic, porcine pancreatic and human salivary amylase in the ratio of 6.5:5:1. The possible practical application of TAI for the purification of α-amylases by affinity chromatography is indicated based on the demonstration of the dissociation of porcine pancreatic amylase from a ‘trypsin-TAI-amylase’ trimer complex in the presence of maltose. The antichymotryptic activity of CTI was less stable than its antitryptic activity at high temperature, acidic pH and on treatment with pepsin. Modification of arginine residues by 1,2-cyclohexane dione led to a preferential inactivation of its antitryptic activity. Treatment of CTI with trinitrobenzene sulphonic acid or pronase, however, caused almost identical loss of both antitryptic and antichymotryptic activities. The mode of inhibition of CTI was non-competitive for trypsin but was uncompetitive for chymotrypsin. The CTI could bind to immobilised trypsin or chymotrypsin but not to immobilised TPCK-chymotrypsin or chymotrypsinogen.     

Biochemical Study on the Nonprotein Trypsin and Chymotrypsin Inhibitors in the Soybean

The chemical properties of nonprotein trypsin inhibitor (NPTI) and nonprotein chymotrypsin inhibitor (NPCTI) in the soybean were studied. NPTI was extracted with 50% methanol and treated with TCA to a final concentration of 16% (W/V). Two dimensional thin-layer chromatography was used to make a peptide map of the methanol extract; 14 spots were observed. To distinguish the active peptides, the sample was mixed with trypsin or chymotrypsin; some peptides disappeared which formed complexes with the enzyme. The relative rate of inhibition indicated that inhibition was stoichiometric for trypsin and chymotrypsin. Dissociation constants of complexes (enzyme and inhibitor) showed that inhibition of chymotrypsin and trypsin were competitive and uncompetitive, respectively.     

Characterisation of trypsin and α-chymotrypsin inhibitors in Australian wattle seed (Acacia victoriae Bentham)

Crude water extracts from Australian wattle seed (Acacia victoriae Bentham) and their salt (ammonium sulphate)-precipitated fractions were analysed for trypsin and α-chymotrypsin (chymotrypsin) inhibitor activity, using gel electrophoresis and spectrophotometric methods. Three different bands with molecular weight 30.20, 38.03 and 39.81 kDa were active, with the 50% salt-precipitated fraction exhibiting highest activity and number of active bands. The same proteins also appeared to be responsible for both trypsin and chymotrypsin inhibitor activity. To establish conditions for the inactivation of these inhibitors, whole seed and uncoated (dehulled) cotyledon were subjected to different heat treatments. Moist heat treatment at 100 °C for 30 s was sufficient to inactivate both protease inhibitors although the trypsin inhibitor was found to be more heat-resistant than was the chymotrypsin inhibitor. Soaking overnight, before thermal treatment, improved the trypsin inhibitor activity but enhanced the efficiency of thermal inactivation in both inhibitors.     

Purification and partial characterisation of trypsin inhibitors from seeds of Clitoria ternatea

Three trypsin inhibitors of 20, 12 and 7 kDa were isolated in pure form from seeds of the vine Clitoria ternatea L of the tribe Glycineae of the Papilionoideae. The 20-kDa inhibitor is a one-chain molecule with arginine in the reactive site. The 12-kDa one is also a one-chain molecule and has lysine in the reactive site. It is also an inhibitor of chymotrypsin. They were tentatively assigned to the Kunitz and Bowman-Birk families, respectively. The small molecular weight inhibitor (7 kDa) also has an arginine in the reactive site and is probably of the Bowman-Birk type.     

Trypsin- und Chymotrypsin-Inhibitoren in Leguminosen

Zusammenfassung Die Aminosäuresequenzen am N- und C-Terminus und um die reaktiven Zentren des Trypsin-Chymotrypsin-Inhibitors PCI 3 aus den Samen von Feuerbohnen (Phaseolus coccineus L.) wurden durch Abbau mit Aminopeptidase O und Carboxypeptidase A vor und nach der enzymatischen Modifizierung mit Trypsin oder Chymotrypsin bestimmt. Beginnend am N-Terminus wurden folgende Sequenzen gefunden: Ser-Glu-Ala-Gly-Gln..., ...-Ile-Tyr-Lys-Ser-Gln-(Pro)-... mit Lys-Ser als reaktivem Zentrum gegen Trypsin, ...-Asp-Val-Ala-Leu-Ser-(Pro)-... mit Leu-Ser als reaktivem Zentrum gegen-Chymotrypsin und ...-Thr-Arg-Ala-Lys-Phe-Leu als C-Terminus. Die Bedeutung des Serinrestes in den reaktiven Zentren für die Spezifität der Inhibitoren wird diskutiert.

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Trypsin and chymotrypsin inhibitors in leguminosaeVII. Partial amino acid sequences of the trypsin chymotrypsin inhibitor PCI 3 from Phaseolus coccineus

Summary The sequences of amino acid residues at the amino and carboxyl terminus and around the reactive sites of the trypsin chymotrypsin inhibitor PCI 3 from the seeds of runner beans (Phaseolus coccineus L.) were estimated by aminopeptidase O and carboxypeptidase A degradation before and after enzymatical modification with trypsin or chymotrypsin. Beginning at the amino terminus the sequences are: Ser-Glu-Ala-Gly-Gln-..., ...-Ile-Tyr-Lys-Ser-Gln-(Pro)-... with Lys-Ser as reactive site against trypsin, ...-Asp-Val-Ala-Leu-Ser-(Pro)-... with Leu-Ser as reactive site against-chymotrypsin, and ...-Thr-Arg-Ala-Lys-Phe-Leu as C-terminus. The importance of the serine residue in the reactive sites concerning the specificity of inhibitors is dicussed.

     

PURIFICATION AND CHARACTERIZATION OF CHYMOTRYPSIN FROM PENAEUS VANNAMEI (CRUSTACEA: DECAPODA)

The purification and characterization of a chymotrypsin from the hepatopan-creas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl-PheCH₂Cl or benzyloxycarbonyl-Phe-CH₂Cl. Protein profiles of the hepatopancreas from two populations varied     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reaction with the human and bovine proteinases.

The reaction between the three Bowman-Birk proteinase inhibitors isolated from fenugreek seeds (TFI-B2, TFI-N2 and TFI-A8) and the human and bovine proteinases was investigated by studying the complexes formed and their properties. TFI-B2, the Lys-Leu trypsin chymotrypsin inhibitor, can bind 1.9 mol human trypsin (HT), 1.3 mol bovine trypsin (BT) and/or 0.4 mol human (HCT) or bovine (BCT) chymotrypsin per mole of inhibitor. HT was bound at the two reactive sites and BT mainly at the lysine-containing trypsin-reactive site, whereas HCT and BCT were only bound at the leucine-containing chymotrypsin-reactive site. TFI-N2, the Arg-Leu trypsin chymotrypsin inhibitor, could bind 1 mol BT and BCT, but 1.3 mol HT and 1.2 mol HCT per mole of inhibitor. In addition to the usual binding, the human enzymes could also be bound at the respective "wrong" reactive site. TFI-A8, the Arg-Arg trypsin inhibitor, binds 2 mol HT or BT per mole of inhibitor at the two trypsin-reactive sites, whereas HCT and BCT (about 0.2 mol/mol) are bound to one of the two "wrong" reactive sites.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

Natural plant enzyme inhibitors. Isolation and characterisation of a trypsin/chymotrypsin inhibitor from indian red wood (Adenanthera pavonia) seeds

A trypsin/chymotrypsin inhibitor was isolated from Indian red wood seeds by extraction with 0.01m HCl, chromatography on diethyl amino ethyl-cellulose, ammonium sulphate fractionation and gel chromatography on Sephadex G-100. The homogeneity of the final product was ascertained by affinity chromatography on trypsin sepharose and chromatography on phenyl sepharose CL-4B columns. During all stages of purification and characterisation the ratio of activities against trypsin and chymotrypsin remained constant at about 1.1:1 indicating that the same factor is responsible for both activities. The size of the inhibitor was found to be 24 000 daltons based on gel chromatographic studies on Sephadex G-100 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molar ratio of interaction between the inhibitor and bovine trypsin for complete inactivation of the enzyme was found to be 1.04:1. Electrophoretic and gel chromatographic studies indicated that the purified inhibitor is capable of undergoing aggregation to form dimers and trimers. Even in the presence of sodium dodecyl sulphate and sodium dodecyl sulphateurea, this phenomenon was discernible. The binding sites on the inhibitor for trypsin and chymotrypsin were not mutually exclusive, based on the data from mixed enzyme studies and on analysis of the inhibitor-enzyme complexes by gel chromatography on Sephadex G-100. Modification of the arginyl residues of the inhibitor resulted in the loss of more of the antitryptic activity than of antichymotryptic activity. Conversely, modification of amino groups resulted in the loss of more of the antichymotryptic activity. The inhibitor was stable to exposure to a wide range of pH (1.0–12.0), but it was completely inactivated on heat-treatment at 100°C for 15 min. The mode of inhibition of trypsin as well as chymotrypsin was non-competitive and K_i values for the inhibitor were 2.92 × 10-¹⁰M and 4.46 × 10-¹⁰M , respectively, for the two enzymes.     

Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.     

Low Trypsin and Chymotrypsin Inhibitor Mutants in Winged Bean(P sophocarpus tetragonolobus(L) DC)

Low trypsin inhibitor (LTI) and chymotrypsin inhibitor (LCTI) mutants have been identified and isolated from the M₅ mutant lines of winged bean. The LTI and LCTI lines demonstrated a true breeding feature in the subsequent generations. Three to five isoinhibitor bands of trypsin and chymotrypsin were absent in different mutants, indicating mutations in the respective gene loci. The LTI and LCTI mutants inhibited insect gut proteases as efficiently as their control counterparts. This could be attributed to a specific group of inhibitors present in all the mutant types.     

Trypsin and chymotrypsin inhibitor activities in plant foods from Vietnam and Hungary

A method for the determination of chymotrypsin inhibitor activity and a screening test for evaluating higher activities of trypsin inhibitor were elaborated. The protease inhibitor activities of the most important plant foods from Vietnam and Hungary were determined. In cereals and legume seeds the activity of chymotrypsin inhibitors is generally lower than that of the trypsin inhibitors. In various soy products (isolates, texturates) the extent of lowering of chymotrypsin inhibitor activity was less than that of trypsin inhibitor related to raw soy bean. The cooking process according to the usual kitchen technique of Vietnam has more effect on the higher trypsin inhibitor level of soy bean than on other legumes of average activity.     

Characterisation of a trypsin/chymotrypsin inhibitor from jack fruit (Artocarpus integrifolia) seeds

Jack fruit seed (Artocarpus integrifolia Hook f) trypsin inhibitor (JSTI) was found to be rich in acidic amino acids and devoid of free thiol groups. The N-terminal and C-terminal amino acids of JSTI were aspartic acid and scrine, respectively. The inhibitor was stable under conditions of extremes of pH (3·0–12·0), at high temperatures and in the presence of denaturing agents. JSTI showed a non-competitive type of inhibition with K_i values of 0·48 ± 0·17 nM and 0·16 ± 0·04 nM for trypsin and chymotrypsin, respectively. The JSTI–trypsin complex exhibited chymotrypsin inhibitory activity suggesting the ‘double-headed’ nature of the inhibitor. Chemical modification of lysine residues resulted in loss of trypsin and chymotrypsin inhibitory activities of JSTI indicating that amino groups are essential for activity.     

Isolation and Heat Stability of Trypsin Inhibitors in Amaranth (Amaranthus hypochondriacus)

Trypsin inhibitors were isolated from seeds of Amaranthus hypochondriacus by extraction at pH 7.5, heat treatment at 70°C for 15 min and affinity chromatography with trypsin-Sepharose 4B. Inhibitors of trypsin and chymotrypsin were identified in polyacrylamide gels following electrophoresis using a stain procedure that employed the chromogenic substrate acetyl-D, L-phenylalanine-2-napthyl ester. Three of the thirteen trypsin inhibitors identified in the electrophoresis gels, also had antichymotryptic activity. The inhibitor preparation was very thermostable retaining 20% of its original activity after 7 hr at 100°C.     

Effects of limited proteolysis on broad bean polyphenoloxidase

Partially purified broad bean polyphenoloxidase was treated with a variety of proteases. Enzyme activity increased after treatment with chymotrypsin, pepsin, subtilisin, thermolysin, trypsin, and Staphylococcus aureus V₈ protease but this activation was masked when assayed in the presence of sodium dodecylsulfate. The enzyme was resistant to proteolysis when treated with chymotrypsin, elastase, pepsin, thermolysin, and V₈ protease. Proteinase K, subtilisin and trypsin treatment resulted in partial degradation of the enzyme but only trypsin generated an active enzyme form with a slightly smaller molecular weight. Electrophoresis, followed by enzyme staining and or Western blotting, showed that trypsin treatment converted the active 45 kd enzyme into a 43 kd enzyme within 20 min. Treatment with proteinase K, subtilisin and trypsin also produced inactive higher molecular weight forms of purified polyphenoloxidase that were identified by Western blotting. The use of antibodies coupled with Western blotting also identified inactive, active, and latent polyphenoloxidase after protease treatment, and suggests that immunological methods can be used to establish the types and amounts of polyphenoloxidase present in a given tissue.     

Isolation and properties of trypsin and chymotrypsin inhibitors from barley grits after storage

The occurrence of antiproteolytic activity in albumins of barley grits has been shown. As a result of storage lasting 6 months at a temperature of ?10 °C the freeze-dried albumin of barley grits lost its antiproteolytic activity and a proteolytic activity appeared. The antiproteolytic activity was partially regained after dissociation of the enzyme–inhibitor complex which had formed. From the reactivated freeze-dried albumins of stored barley grits two thermolabile protease inhibitors were isolated and partially purified. The first trypsin–chymotrypsin inhibitor B_7.1 inhibits, at a concentration of 2.5 μg, 1 μg of crystalline trypsin and 1.9 μg of this inhibitor inhibits 1 μg of crystalline alpha-chymotrypsin. Lower activity was demonstrated for the second inhibitor of chymotrypsin B_7.4; 4.8 μg of this inhibitor is needed to inhibit the activity of 1 μg of crystalline alpha-chymotrypsin. The occurrence of both inhibitors has been confirmed by means of starchgel electrophoresis. Their specific activity was small in comparison with that of some commercial crystalline preparations of inhibitors. The capacity to inhibit native proteases shows their important physiological function, based mainly on the control of proteolytic activity of endogenous enzymes. However it seems unlikely that they could have a negative influence on the nutritive value of proteins in barley grains or grits.