α2-Macroglobulin-protease reactions: Relationship of covalent bond formation,methylamine reactivity,and specific proteolysis

Experiments were performed to define the relation between covalent binding of enzymes to β₂-macroglobulin (α₂M), the specific proteolysis of α₂M subunits to 85K fragments, and the reactivity of the methylamine site on α₂M. We studied the reaction of α₂M with native trypsin, anhydrotrypsin, and two active lysyl-blocked derivatives, methyl-trypsin and dimethylmaleyl-trypsin, the last with reversibly modified amino groups that can be regenerated at low pH. The results were: (1) All enzymes tested reacted with α₂M but only native trypsin formed covalent complexes (not dissociable by sodium dodecyl sulfate). Trypsin and the lysyl-blocked enzymes caused complete proteolysis of the α₂M subunits, in agreement with previous studies. (2) The dimethyl-maleyl-trypsin became covalently bound to α₂M only after removing the blocking groups of the bound enzyme, indicating that sequential proteolysis and covalent bond formation is possible. Under the conditions used for deblocking, there was no change in the covalent/noncovalent binding ratio of native trypsin, anhydrotrypsin, or the other lysyl-blocked derivative, methyl-trypsin. (3) Native trypsin or anhydrotrypsin displaced methyl- or dimethylmaleyl-trypsin from their α₂M complexes but the newly bound enzymes with free amino groups did not form covalent bonds indicating that enzymes must remain in association with the inhibitor for the bond to form. (4) Methylamine reacts with noncovalent α₂M complexes but not with covalent complexes. (5) Methylamine-treated α₂M can still form complexes with trypsin but at a drastically reduced rate and only noncovalent complexes are formed. In summary, sequential proteolysis and covalent bond formation is possible under certain conditions, and there is a strong correlation between covalent binding and loss of methylamine reactivity. The latter observation is suggestive evidence for the identity of the covalent binding site of α₂M and the putative thiol ester of the methylamine site. The enzyme lysyl amino groups, are likewise possible candidates for attacking nucleophile at that site.     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Structure and specific binding of trypsin: comparison of inhibited derivatives and a model for substrate binding

The high-resolution structure of bovine trypsin inhibited with DFP² was determined by Stroud et al. (1971 and R. M. Stroud, L. M. Kay, A. Cooper &; R. E. Dickerson, Abstr. 8th Int. Congr. Biochem. 1970). The experiments reported here were designed to study the specific side-chain binding pocket of trypsin using benzamidine, which is a competitive, specific inhibitor of trypsin. High-resolution electron density syntheses and difference syntheses unambiguously identify the side-chain binding pocket, which normally recognizes and binds the side chains of arginine or lysine during proteolysis. Several important conformational differences in the protein structure are apparent between DIP- and BA-trypsins, and these are discussed with particular reference to inhibition, the binding of lysine and arginine, subsequent orientation of the target at the active site, and the enhancement of tryptic activity towards non-specific substrates seen on binding small alkyl amines or guanidines in the specific binding pocket.The BA-trypsin structure provides a good model for the binding of real substrate side chains to trypsin during catalysis, explaining the sharp trypsin specificity for lysine or arginine side chains (Weinstein &; Doolittle, 1972) and the lack of specificity for stereochemically different basic side chains. Benzamidine is shown to inhibit trypsin by steric interference with the inferred position of good substrates, even when they do not carry any side chain.Apart from the substitution of benzamidine and DIP, the most significant differences between DIP-trypsin and BA-trypsin involve complete repositioning of the side chain of Gln192, alterations in the side chains of Asp102, His57 and Ser195 at the active site, and changes in the solvent structure around this region. The carboxyl group of Asp189, which is responsible for trypsin specificity, shows no movement on binding benzamidine. The amidinium cation of benzamidine forms a salt bridge with Asp189 in BA-trypsin; a similar salt bridge can be constructed between the side chains of model substrates with lysyl or arginyl side chains and Aspl89. The γ-oxygen of Ser190 is displaced by a 120 ° rotation about its αβ bond on binding benzamidine and the binding pocket closes to sandwich the inhibitor ring between the peptide planes of 190–191 and 215–216. These contacts are presumably found in the enzyme-substrate complex with specific substrates.The active site structure at pH 8.0 is discussed with particular reference to the microscopic pK_a values of Asp102 and His57, the pK_a of the Asp-His system, and the mechanistic consequences of these assignments.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Isolation,purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>

Objectives

An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina.

Results

BTPI-301was purified to homogeneity by (NH₄)₂SO₄, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5 × 10^?10 M. It was stable and active at pH 4–12 and also at 4–90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin.

Conclusion

A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.

     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo)

A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of ∼8 kDa correlating to the peak 8041.5 Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K_i of 309.8 nM) and chymotrypsin (K_i of 10.7 μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 °C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 °C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.     

Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Reactions of human plasma α₁-antitrypsin (α₁-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α₁-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (?-NH₂ groups of lysine residues) in human plasma α₁-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α₁-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α₁-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α₁AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α₁-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α₁-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α₁AT, all of the other chemically modified α₁-AT derivatives studied presently retained their immunological reactivities against antisera to native α₁-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α₁-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α₁-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α₁-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α₁-AT molecule. Based on these modification studies, it is concluded that α₁-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α₁-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).     

Structural basis for the design of novel Schiff base metal chelate inhibitors of trypsin

The crystal structures of the complexes of bovine trypsin with m-guanidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 1), [N,N′-bis(m-guanidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 2), and [N,N′-bis(m-amidinosalicylidene)ethylenediaminato]copper(II) (inhibitor 4) have been determined. The guanidine-containing trypsin-inhibitors (1 and 2) bind to the trypsin active site in a manner similar to that previously reported for amidine-containing inhibitors, for example, m-amidinosalicylidene-l-alaninato(aqua)copper(II) hydrochloride (inhibitor 3). However, the binding mode of the guanidino groups of inhibitors 1 and 2 to Asp189 in the S1 pocket of trypsin was found to be markedly different from that of the amidino group of inhibitor 3. The present X-ray analyses revealed that the interactions of the metal ion of the inhibitors with the active site residues of trypsin play a crucial role in the binding affinity to the trypsin molecule. These structural information and inhibitory activity data for amidine- and guanidine-containing Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.     

A new lysine derived glyoxal inhibitor of trypsin,its properties and utilization for studying the stabilization of tetrahedral adducts by trypsin

New trypsin inhibitors Z-Lys-COCHO and Z-Lys-H have been synthesised. K_i values for Z-Lys-COCHO, Z-Lys-COOH, Z-Lys-H and Z-Arg-COOH have been determined. The glyoxal group (–COCHO) of Z-Lys-COCHO increases binding ~300 fold compared to Z-Lys-H. The α-carboxylate of Z-Lys-COOH has no significant effect on inhibitor binding. Z-Arg-COOH is shown to bind ~2 times more tightly than Z-Lys-COOH. Both Z-Lys-¹³COCHO and Z-Lys-CO¹³CHO have been synthesized. Using Z-Lys-¹³COCHO we have observed a signal at 107.4 ppm by ¹³C NMR which is assigned to a terahedral adduct formed between the hydroxyl group of the catalytic serine residue and the ¹³C-enriched keto-carbon of the inhibitor glyoxal group. Z-Lys-CO¹³CHO has been used to show that in this tetrahedral adduct the glyoxal aldehyde carbon is not hydrated and has a chemical shift of 205.3 ppm. Hemiketal stabilization is similar for trypsin, chymotrypsin and subtilisin Carlsberg. For trypsin hemiketal formation is optimal at pH 7.2 but decreases at pHs 5.0 and 10.3. The effective molarity of the active site serine hydroxyl group of trypsin is shown to be 25300 M. At pH 10.3 the free glyoxal inhibitor rapidly (t_1/2=0.15 h) forms a Schiff base while at pH 7 Schiff base formation is much slower (t_1/2=23 h). Subsequently a free enol species is formed which breaks down to form an alcohol product. These reactions are prevented in the presence of trypsin and when the inhibitor is bound to trypsin it undergoes an internal Cannizzaro reaction via a C2 to C1 alkyl shift producing an α-hydroxycarboxylic acid.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P₁ position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4-nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K_a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe⁵]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe(p-F), Phe(p-NH₂) and Phe(p-CH₃) in this position retained the affinity towards this proteinase, whereas Phe(p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues.     

Micrococcus lysodeikticus membrane ATPase. effect of trypsin on stimulation of a purified form of the enzyme and identification of its natural inhibitor

A soluble purified form of Micrococcus lysodeikticus ATPase (form B_AT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125–150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25 000 (ε subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the α subunit was also observed. The interaction between the ε subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.     

Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium <Emphasis Type="Italic">Oceanimonas</Emphasis> Sp. BPMS22 and its Kinetics

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, K_i, of 3.44?±?0.13 μM and second-order association rate constant, k_ass, of 1.08?×?10³ M^?1 S^?1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.     

Demonstration of protease activity for epidermal growth factor-binding protein

The epidermal growth factor can be isolated from the male mouse submaxillary gland as part of a high molecular weight complex. The complex is composed of two molecules of epidermal growth factor and two molecules of epidermal growth-factor binding protein (J.M. Taylor, W.M. Mitchell, and S. Cohen, 1974, J. Biol. Chem.249, 3198–3203). The proteolytic activity of epidermal growth-factor binding protein was demonstrated by its self-proteolysis in moderate (3–7 m) concentrations of urea, and, its inhibition by formation of a complex with pancreatic trypsin inhibitor. This complex was characterized by its pI and by its ability to yield pancreatic trypsin inhibitor and epidermal growth factor-binding protein in sodium dodecyl sulfate-urea gel electrophoresis. The association equilibrium constant was determined to be 3.6 × 10⁷m^?1 by inhibition studies of the esteropeptidase. These results, which indicate that epidermal growth factor-binding protein is capable of autodigestion and of forming a stable complex with a macromolecular inhibitor of trypsin, lend strong support to the hypothesis that epidermal growth factor-binding protein is capable of cleaving a larger precursor by its proteolytic action.     

A kidney bean trypsin inhibitor with an insecticidal potential against Helicoverpa armigera and Spodoptera litura

In the present study, trypsin inhibitor extracts of ten kidney bean seed (Phaseolus vulgaris) varieties exhibiting trypsin and gut trypsin-like protease inhibitor activity were tested on Helicoverpa armigera and Spodoptera litura. Trypsin inhibitor protein was isolated and purified using multi-step strategy with a recovery of ~15 % and purification fold by ~39.4. SDS-PAGE revealed a single band corresponding to molecular mass of ~15 kDa and inhibitory activity was confirmed by reverse zymogram analyses. The inhibitor retained its inhibitory activity over a broad range of pH (3–11), temperature (40–60 °C) and thermostability was promoted by casein, CaCl₂, BSA and sucrose. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.85 μM. The purified trypsin inhibitor protein was further incorporated in the artificial diet and fed to second instar larvae. A maximum of 91.7 % inhibition was obtained in H. armigera, while it was moderate in S. litura (29 %) with slight varietal differences. The insect bioassay showed 40 and 22 % decrease in larval growth followed by 3 and 2 days delay in pupation of H. armigera and S. litura, respectively. Some of the adults emerged were deformed and not fully formed. Trypsin inhibitor protein was more effective against H. armigera as it showed 46.7 % mortality during larval growth period compared to S. litura (13.3 %).     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasiamacrorrhiza)

Giant taro (Alocasiamacrorrhiza) contains a protein which inhibits both trypsin and chymotrypsin. This trypsin/chymotrypsin inhibitor exists as a dimer of two identical monomers each with slight polymorphism and is an attractive candidate for conferring insect resistance in transgenic plants. The 184 amino-acid sequence (molecular mass of 19774 Da for the Met-24, Glu-50 form) has been determined and is compared with those of other Kunitz-type trypsin, chymotrypsin and subtilisin inhibitors. There appears to be greater ‘homology’ between the giant taro inhibitor and those inhibitors from other monocotyledons than inhibitors from dicotyledons. The P₁ loop region is different from that of other Kunitz-type inhibitors and contains a sequence Leu-Ala-Phe-Phe-Pro at residues 56–60. This section of sequence differs only by a Leu/Ile replacement to a tight binding inhibitor of neutrophil elastase, Recently produced by genetic engineering. The most likely candidate for the P₁ residue in the giant taro trypsin/chymotrypsin inhibitor is Leu-56.