Trypsin Molecules Research Articles

Structures of porcine β-trypsin–detergent complexes: the stabilization of proteins through hydrophilic binding of polydocanol

Polydocanol has a wide range of medical applications, especially in sclerotherapy of many diseases such as gastrointestinal antiplastia, oesophageal haemangioma etc. It is of interest to study the mode of binding of this medically important detergent and its subsequent action on proteins. Here, three crystal structures of serine protease trypsin are reported in the presence of varying concentrations of polydocanol in order to elucidate its mode of binding and interactions with proteins. Polydocanol binds to the protein with its hydrophilic head rather than the hydrophobic tail as is the case with other detergents such as SDS and MEGA-8. This hydrophilic binding mode results in the binding sites of polydocanol being distributed on the surface of the enzyme. There are at least 11 binding sites for polydocanol in trypsin. Polydocanol forms part of the large-scale water networks which connect distant regions of the enzyme, thereby stabilizing it. The hydrophilic binding of polydocanol also results in cross-linked pairs of trypsin molecules.

Immobilization of trypsin on polyester fleece via different spacers

The immobilization of trypsin directly on polyester fleece FO 2413 as well as via PEG-diamine, aldehyde dextran, amino dextran and bovine serum albumine (BSA) spacers was investigated. It is well established that longer spacers are required for efficient immobilization of biospecific molecules to solid support materials. These minimize steric hinderance and environmental effects imposed by the surface properties of the matrices. The hydrophobic surface of the polyester fleece causes inactivation of the immobilized enzyme. Therefore, the above-mentioned spacers were employed not only to achieve a high amount of bound trypsin but also to immobilize trypsin in its active conformation. The trypsin activity was tested with the N-benzoyl-DL-arginine-p-nitroanilide hydrochlorid (BAPNA) substrate as well as with the azocasein substrate, whereas the amount of fixed trypsin was determined by BCA method. To characterize the properties of the different linked trypsin molecules, the thermal stability, pH stability and the behaviour in ethanol/water mixtures was investigated. Due to the highest activity values obtained with the BSA spacer, this system was characterized more thoroughly by testing the activity behaviour after repetitive use and various storage conditions. Immobilized trypsin shows a high thermal and pH stability. Furthermore it can be stored for several months without loosing its activity.

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin ( M r 26 000) and four trypsins (T1–T4) with the following properties: T1, K m 0.3 μM, M r 70 000 ; T2, K m 0.4 μM, M r 67 000; T3, K m 2.4 μM, M r 29 000; T4, K m 15 μM, M r 17 000. Diet-fed larvae have a chymotrypsin ( M r 26 000) and a major trypsin ( K m 2.9 μM, M r 29 000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K m values) and a putative decreased affinity for PIs.

Isolation and characterization of an α-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment

A proteinase inhibitor with Mr 697 000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-α-benzoyl-dl-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an α-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate α-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II.

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.

L-Isoaspartate 115 of porcine beta-trypsin promotes crystallization of its complex with bdellastasin.

Bdellastasin is a 59-amino-acid, cysteine-rich, antistasin-type inhibitor of sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hirudo medicinalis. The complex formed between bdellastasin and porcine beta-trypsin has previously been crystallized in the presence of PEG in a tetragonal crystal form of space group P4(3)2(1)2 and has now been found to crystallize under high-salt conditions in the enantiomorphic space group P4(1)2(1)2. These structures have been solved and refined to 2.8 and 2.7 A resolution, respectively. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure. In the second new crystal form, the flexible N-terminal subdomain is rotated with respect to the C--terminal subdomain by about 90 degrees, fitting into a cavity formed by symmetry-related trypsin molecules. The canonical inhibitor-proteinase interaction is restricted to the primary binding loop comprising residues Leu31-Lys36 of bdellastasin. During the refinement, a bound sodium ion occupying the calcium-binding site of the porcine beta-trypsin component was discovered. This sodium ion is coordinated in a tetragonal-pyramidal manner, with the geometry of the enclosing loop slightly changed compared with the loop in the presence of calcium. In the crystal form of space group P4(3)2(1)2, the electron density for residue 115 of porcine beta-trypsin clearly indicates the presence of a beta-isomerized L-aspartic acid, which is placed in spatial proximity to segment Thr144--Gly148 of a symmetry-related trypsin molecule. This is the first structurally observed example of an L-isoaspartate in beta--trypsin originating from Asn. A comparison with other known crystal structures of porcine beta-trypsin-macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step in crystallization.

Differential mechanisms for structural and functional alterations of trypsin by heparin, evidence for a specific, radical-generating mechanism at low heparin concentrations

The oxidative mechanism whereby heparin may interact with various proteins was investigated in detail in this work by addressing the role of doses of heparin on the nature and effects of its binding to bovine trypsin, taken as reference protein. Unfractionated heparin was used at concentrations ranging from 6 to 400 μg/ml with a fixed trypsin concentration (250 μg/ml). At concentrations of up to 60 μg/ml, equivalent to trypsin/heparin molar ratios of between 30 and 3, increasing inhibition of amidolytic activity and radical-dependent peptide bond cleavage of the enzyme was observed, with the appearance in the electrophoretic pattern of new bands of trypsin fragments to which heparin was demonstrated to be bound specifically. Structural modifications were also revealed by increases in fluorescence emission spectra. On the whole, however, the alterations induced by these heparin concentrations only involved a limited number of trypsin molecules. At concentrations from 120 to 400 μg/ml (equivalent trypsin/heparin molar ratios of 1.5–0.46), heparin binding to trypsin appeared to cause more profound and generalized alterations of enzyme structure and function, with dose-dependent quenching of fluorescence emission and almost complete loss of amidolytic activity, although evidence of radical production was lacking. Collectively, the results stress the crucial role of heparin dose on both the nature and effects of its binding to trypsin. The change in heparin effects which reflects distinct underlying molecular mechanisms occurs dramatically at a critical concentration threshold. While a specific, radical-generating mechanism operates at low concentrations, less specific ionic linkages, apparently independent of radical production, best explain the effects of high heparin concentrations.

Crystal structure at 1.63 Å resolution of the native form of porcine β-trypsin: revealing an acetate ion binding site and functional water network

The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine β-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 Å resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-H…O hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cβ-Oγ-His-57 Nϵ angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.

Crystal structure of a 16 kda double-headed bowman-birk trypsin inhibitor from barley seeds at 1.9 Å resolution

The Bowman-Birk trypsin inhibitor from barley seeds (BBBI) consists of 125 amino acid residues with two inhibitory loops. Its crystal structure in the free state has been determined by the multiwavelength anomalous diffraction (MAD) method and has been refined to a crystallographic R-value of 19.1 % for 8.0–1.9 Å data. This is the first report on the structure of a 16 kDa double-headed Bowman-Birk inhibitor (BBI) from monocotyledonous plants and provides the highest resolution picture of a BBI to date. The BBBI structure consists of 11 β-strands and the loops connecting these β-strands but it lacks α-helices. BBBI folds into two compact domains of similar tertiary structure. Each domain shares the same overall fold with 8 kDa dicotyledonous BBIs. The five disulfide bridges in each domain are a subset of the seven disulfide bridges in 8 kDa dicotyledonous BBIs. Two buried water molecules form hydrogen bonds to backbone atoms in the core of each domain. One interesting feature of this two-domain inhibitor structure is that the two P1 residues (Arg17 and Arg76) are approximately 40 Å apart, allowing the two reactive-site loops to bind to and to inhibit two trypsin molecules simultaneously and independently. The conformations of the reactive-site loops of BBBI are highly similar to those of other substrate-like inhibitors. This structure provides the framework for modeling of the 1:2 complex between BBBI and trypsin.

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.

Conformations of trypsin-bound amidine inhibitors of blood coagulant factor Xa by double REDOR NMR and MD simulations.

Double rotational-echo double resonance (double REDOR) has been used to investigate the bound conformations of (13)C,(15)N,(19)F-labeled factor Xa inhibitors to bovine trypsin. Carbon-fluorine dipolar couplings were measured by (13)C{(19)F} REDOR with natural-abundance background interferences removed by (13)C{(15)N} REDOR. The conformations of the bound inhibitors were characterized by molecular dynamics (MD) simulations of binding restrained by double REDOR-determined intramolecular C-F distances. A symmetrical bisamidine inhibitor and an asymmetrical monoamidine-monoamine inhibitor of the same general shape had distinctly different conformations in the bound state. According to the MD models, these differences arise from specific interactions of the amidine and amine groups with the active-site residues of trypsin and nearby water molecules.

Some haemato-immunological parameters in the mussel Perna perna

The aim of this study was to establish the normal pattern of some haemato-immunological parameters in the mussel Perna perna to serve as a reference for further studies using this species as a biological indicator of environmental contaminants. We investigated the morphology and cytochemistry of the circulating haemocytes of P. perna , their role in phagocytosis and superoxide anion production, their numbers and composition, the agglutinin concentration in the plasma and the presence of phenoloxidase activity. Two blood cell types, hyalinocytes (HY) and granulocytes (GR), were found in the haemolymph of P. perna . HY were agranular cells with a basophilic cytoplasm, whereas GR contained abundant eosinophilic granules. HY comprised approximately 60% of the blood cell types in the circulation. Both haemocyte types, but especially the GR were actively phagocytic and contained acid phosphatase. After stimulation with zymosan, the blood cells were able to reduce NBT to formazan, suggesting the generation of the superoxide anion. Glycogen was not histochemically detectable in either haemocyte type. The number of cells in circulation (THC) did not significantly vary with sex or size of the mussels (3,145·5 and 3,668·5 cells/mm3in males and females respectively). The mussel plasma had an agglutinating activity against different mammalian erythrocytes. A phenoloxidase activity was not encountered either in the haemocyte fraction or in the mussel plasma, even after induction with the protease trypsin or non-self molecules, such as LPS from Gram-negative bacteria and β-1, 3-glucans from fungi.

Interaction of second and third domains of Japanese quail ovomucoid with ten mammalian trypsins

Second and third domains were prepared from Japanese quail ovomucoid and association equilibrium constants, K as, were measured at 25°C and pH 8 for these domains with trypsins from ten mammalian species: cat, cow, dog, guinea pig, hog, horse, man, rabbit, rat, and sheep. The values ranged from 10 8 M −1 to 10 10 M −1 for the second domain–trypsin associations and from 10 6 M −1 to 10 8 M −1 for the third domain–trypsin associations. Changes in K a values for the interactions between the trypsins and each domain are attributed to slight changes in surface conformation caused by the residue changes in the inhibitor-binding region other than the S 1 pocket of the trypsin species. The representative of such residue changes is assumed to be the one observed at residue 217 of trypsin molecule. Concerning each trypsin, the K a value with the second domain was always higher than that with the third domain. However, the ratios between the two equilibrium constants varied from 3 to 60 depending upon trypsin species. This means that amino acid changes in enzyme-contact residues other than the P 1 site of the Kazal-type inhibitor can make it possible to recognize even a slight difference in inhibitor-binding surface among the enzymes with the same S 1 pocket and highly similar overall three-dimensional structure.

Anti-autolysis of Trypsin by Modification of Autolytic Site Arg117

In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30° and 70°C: T50 values were 59°C and 46°C, respective. EG trypsin's half-life of 25 min at 55°C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-l-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-l-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:366–373, 1998.

Unusual properties of thermally sensitive oligomer–enzyme conjugates of poly(N‐isopropylacrylamide)–trypsin

Reversible soluble–insoluble oligomer–enzyme conjugates have been prepared by conjugating a thermally sensitive oligomer, poly(N-isopropylacrylamide) [poly(NIPAAm)] to trypsin. The conjugates can catalyze enzymatic reactions in solution and then may be separated from the solution by thermal precipitation. One special feature of the conjugates is that every poly(NIPAAm) chain has only one end attachment to the enzyme, so that the loss of enzymatic activity due to steric hindrance should be minimized. Conjugates with various numbers of oligomer chains per trypsin molecule were prepared. Surprisingly, the conjugates increased in enzymatic activity with increasing oligomer conjugation to the native trypsin. The trypsin active sites in the conjugates were accessible to large molecules, such as soybean trypsin inhibitor (MW = 21,500). The enzyme conjugates were more stable than native trypsin, both in solution and in the precipitated phase. On the other hand, the conjugates lost enzymatic activity faster than native trypsin when the temperature was repeatedly cycled through the lower critical solution temperature (LCST) of the poly(NIPAAm). The recovery of the conjugates by thermal precipitation in each cycle was over 95% even after 14 cycles through the LCST. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 498–505, 1998.

Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin.

Reversible soluble-insoluble oligomer-enzyme conjugates have been prepared by conjugating a thermally sensitive oligomer, poly(N-isopropylacrylamide) [poly(NIPAAm)] to trypsin. The conjugates can catalyze enzymatic reactions in solution and then may be separated from the solution by thermal precipitation. One special feature of the conjugates is that every poly(NIPAAm) chain has only one end attachment to the enzyme, so that the loss of enzymatic activity due to steric hindrance should be minimized. Conjugates with various numbers of oligomer chains per trypsin molecule were prepared. Surprisingly, the conjugates increased in enzymatic activity with increasing oligomer conjugation to the native trypsin. The trypsin active sites in the conjugates were accessible to large molecules, such as soybean trypsin inhibitor (MW = 21,500). The enzyme conjugates were more stable than native trypsin, both in solution and in the precipitated phase. On the other hand, the conjugates lost enzymatic activity faster than native trypsin when the temperature was repeatedly cycled through the lower critical solution temperature (LCST) of the poly(NIPAAm). The recovery of the conjugates by thermal precipitation in each cycle was over 95% even after 14 cycles through the LCST.

High desorption—ionization yields of large biomolecules induced by fast C 60 projectiles

C 60 molecules accelerated to MeV energies (20 MeV) have been used to induce the desorption-ionization of large biomolecules from solid samples. For a sample consisting of trypsin molecules, the secondary molecular ion emission yield is about two orders of magnitude larger than with MeV atomic ions. This is a consequence of the very high energy density deposited in solids by 20 MeV C 60 projectiles that gives rise to a large amount of matter ejected after each impact. Although time-of-flight mass spectra can be recorded within a few seconds, it is more the acquisition of knowledge on mechanistic aspects, in comparison with other particle-induced desorption methods, which is the objective of obtaining these first results with energetic fullerenes.

Two Reactive Site Locations and Structure−Function Study of the Arrowhead Proteinase Inhibitors, A and B, Using Mutagenesis

The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges. Both their primary structures and cDNA sequences have been elucidated [Yang, H. L., Luo, R. S., Wang, L. X., Zhu, D. X., & Chi, C. W. (1992) J. Biochem. 111, 537; Xu, W. F., Tao, W. K., Gong, Z. Z., & Chi, C. W. (1993) J. Biochem. 113, 153; Luo, M. J., Lu, W. Y., & Chi, C. W. (1997) J. Biochem. (in press)]. Though they share 91% homology, they are different in inhibitory activities. Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing. To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78. Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one. The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed. The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed. As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors. The gene coding for inhibitor A, which for some reason could not be expressed in S. cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body. After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography. Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively. As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant. Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one. The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed.