Two simple quantitative assays for trypsin inhibitors using immobilized trypsin.

Two new methods for quantitative assay of trypsin inhibitors, suitable for large numbers of samples, are described. The assay methods use trypsin-Sepharose conjugates incorporated into agarose gel slabs. Trypsin inhibitors are allowed to diffuse into, or are electrophoretically moved through, the slabs, and the consequent areas of inactivation of immobilized trypsin are visualized using a histochemical enzyme substrate. Quantitation of the trypsin inhibitor content of samples can be made on the basis of the inactivated areas. The limit of detection is 1–2 μg of soybean trypsin inhibitor and determinations are reproducible to 10% or better. Measured trypsin inhibitor contents of several legume species and varieties agree with spectrophotometric determinations.     

Characterization of agarose-bound trypsin.

Agarose-bound trypsin (EC 3.4.21.4) was prepared and its properties were compared with those of soluble trypsin. The bound form of the enzyme was found to be equally available to large and small molecular weight substrates as the soluble form. In addition, the bound form of the enzyme showed the same specificity towards protein substrates as the soluble enzyme. However, the agarose-bound trypsin showed greater stability than the soluble trypsin to denaturing conditions for prolonged period of time.     

Adenylate cyclase stimulation by trypsin.

Adenylate cyclase activity of a rat embryo fibroblast cell line (F111) is markedly increased by brief treatment with 1:300 trypsin. The degree of stimulation depends upon the length of time the cells are treated and the concentration of trypsin. Crystalline trypsin produced a stimulation similar to that obtained with 1:300 trypsin. Further, the addition of soybean trypsin inhibitor blocked the stimulation of adenylate cyclase by 1:300 trypsin. Trypsin-treated adenylate cyclase responds to PGE1, but there is no increase over that of untreated enzyme. This result and the increase in fluoride-stimulated levels of activity suggest that the trypsin is acting upon the catalytic unit of the enzyme.     

Coaggregate of trypsin and chymotrypsin

The method of chemical aggregation of enzymes has the advantage of yielding an immobilized enzyme preparation wherein reactor volume can be significantly reduced because of the absence of an inert carrier. A coaggregate of trypsin and chymotrypsin formed by extensive cross-linking with glutaraldehyde is described. A significant property of this aggregate is the reduced autolysis of the trypsin component of the coaggregate.     

13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

NMR studies of the complex between trypsin and soybean trypsin inhibitor with 1-¹³C-arginine and modified inhibitor with 1-¹³C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsin/¹³C-Lys-inhibitor at pH 6.5 and 8.1 and for trypsin/¹³C-Arg-inhibitor at pH 5.0. At pH 7.1 for trypsin/¹³C-Arg-inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor.     

Expression and purification of a cold-adapted group III trypsin in Escherichia coli

The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.     

Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.     

Synthesis and characterization of a water-soluble affinity polymer for trypsin purification

A specific ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine, a strong trypsin inhibitor, and acrylamide in the absence of oxygen. Kinetic studies on the trypsin inhibition revealed that there was a strong binding between this enzyme and the polymer and the mechanism was of a competitive manner with an inhibition constant of 0.6 x 10(-3)M. Such an affinity polymer was also very effective in preventing trypsin from auto-digestion at 4 degrees C.Based on this finding and the principle of cross flow filtration, a new process has been developed for purification of trypsin from a solution containing chymotrypsin. The experimental data indicated that trypsin was bound to the polymer (MW > 10(5)) and remained in the retentate while unbound chymotrypsin was collected in the filtrate. This purification process has a capability of recovering 98% pure trypsin at 90% yield.     

Effect of alpha2 macroglobulin on some kinetic parameters of trypsin.

1. Complex formation of trypsin with alpha2 macroglobulin results in marked changes of the Michaelis-Menten constant, pH optimum and sensitivity to ionic strength in a system using N-carbobenzoxy-glycylglycyl-L-arginine-2-naphthylamide as substrate. 2. In contrasts to the inhibition (50%) observed when alpha2 macroglobulin-bound trypsin is assayed under conditions optimal for the free enzyme, there is minimal reduction of activity when determinations are performed at a substrate concentration and pH optimal for the bound enzyme. 3. The changes in substrate concentration and ionic environment required for maximum activity of alpha2 macroglobulin-bound trypsin are similar to those observed with enzymes embedded in polyelectrolyte matrices and may reflect alterations in the microenvironment of the enzyme resulting from conformational changes of the macromolecule during interaction with trypsin. 4. Enzymatic activity of trypsin towards casein is greatly reduced by alpha2 macroglobulin, even under assay conditions optimal for the bound enzyme, confirming previous findings that access to the active center for high-molecular weight substrates is sterically hindered by alpha2 macroglobulin.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Some peculiarities of trypsin and heparin interaction]

The interaction of trypsin with an acid polysaccharide, heparin, at pH 4.2 and 8.0 is studied. Heparin is found to destabilize the enzyme under condition of both autolytic denaturation (pH 8.0) and thermoinactivation (pH 4.2). Data on trypsin inactivation kinetics suggest that the stage of forming molecular complexes with different contents of trypsin and heparin precedes the stage of the enzyme denaturation. Maximal trypsin inactivation rate takes place under equimolar enzyme:heparin ration.     

Mechanism of gold nanoparticles-induced trypsin inhibition: a multi-technique approach

The binding interactions of gold nanoparticles with trypsin were investigated using multi-spectra methods and molecular modeling. The experiment data showed that trypsin modified the surface of gold nanoparticles. The fluorescence intensity of trypsin was quenched by gold nanoparticles that strongly associated with protein and induced the inhibition of enzyme activity. The electrostatic and hydrophobic interactions were the primary contributors to the binding forces between trypsin and gold nanoparticles. The covalent interactions might be also involved in the binding process. The modeling calculated results indicated that the binding site was near to the primary substrate-binding pocket and the active site of the enzyme substrate. This work elucidated the interaction mechanism of trypsin with gold nanoparticles from the theoretical and experimental angle.     

Radioimmunoassay of human plasma trypsin.

A radioimmunoassay has been developed for the determination of human trypsin (3.4.21.4) in plasma. It allows the measurement of trypsin concentration in spite of the presence of plasma or pancreatic inhibitors. The human trypsin used as a standard and for labelling was isolated from pancreatic tissue and purified by affinity chromatography. The antiserum was obtained from guinea-pigs immunized with partially purified human trypsin. In the radioimmunoassay, the values of trypsin in serial dilutions of plasma were parallel to those of the standard curves. The assay was shown to be reproducible, sensitive and specific. However, the two antisera used did not distinguish between the enzyme and its proenzyme. In normal subjects, plasma values were found to be around 400 ng/ml. They were 10-40 times higher in patients with acute pancreatitis. The method appears to be much more specific for the diagnosis of acute pancreatitis than the current determinations of amylase and lipase activity.     

Kinetics of trypsin inhibition by its specific inhibitors

The kinetics of inhibition of trypsin by its specific inhibitors, pancreatic trypsin inhibitor, ovomucoid trypsin inhibitor, and soybean trypsin inhibitor, has been studied by following the hydrolysis of benzoylarginine ethyl ester in the presence of the inhibitor, and the results have been analyzed with the method described previously [Tian & Tsou (1982) Biochemistry 21, 1028]. The results obtained are consistent with the following: (a) The enzyme binds with the pancreatic inhibitor irreversibly to form an inactive complex. (b) The binding with the ovomucoid inhibitor to form the inactive complex is reversible. (c) An intermediate is formed before the relatively stable inactive complex with the soybean inhibitor, and both steps are reversible. The respective microscopic rate constants are determined by suitable plots of the apparent rate constants under different substrate and inhibitor concentrations. The second-order rate constants for the initial binding step thus obtained are in accord with the apparent inactivation rate constants determined by measuring the activity remaining with a stopped-flow apparatus equipped with a multimixing system after the enzyme-inhibitor mixture has been incubated for different time intervals.     

Specific modification of fatty acid synthetase from lactating rat mammary gland by chymotrypsin and trypsin.

Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme.     

Calcium-binding constants of trypsin and trypsinogen. A reassessment.

The Ca2+-binding constants for trypsin and trypsinogen have been reassessed by using enzyme that has been purified by affinity chromatography and measuring the distribution of 45Ca2+ between the protein and a cation exchanger. The pKCa2+ value of 4.5 for the high-affinity site on trypsin was 1 logarithmic unit greater than that previously reported.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

Preparation and properties of trypsin from the pyloric caeca of Pacific Ocean salmon

Trypsin from pyloric caeca of Pacific salmon was purified by affinity chromatography of the water extract on hexamethylenediamine-glycidylmethacrylate-cellulose. A protein band with a molecular weight of 22.5 kDa was found on SDS-electrophoresis in PAG. The protein band was homogeneous according to isoelectrofocusing in PAG (pI 4.0). The amino acid composition of the enzyme is typical of trypsin anionic forms; the major difference from the cationic forms is the lower content of lysine. The differences in properties caused by change of the enzyme molecule charge are similar to those observed in cationic trypsin when the lysine epsilon-amino groups of the latter are modified (change of pI, shift of the pH-optimum towards basic values, increase of stability to autolysis). Some natural trypsin inhibitors of the different origin suppressed the enzyme activity of trypsin from Pacific salmon in typical stoichiometric ratios. An unusual interaction of the enzyme with the specific inhibitor N-L-tosyl-L-lysine chloromethyl ketone was observed.     

Activation of calmodulin-dependent NAD+ kinase by trypsin

Sea urchin egg NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23), a calmodulin-dependent enzyme, can be activated by a moderate treatment with trypsin in a similar fashion to calmodulin. Stimulation by trypsin is dependent on its concentration (half-maximal dose: 1.5 microgram/ml) but independent of the presence of calcium. This suggests that limited proteolysis is able to activate NAD+ kinase as described for several other calmodulin-activated enzymes and that these enzymes may interact with calmodulin in a similar way.     

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.