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.     

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.     

Stability of the allergenic soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.     

Reversible denaturation of the soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.     

A soybean trypsin inhibitor. Crystallization and x-ray crystallographic study

Five trypsin and alpha-chymotrypsin inhibitors which have low molecular weights (ranging from 6800 to 8600) and are present in soybean seeds of the Tracy variety have been isolated and purified, and single crystals which give x-ray diffraction data beyond 3-A spacings have been obtained from one of them. The trypsin inhibitor crystallizes in a monoclinic unit cell of symmetry P2(1) and dimensions a = 25.919(7) A, b = 43.23(1) A, c = 19.905(5) A, and beta = 103.63(2) degrees. The assymmetric unit contains 1 molecule of molecular weight 6800. The crystal, which has been found to be unusually stable to x-radiation, has solvent content of approximately 26% by volume.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor-trypsin complex.

The hydrogen exchange kinetics of the complex of trypsin-soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins.     

Studies on soybean trypsin inhibitors. X. Isolation and partial characterization of four soybean double-headed proteinase inhibitors

Four Bowman-Birk type double-headed inhibitors (B, C-II, D-II, and E-I) were isolated from soybeans. Inhibitor B was different from Bowman-Birk inhibitor only in chromatographic behavior. One mole of C-II inhibited one mole each of bovine trypsin and bovine alpha-chymotrypsin, probably at the same site, and porcine elastase at another reactive site. In the ordinary assay system D-II and E-I inhibited only trypsin activity at a non-stoichiometric inhibitor-enzyme ratio of 1:1.4, and the complexes had rather high dissociation constants. These inhibitors were all inactive toward subtilisin BPN'.