Effect of polysaccharides on the stability and renaturation of soybean trypsin (Kunitz) inhibitor

Thermal denaturation and renaturation of soybean trypsin (Kunitz) inhibitor (STI) were studied by high-sensitivity differential scanning calorimetry in the presence of polysaccharides (dextran, ι- and κ-carrageenans, gum arabic, pectins and dextran sulfate). This study was carried out under conditions of both thermodynamic incompatibility and complex formation of STI and polysaccharides. The presence of polysaccharides did neither influence the denaturation temperature nor the denaturation enthalpy of STI under conditions of their incompatibility with the protein. No polysaccharide (except gum arabic) affected the ability of STI to renature and recover its inhibitory activity after thermal denaturation. At acidic pH values, the protein was shown to form electrostatic complexes with pectins and dextran sulfate. Substantial destabilisation of STI bound to dextran sulfate was observed. In the case of STI/pectin complexes, either a decrease or increase in the stability of STI was observed depending on the complex composition and esterification degree of pectin. The mechanism behind the changes in stability of STI bound to the polysaccharide matrix is discussed. Thermal denaturation of STI in complexes with dextran sulfate and pectin was completely irreversible. This observation indicates a possibility of suppressing antinutritional activities of trypsin inhibitors in soy products.

Schematic presentation of the denaturation of a protein (P) bound to a polymer matrix (M): (A) loose protein occupancy, (B) dense protein occupancy.     

Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods.

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughout. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 microns). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-microns preparative column, led to the conclusion that the overloaded 20 microns column gave a higher throughput than the 40-microns column.     

Uncertainty analysis of the microtiter plate method for determining trypsin inhibitor activity

Protease inhibitor activity is frequently measured in legume seeds as protease inhibitors are thought to have anti-nutritional as well as anti-carcinogenic properties. Trypsin inhibitor activity (TIA) can be measured using different methods. The microtiter plate method is very convenient and routinely used; therefore, in this study, we analyzed the measurement uncertainty of the microtiter plate method to understand what affects the measurement results, as well as to compare TIA values obtained by similar and different methods. For uncertainty analysis of TIA measurement, we used the soybean variety ‘Vojvodjanka,’ which is known to have TIA greater than 80 trypsin units inhibited (TUI) per mg of seed. We followed the Guide to the Expression of Uncertainty in Measurement (GUM) for our uncertainty analysis of the microtiter plate method for TIA testing, which we present in the form of an uncertainty budget. Absorbance measurement and preparation of sample reaction mixture took the largest percent (71 %) of overall uncertainty of TIA value. The TIA of soybean variety ‘Vojvodjanka’ was (94.1 ± 8.4) TUI/mg, and this result is consistent with those obtained by other authors. The microtiter plate method is a reliable method for TIA measurement, making seed quality testing more efficient.     

Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein

We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.     

The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein

The pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor, is one of the most extensively studied globular proteins. It has proved to be a particularly attractive and powerful tool for studying protein conformation as well as molecular bases of protein/protein interaction(s) and (macro)molecular recognition. BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase-like serine (pro)enzymes endowed with very different primary specificity. BPTI reacts rapidly with serine proteases to form stable complexes, but the enzyme: inhibitor complex formation may involve several intermediates corresponding to discrete reaction steps. Moreover, BPTI inhibits the nitric oxide synthase type-I and -II action and impairs K+ transport by Ca2+-activated K+ channels. Clinically, the use of BPTI in selected surgical interventions, such as cardiopulmonary surgery and orthotopic liver transplantation, is advised, as it significantly reduces hemorrhagic complications and thus blood-transfusion requirements. Here, the structural, inhibition, and bio-medical aspects of BPTI are reported.     

Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles

Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH 8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50 cm bare fused-silica capillary was employed for obtaining adequate separations in about 12 min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples.     

Spectral-fluorescent properties of maize grain trypsin inhibitor

The trypsin inhibitor isolated from maize grain has been investigated by the methods of IR, UV, fluorescence, derivative, and differential spectroscopy. Its spectral characteristics have been determined and the influence of the temperature and of a detergent on the structure of the protein has been studied.Scientific-Research Institute of Biology, Dnepropetrovsk State University, Dnepropetrovsk. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 375–381, May–June, 1989.     

Total synthesis of Trichosanthes trypsin inhibitor and its analogue

Trichosanthes trypsin inhibitor (TTI) is a peptide consisting of 27 amino acid residues with three pairs of disulfide bonds. This paper reports the total synthesis and disulfide bond refolding of this inhibitor and its analogue. After purification, the amino acid sequence and stoichiometrical inhibitory activity against trypsin of the synthetic inhibitor were compatible with those of the natural inhibitor. The analogue of this inhibitor in which residue Met in position 6 was replaced by Ala was also synthesized. The antitrypsin activity of this synthetic analogue was also approximate to that of the natural inhibitor.     

Variety characteristics of soybean seeds in relation to protein and oil contents and activities of proteinase inhibitors

The protein and oil contents and the activity of proteinase inhibitors in six varieties of soybean have been studied. It has been found that the specific amidase activity of trypsin inhibitors ranges from 170 to 320 nominal units. Electrophoretic results indicates the presence in the water-soluble fraction of seven or eight components possessing inhibitor activity in relation to trypsin and chymotrypsin.     

Variety characteristics of soybean seeds in relation to protein and oil contents and activities of proteinase inhibitors

The protein and oil contents and the activity of proteinase inhibitors in six varieties of soybean have been studied. It has been found that the specific amidase activity of trypsin inhibitors ranges from 170 to 320 nominal units. Electrophoretic results indicates the presence in the water-soluble fraction of seven or eight components possessing inhibitor activity in relation to trypsin and chymotrypsin.Institute of Plant Physiology and Biophysics, Academy of Sciences of the Tadzhik SSR, Dushanbe. Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 761–765, November–December, 1984.     

Predigestion of soybean proteins with immobilized trypsin for infant formula

Soybean protein isolates of low soybean trypsin inhibitor (STI) residue were prepared by acidic precipitation of soybean flour water extracts (0.8–1.2%) at pH 5.0, followed by acidic washing at this pH and affinity adsorption of residual STI with immobilized trypsin on polystyrene anion-exchange resin GM 201. After heat treatment, soybean protein isolates were subjected to controlled hydrolysis with the immobilized trypsin. Then, the predigested soybean protein was prepared. The predigested soybean protein was free of STI activity, and its solubility at acidic pH range was greatly increased. Sedimentation test showed that it formed a much finer clot at pH 4.5 than that of untreated soybean protein. The pepsin digestibility index at pH 4.0 and chymotrypsin digestibility index at pH 8.0 were obviously improved. These results suggested that the predigested soybean protein prepared by this method may be used in infant formulas.     

Phenolic compounds and the antioxidant activity of the bran,flour and whole grain of different wheat varieties

Total phenolic content and DPPH radical scavenging capability of the bran layer, flour made from endosperm and whole grain of wheat were determined. Fifteen different wheat samples of ten spring and five winter wheat varieties were analyzed. The spring wheat varieties were grown in both conventional and organic conditions. The total phenolic content of the bran layer found to be the highest (1258-3157 μg/g), followed by that of grains (168 - 459 μg/g) and the lowest of flour (44 - 140 μg/g). The bound phenolic acids were quantified by CE-DAD analysis after alkaline hydrolysis. Ferulic acid was a major compound among phenolic acids found in wheat varieties.     

Biodiesel from soybean oil,castor oil and their blends

Even not being described in the EN 14112 standard, PDSC has been used for the determination of the biodiesel oxidative stability, by OIT and OT measurements. In this study, biodiesel blends were obtained by mixing soybean (BES) and castor (BEM) ethyl esters and its induction periods were measured by Rancimat and PDSC. The blends (BSM _X ) showed intermediate values of OSI, OT, and OIT, compared with BES and BEM. Although, the molar fraction of the components varied linearly in BSM _X , OSI, OT, and OIT values increased exponentially in relation to the castor biodiesel amount in the blends. Introduction of castor oil biodiesel increased the blend stability, so the BSM₃₀ blend reached the OSI limit of 6 h. OSI, OIT, and OT showed a high-linear correlation, pointing out that PDSC can be used in the analysis of this kind of biodiesel, with a smaller sample and analysis time, as compared to Rancimat. The use of biodiesel blends was a good alternative in the correction of the oxidative stability of the final product without the need of antioxidant addition.     

13C spin relaxation studies of the basic pancreatic trypsin inhibitor

The longitudinal ¹³C spin relaxation times T₁ and the ¹³C{¹H} nuclear Overhauser enhancement were measured in a concentrated aqueous solution of the basic pancreatic trypsin inhibitor. The correlation time for overall rotational motions of the basic pancreatic trypsin inhibitor molecules was found to be τ_R ≈ 2 × 10^?8 s. In connection with previous ¹H n.m.r. studies of intramolecular motions of the aromatics, we were particularly interested in the correlation times τ_G for intramolecular segmental motions of the aromatic rings. The present experiments revealed no manifestation of intramolecular motions of the aromatics, indicating that τ_G ? 2 × 10^?8 s for the aromatic ring carbon atoms. On the other hand, rapid segmental motions were evidenced for the peripheral carbon atoms of aliphatic amino acid sidechains. Comparison of the ¹H and ¹³C n.m.r. data on the basic pancreatic trypsin inhibitor indicates that the time scale of high resolution ¹H n.m.r. at high fields may in many instances be more appropriate for studies of the molecular dynamics in globular proteins than the time scale of spin relaxation measurements.     

Membrane affinity chromatography used for the separation of trypsin inhibitor.

Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability.     

Affinity purification of urinary trypsin inhibitor from human urine

Affinity protocols for the purification of urinary trypsin inhibitor (UTI) were developed. To imitate the substrate/inhibitor-binding domain (S1 domain) of trypsin and chymotrypsin, the key amino acid residues were composed to sorbents. The sorbents were then subjected to adsorption analysis with UTI. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. The purified enzyme was subjected to SDS-PAGE, trypsin inhibitor activity and peptide map fingerprinting analysis. As calculated, the theoretical maximum adsorption (Q(max)) of two affinity sorbents entitled as S-D-G and S-S-G were 31.7 and 30.1 mg/g, respectively; the desorption constants K(d) of the two sorbents were 8.9 and 18.6 μg/mL, respectively. After the separation of UTI with S-D-G and S-S-G, reducing SDS-PAGE analysis revealed that the protein was a single polypeptide with the mass of ~66 kDa, and the purified proteins were ~95 and 97% pure, respectively; the band on gel was further confirmed with peptide map fingerprinting analysis. Protein and bioactivity recoveries were 1.3 and 75.9% with S-D-G, 1.0 and 70.2% with S-S-G, respectively.     

HPLC-method for the determination of a low molecular weight trypsin inhibitor in urine

Summary A method to analyse a low molecular weight trypsin inhibitor (UTI-L, apparent molecular weight: 6200) in human urine is described. The procedure consists of an enrichment step and a separation by RP-HPLC. The urine sample with aprotinin as internal standard is adjusted to pH 8.3 and percolated through a trypsin-Sepharose 4B column; the inhibitors are eluted with acid solution. This eluate is analysed by RP-HPLC with ultraviolet detector (200 nm). Starting with a known sample volume (ca. 200 ml) the detection limit of the whole analytical procedure is 0.02 IU _bapna 1^–1 of urine.

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HPLC-Methode zur Bestimmung eines niedermolekularen Trypsin-Inhibitors in Harn