Exploring bovine pancreatic trypsin inhibitor phase transitions

This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the T(L-L) boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the T(L-L) boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 A). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation.     

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.     

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.     

Purification and some properties of a protein inhibitor of sucrose synthetase from ripening wheat grain

A heat-stable protein with a molecular mass of 30 kDa inhibiting the reverse reaction of sucrose synthetase has been isolated from ripening wheat grain and purified to the homogeneous state by the methods of gel filtration on Sephacryl S-200, ion-exchange chromatography on DEAE-cellulose 52, and chromatofocusing in a pH 6–4 gradient. The influence of this protein on K_M for sucrose has been shown which is expressed in a decrease in the affinity of the substrate for the enzyme. An inverse correlation has been found between the activity of sucrose synthetase and the inhibitor in the process of ripening of the wheat grain. On the basis of the results obtained, a hypothesis has been put forward of the participation of the inhibitor protein in the regulation of sucrose synthetase in the ripening of wheat grain.M. A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Kazakh SSR Academy of Sciences, Alma-Ata. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 381–383, May–June, 1989.     

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.     

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.     

Composition of lipids of the grain of opaque-2 maize

It has been established that the change in the composition of the total lipids and the fall in the fatty acid content depend on the initial maize genotype. A decrease in the amount of unsaponifiable substances in the grain of mutants is accompanied by qualitative changes in the composition regardless of the nature of the genotype.Scientific-Research Institute of Biology Dnepropetrovsk State university. Translated from Khimiya Prirodnykh Soedinenii. No. 3. pp. 360–363. May–June, 1988.     

Absorption spectra of the grain of maize with different phenotypes

Scientific-Research Institute of Biology, Dnepropetrovsk State University. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 858–859, November–December, 1991.     

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.     

Hydrophobic properties of maize zein

The proteins of the zein complex from maize endosperm have been studied with the aid of hydrophobic chromatography. Their best separation was achieved on a column with TSK gel HW-65f. By comparing the results of fractionation by hydrophobic chromatography with those of electrophoresis, it was found that electrophoresis in PAAG under denaturing conditions separates the zein protein into groups according to their surface hydrophobicity. The most hydrophobic is the high-molecular-mass group of zein polypeptides.V. R. Vil'yams Kazakh Scientific-Research Agricultural Institute, Alma-Ata Province. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 105–108, January–February, 1991.     

Stabilization of phospholipase C fromClostridium perfringens by conjugation with a trypsin inhibitor

A new approach for the stabilization of phospholipase C by conjugation with a proteinase inhibitor is proposed. A heat-stable fraction obtained from a conjugate retained its initial activity for an hour at an incubation temperature of 50°C. An appropriate method of conjugation using an affinity adsorbent preventing the appearance of inactive forms of the conjugate has been developed.Mirzo Ulugbek Tashkent State University, fax (3712) 46 24 72. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 115–117, January–February, 1998.     

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.     

Structural analysis of the N-linked oligosaccharides from human urinary trypsin inhibitor.

The N-linked oligosaccharides from human urinary trypsin inhibitor were purified and their structures were investigated by compositional analysis, the two-dimensional sugar map method and 500 MHz 1H-NMR. The results revealed that they were composed of disialosyl, monosialosyl and asialosyl oligosaccharides, which have the common biantennary core structure; Gal1-4GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)M an1-4GlcNAc1-4GlcNAc.