Bowman-Birk Soybean Proteinase Inhibitor Research Articles

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to (31)P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).

Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity

Here we investigated encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and between the bilayers. The biological activity (antitrypsin and antichymotrypsin) assay of the protein entrapped in liposomes showed its active sites were spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI confirmed this hypothesis and suggested that this shielding is reversible. The liposomes stability in three various media-artificial gastric juice and intestinal fluids was also examined. The liposomes prepared seem to be promising formulations for BBI and BPTI delivery.

Amphiphilic poly-N-vinylpyrrolidone nanocarriers with incorporated model proteins

New nanoscaled polymeric carriers have been prepared onthe basis of different amphiphilic water-soluble derivatives ofpoly-N-vinylpyrrolidone (PVP). The polymer self-assembly and interaction with model proteins(Bowman–Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) werestudied in aqueous media. The possibility of inclusion of both BBI and hydrophobized oleicacid derivatives of BBI in amphiphilic PVP aggregates was investigated. It was ascertainedthat polymeric particles of size 50–80 nm were formed in certain concentrations ofamphiphilic PVP and poorly soluble dioleic acid derivatives of BBI. Such polymericaggregates are capable of solubilization of dioleoyl BBI with a concomitant prevention of itsinactivation at low pH values.

Encapsulation of Bowman-Birk soybean proteinase inhibitor within zwitterionic phospholipid multilamellar vesicles

In the present document we describe the possibility for encapsulating a water-soluble protein lacking α-helices into multilayer liposomes made of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) in order to prepare proteoliposomes. The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) into liposomes was studied. Increase in ionic strength did not inhibit the proteoliposome formation process. BBI encapsulation did not affect liposome sizes, surface charge and structural phospholipid organization. Confocal laser scanning microscopy showed that BBI was located in the central part of the spherical particle and between bilayers. The fluorescence intensity after anthracene-labelled phospholipids interaction with Rhodamin B-labelled BBI confirmed that the protein is deeply immersed into liposomes. The biological activity (antitrypsin and antichymotrypsin) estimation of the protein entrapped in liposomes showed that the protein active centers are spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI confirms this hypothesis and suggests that this shielding is reversible. The proteoliposomes prepared seem to be promising formulations for BBI delivery.

Interaction of native Bowman—Birk soybean protease inhibitor and its hydrophobized derivative with multilamellar vesicles of soybean phospholipids

The interaction of native Bowman-Birk soybean protease inhibitor (BBI) and its hydrophobized derivative with multilamellar vesicles of various soybean phospholipids was investigated. Decrease in pH and introduction of negatively charged components to the lipid mixture increased BBI content in the protein-lipid complex. This suggests a contribution of electrostatic forces in the protein-lipid interaction. Protein hydrophobization insignificantly influenced BBI binding to lipids. In the complex with lipids, both proteins (BBI and its hydrophobized derivative) retained high anti-chymotrypsin activity (75-100%), which was not influenced by the presence of the ionic detergent sodium deoxycholate.

Effect of membranotropic and mucoadhesive formulations of protein proteinase inhibitors on bovine herpes virus-1 reproduction

The lipidized derivatives of Bowman–Birk soybean protease inhibitor (BBI) containing one to three oleoyl groups were synthesized and characterized. The (ole) 1- and (ole) 2BBI were demonstrated to have 200- and 100-fold higher uptake into Caco-2 cell monolayers compared to native BBI. The acylated BBI had increased affinity to elastase-like proteases. Aprotinin-loaded starch/bovine serum albumin microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Various formulations of protein proteinase inhibitors were tested for their influence on BHV-1 reproduction in cell cultures. Native aprotinin possessed palpable dose-dependent effect inhibiting the virus reproduction up to 4.0 lg (10,000-fold). The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective decreasing virus infectious titer up to 4.0 lg and delaying the cytopathic effect up to 144 h in lesser doses of aprotinin. The lipophilic derivative (ole) 1BBI was shown to exhibit effective inhibition (>100-fold) of BHV-1 reproduction unlike native BBI.

Self-assembling systems based on amphiphilic poly-N-vinylpyrrolidone and their interaction with model proteins.

Polymeric particles formed by stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of M(n) = 2600 were obtained in aqueous solution, and their shape and size distribution were characterized. The size of the particles was shown to decrease with an increase in the ionic strength of the solution. Interaction of PVP-stear and its aggregates with model proteins (Bowman-Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) was studied. The possibility of inclusion of both native BBI and oleoylic derivative of BBI in the PVP-stear polymeric aggregates was investigated. It was established that polymeric particles with a diameter of 30 nm formed under certain concentration ratios between PVP-stear and poorly soluble dioleoyl BBI are capable of solubilization of dioleoyl BBI as well as prevention of its inactivation at low pH values.

Study of antiproteinase activity of acylated derivatives of Bowman-Birk soybean proteinase inhibitor.

The effect of acylation of Bowman-Birk soybean proteinase inhibitor (BBI) by derivatives of various unsaturated fatty acids on inhibition of trypsin, alpha-chymotrypsin, and human leukocyte elastase was investigated. Inhibition (K(i)) and kinetic (k(ass), k(diss)) constants of interaction between proteases and acylated BBI derivatives were determined. For mono-, di-, and triacylated BBI derivatives, insertion of two oleic residues into the BBI molecule was demonstrated to be more potent for exhibiting antiproteinase activity.

Potential of block copolymer- and immuno-conjugates for tumor-targeted delivery of Bowman–Birk soybean proteinase inhibitor

The present work reports the effect of conjugation of the anticarcinogenic and antitumor soybean Bowman–Birk protease inhibitor (BBI) with amphiphilic block copolymer of ethylene oxide and propylene oxide (PEO-PPO) as well as with monoclonal antibody via clinical dextran (D) on tumor-targeted delivery of BBI.

Acylation of Bowman-Birk soybean proteinase inhibitor by unsaturated fatty acid derivatives.

A procedure was developed for acylation of Bowman-Birk soybean proteinase inhibitor (BBI) by N-hydroxysuccinimide esters of oleic, linoleic, and alpha-linolenic acids in a dimethyl sulfoxide-dioxane-pyridine mixture. BBI derivatives containing two acylated amino groups were prepared with high yield. The use of the reversible modifier citraconic anhydride in the first stage of synthesis permitted the synthesis of hydrophobized BBI derivatives retaining high antitrypsin and antichymotrypsin activities. It was found that the insertion of two long-chain moieties in the BBI molecule decreases its thermostability.

Conjugation of the Bowman-Birk soybean proteinase inhibitor with hydroxyethylstarch.

The classical Bowman-Birk soybean proteinase inhibitor was modified by hydroxyethylstarch. The modified inhibitor retained the capacity for simultaneous binding of trypsin and human leukocyte elastase. The inhibition constants, Ki, of bovine trypsin, alpha-chymotrypsin and human leukocyte elastase (HLE) increased not more than 10-, 1.5-, and 20-fold, respectively, on modification of the inhibitor. The less effective inhibition is presumably due to the steric hindrance brought about by the conjugation with polysaccharide molecules. The results obtained indicate the pronounced structure differences of the binding regions for trypsin and chymotrypsin/leukocyte elastase in the modified preparation.

Synthesis and biodistribution of Bowman-Birk soybean protease inhibitor conjugate with amphiphilic polyester.

The modification of Bowman-Birk soybean protease inhibitor (BBI) with the monoaldehyde derivative of block copolymer of ethylene oxide and propylene oxide (PE), M(r) 2,000 is described. The conjugate contains five covalently bound polymer chains per protein molecule, and retains the ability to inhibit trypsin and chymotrypsin-like proteinases. The distribution of native BBI and the BBI-PE conjugate was examined in mice. After i.v. injection of [125I]BBI and [125I]BBI-PE, both inhibitors distributed very rapidly to the liver, kidney, and lungs, and more slowly to the brain. At the same time-points (up to 24 h), radioactivity in the blood and organs of mice injected with modified inhibitor was higher than that of the native inhibitor. The blood concentration time profile following i.v. administration of two BBI preparations at a dose 3 mg/kg was reasonable well described by a two-compartment open model with first-order elimination kinetics. The total clearance of BBI-PE decreased by a factor of 8, body mean residence time increased by a factor of 5 in comparison with BBI. A physiological pharmacokinetic model was developed to describe the tissue-to-blood distribution of two inhibitors. One-compartment physiological organ model (flow limited) was used to describe of time-course profiles of BBI concentration in organs. A two-compartment physiological organ model (membrane limited) was used to predict tissue-to-blood distribution of conjugated BBI in some organs of mice (liver, lungs). The predicted concentration curves of BBI and BBI-PE in blood and organs in mice (with the exception of kidney) showed good agreement with the observed values.

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds

Three fenugreek inhibitors (TFI-A8, TFI-N2, and TFI-B2) were isolated from an inhibitor preparation by anion exchange chromatography and subsequent preparative isoelectric focusing using immobilized pH gradients and the canal technique. The purified inhibitors inhibited the enzymes tested differently: TFI-A8 exhibited a high inhibition of trypsin (8.2 mg human trypsin/mg and 8.1 mg bovine trypsin/mg) and a very low inhibition of chymotrypsin (0.8 mg human chymotrypsin/mg and 1.0 mg bovine chymotrypsin/mg). TFI-N2 inhibited the four enzymes to about the same extent (5.0 mg/mg human and 4.1 mg/mg bovine trypsin; 4.9 mg/mg human and 3.7 mg/mg bovine chymotrypsin). TFI-B2 displayed a high inhibition of trypsin (7.5 mg/mg human and 5.1 mg/mg bovine) and a low inhibition of chymotrypsin (1.8 mg/mg human and 1.9 mg/mg bovine). On average, the human enzymes were inhibited better than the bovine ones by the purified inhibitors. The inhibitors contained high amounts of cystine (five or six disulfide bridges per molecule), aspartic acid, threonine, serine and proline, no valine and methionine and two of them also no tryptophan. Their molecular masses were about 6 kDa. Their inclusion into the Bowman-Birk soybean proteinase inhibitor family is discussed.

Purification and characterization of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

A proteinase inhibitor was purified from the seeds of faba bean (Vicia faba L.) to an electrophoretically homogeneous protein by extraction, salting-out, and column chromatographies on CM-Sephadex C-25, Sephadex G-75, and QAE-Sephadex A-25. The inhibitor (FBPI) was a basic protein with a molecular weight of 7000 and an isoelectric point of 8.7. The amino acid composition had a fairly high content of half-cystine and lacked of methionine, isoleucine, and tryptophan. FBPI inhibited bovine trypsin and α-chymotrypsin in a 1:1 (m/m) stoichiometry: the Ki’s were 6.1 × 10 9 m and 4.4 × 10 8 M, respectively. The inhibitor was stable in acidic and neutral pH, but it lost its activity upon heat treatment at alkaline pH. These properties indicate that FBPI belongs to the Bowman-Birk soybean proteinase inhibitor family.

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors.

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.

Nanomolar concentrations of Bowman-Birk soybean protease inhibitor suppress x-ray-induced transformation in vitro.

Experiments reported here indicate a crude soybean extract, if defatted with acetone, effectively blocks cell transformation in vitro. An active component of this crude extract is the Bowman-Birk trypsin and chymotrypsin inhibitor. The chymotrypsin-inhibitory region of the Bowman-Birk inhibitor is responsible for suppressing in vitro transformation. Another low molecular weight soybean trypsin inhibitor does not significantly suppress transformation. The Bowman-Birk inhibitor (i) has an irreversible effect on the transformation process, (ii) can suppress radiation-induced transformation even when added to cultures many days after the carcinogen exposure, and (iii) is effective in its ability to suppress transformation when present in the medium at a concentration as low as 0.125 nM.

Lima bean proteinase inhibitor. Origins of circular dichroism bands and modification by Br2- and (CNS)2-.

Origins of CD bands in lima bean proteinase inhibitor were deduced from an acetylation-deacetylation study of the sole tyrosyl residue in the protein (Tyr 69), and by analogy with Bowman-Birk soybean proteinase inhibitor, a homologous protein with similar spectral properties. Tyr 69 is relatively inaccessible to N-acetylimidazole; 100-fold molar excess of the reagent in the presence of 6 M guanidine hydrochloride elicited about 70 to 80% O-acetylation. A broad negative CD band centered around 280 nm arises mainly from the longest wavelength transition of cystinyl side chains (epsilon L--epsilon R approximately equal to -0.8 M-1 cm-1 per disulfide). The second cystinyl transition gives rise to a positive CD band of a comparable intensity at 247 nm. The Lb vibronic transition of Tyr 69 has negative CD around 280 nm, contributing approximately 10% of the total CD intensity at 278 nm (epsilon L--epsilon R approximately equal to -0.5 M-1 cm-1). The 232 nm positive shoulder is from the La vibronic transition of Tyr 69. Radical anions, Br2- and (CNS)2-, generated by the irradiation of N2O-saturated inhibitor solutions containing KBr or KCNS, reduced tyrosyl CD without affecting disulfide CD bands, indicating that the radical anions damaged Tyr 69 without altering protein conformation. The inhibitor modified at Tyr 69 by Br2- and (CNS)2- retained full activity toward trypsin and chymotrypsin. The irradiation of the inhibitor in the air-saturated solution led to loss in tyrosyl as well as cystinyl CD bands and decline in both antiproteinase activities.

γ Irradiation of Bowman-Birk Soybean Proteinase Inhibitor

WANDELL, J. L., AND KAY, E. 7 Irradiation of Bowman-Birk Soybean Proteinase Inhibitor. Radiat. Res. 72, 414-426 (1977). Radiation damage to Bowman-Birk soybean proteinase inhibitor was studied in dilute aqueous solutions containing: (a) air, (b) N20 plus KBr, and (c) NO20 plus KCNS. The degradation of tyrosines, monitored by changes in ultraviolet absorption and circular dichroism (CD) spectra of irradiated inhibitor, led to the loss of chymotrypsininhibitory activity without affecting antitrypsin activity. Of two tyrosyl residues in the inhibitor, Tyr 45, located next to the antichymotrypsin site, was shown to be essential to chymotrypsin-inhibitory activity. Radiation damage to Tyr 59 had no effect on either of the antiproteinase activities. The loss of trypsin-inhibitory activity paralleled decrease in disulfide CD. The breakage of disulfide bonds was confirmed by the p-chloromercuribenzoate assay for sulfhydryl groups. However, the exact role of disulfide bonds in antitryptic activity, other than the maintenance of conformational integrity, is not apparent. It seems more likely that the loss of trypsin-inhibitory activity is due to damage in other amino acid (s), although the degradation of tyrosine (2 residues) and histidine (1 residue) did not affect anti

Studies on soybean trypsin inhibitors. IV. Complete amino acid sequence and the anti-proteinase sites of Bowman-Birk soybean proteinase inhibitor.

Bowman-Birk inhibitor was partially hydrolyzed with chymotrypsin [EC 3. 4. 4. 5] at pH 3.0, and the subsequent reduction, S-carboxymethylation and gel filtration of the product yielded two fragments. One fragment containing a methionine residue was further degraded into two fragments with cyanogen bromide. By the amino acid determination and the terminal sequence analysis of these three fragments as well as of the whole protein, the tryptic peptides were aligned into the complete amino acid sequence of the inhibitor. The anti-chymotrypsin and the anti-trypsin sites of the inhibitor were also identified by the limited hydrolysis with these proteinases. The sequence of this inhibitor was found to be very similar to that of lima bean inhibitor IV (Tan and Stevens, 1971), and consisted of two homologous peptide regions, one includes the inhibitory site for trypsin [EC 3. 4. 4. 4] and the other that for chymotrypsin. Comparison with other leguminosae inhibitors was also discussed.

Scanning Density Gradient Isoeiectric Separation of Proteins on a Microscale

Abstract An attachment for the Linear Transport System of the Gilford 2000 recording spectrophotometer is described which allows in situ continuous scanning of protein zones subjected to isoelectric focusing in sucrose gradients. Microgram quantities of protein samples can be analyzed in a short time (1.5 to 3 hr) in a quartz microcolumn. Resolution of separated components is excellent. Application of the method to the isoelectric separation of the commercially available proteins ovalbumin, beta lactoglobulin, myoglobin, Bowman-Birk soybean protease inhibitor, and rabbit gamma globulins is reported. The method provides for scanning at different wavelengths for differentiation of colored proteins, or proteins lacking tryptophan. The described procedure offers an opportunity for the studying of the kinetics of isoelectrofocusing in sucrose gradients.