Sequence Analysis and Biochemical Characteristics of Two Non-specific Lipid Transfer Proteins from Tartary Buckwheat Seeds.

INTRODUCTION: Plant non-specific lipid transfer proteins (nsLTPs) play an important role in plant resistance to various stresses, and show potential applications in agriculture, industrial manufacturing, and medicine. In addition, as more and more nsLTPs are identified as allergens, nsLTPs have attracted interest due to their allergenicity. Two nsLTPs from Tartary buckwheat have been isolated and identified. There is a need to study their biochemical characteristics and allergenicity. OBJECTIVE: The study aims to investigate the biochemical characteristics of two nsLTPs from Tartary buckwheat seeds and evaluate their potential allergenicity. METHODS: Two nsLTPs derived from Tartary buckwheat, namely FtLTP1a and FtLTP1b, were produced by gene cloning, expression, and purification. Sequence analysis and biochemical characteristics of the proteins, including lipid binding ability, α-amylase inhibition activity, antifungal activity, and allergenic activity, were investigated. RESULTS: High-purity recombinant FtLTP1a and FtLTP1b were obtained. FtLTP1a and FtLTP1b exhibited similar lipid binding and antifungal properties. Only FtLTP1b showed weak inhibitory activity against α-amylase. CONCLUSION: FtLTP1b could specifically bind IgE in the serum allergic to buckwheat and cross-react with pollen (w6). FtLTP1b is a novel allergenic member of the lipid-transfer protein 1 family found in Tartary buckwheat.

Regulating inhibitory activity of potato I-type proteinase inhibitor from buckwheat by rutin and quercetin.

This study aims to investigate the effects of two flavonoids, rutin and quercetin, on inhibitory activity of recombinant buckwheat trypsin inhibitor (rBTI). We found that rutin and quercetin could quench the florescence of rBTI through the static quenching process. We also observed that upon binding to rutin or quercetin, rBTI underwent conformational changes. The results also suggested that rutin and quercetin bind to two different sites on rBTI through different interactions: rutin binds to rBTI through van der Waals forces and hydrogen bonds, whereas quercetin binds through hydrophobic interactions. Rutin and quercetin also markedly deactivated the trypsin inhibitory activity (TIA) of rBTI, while quercetin exhibited higher inactivation effect on rBTI than rutin due to its structure. Finally, the molecular docking revealed the molecular binding between the flavonoids and rBTI. These findings can be useful for the understanding of how flavonoid affects the inhibitory of rBTI.

Formulation of a Self-Nanoemulsifying Drug Delivery System of Buckwheat Flavonoids and Evaluation of Its Antimicrobial Activity.

This study was aimed at constructing a self-nanoemulsifying drug delivery system of buckwheat flavonoids and evaluating its antimicrobial activity. The construction of the nanoemulsion followed a pseudo-ternary phase diagram, and its particle properties (particle size, zeta potential, and surface morphology) and physicochemical parameters (turbidity, surface tension, pH value, conductivity, encapsulation efficiency, and stability) were evaluated. The antimicrobial potential of buckwheat flavonoids nanoemulsion was determined against Staphylococcus aureus, Escherichia coli, and Candida albicans and compared to the buckwheat flavonoids suspension. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) exhibited that the antimicrobial activity of the nanoemulsions and suspension increased while enhancing the drug concentration, and the antimicrobial activity of nanoemulsion was significantly higher than that of the suspension against those three bacteria. Agar disc diffusion test demonstrated that the inhibition zone diameter of the suspension was about 50% of the nanoemulsion against three bacteria. The time killing assay indicated that the IC50 of the nanoemulsion was significantly lower than that of the suspension. These results indicate that nanoemulsion is a promising drug delivery system, which can improve the antimicrobial activity of buckwheat flavonoids.

Preparation, characterization, and evaluation of antioxidant activity and bioavailability of a self-nanoemulsifying drug delivery system (SNEDDS) for buckwheat flavonoids.

The self-nanoemulsifying drug delivery system has shown many advantages in drug delivery. In this study, a self-nanoemulsifying drug delivery system of buckwheat flavonoids was prepared for enhancing its antioxidant activity and oral bioavailability. A nanoemulsion of buckwheat flavonoids was developed and characterized, and its antioxidant, in vitro release, and in vivo bioavailability were determined. The nanoemulsion was optimized by the central composite design response surface experiment, and its particle size, polymer dispersity index (PDI), zeta potential, morphology, encapsulation efficiency, and stability were evaluated. The antioxidant activity was tested by measuring its 2,2-diphenyl-1-picrylhydrazyl scavenging activity, hydroxyl radical scavenging activity, and superoxide anion scavenging ability. In vitro release of buckwheat flavonoids nanoemulsion showed a higher cumulative release than the suspension, and the release fitting model followed the Ritger-Peppas and Weibull models. The effective concentration of the nanoemulsion was evaluated in vivo using a Wistar rat model, and the area under the plasma concentration-time curve of the buckwheat flavonoids nanoemulsion was 2.2-fold higher than that of the buckwheat flavonoid suspension. The Cmax of the nanoemulsion was 2.6-fold greater than that of the suspension. These results indicate that the nanoemulsion is a promising oral drug delivery system that can improve the oral bioavailability to satisfy the clinical requirements.

Recombinant buckwheat trypsin inhibitor decreases fat accumulation via the IIS pathway in Caenorhabditis elegans.

Buckwheat trypsin inhibitor (BTI) is a low molecular weight polypeptide that can help to prevent metabolic diseases such as obesity, hyperglycemia and hyperlipidemia. Herein, the effects of recombinant BTI (rBTI) on fat accumulation in Caenorhabditis elegans were studied. rBTI prevented fat accumulation under normal and high glucose conditions, and led to significantly shorter body widths without affecting C. elegans feeding behavior. Results also indicate that rBTI altered fat breakdown, synthesis, and accumulation by altering the transcription, expression and activity of key enzymes in lipolysis and fat synthesis. In daf-2 and daf-16 mutants, rBTI did not prevent fat accumulation, indicating that rBTI activity relies on the insulin/insulin-like growth factor (IIS) pathway. Overall rBTI may regulate changes in lipolysis and fat synthesis by down-regulating the IIS pathway, which can affect fat accumulation. These findings support the application of rBTI in preventing obesity, hyperglycemia and hyperlipemia.

Recombinant Buckwheat Trypsin Inhibitor Improves the Protein and Mitochondria Homeostasis in Caenorhabditis elegans Model of Aging and Age-Related Disease.

BACKGROUND: With the acceleration of aging process in human society, improvements of the physical functionality and life quality in the elderly population are more meaningful than pure longevity. Buckwheat trypsin inhibitor is a low molecular weight polypeptide extracted from buckwheat, which is a beneficial food for improving the health in the elderly. OBJECTIVES: The aim of the current study was to evaluate the potential beneficial effects of recombinant buckwheat trypsin inhibitor (rBTI) on age-dependent function decline and the primary mechanism. METHOD: Day 10 N2 Caenorhabditis elegans and day 6 AM140 C. elegans cultured at 25°C were used as models of aging and age-related disease, respectively. Motor function was as an indicator of age-dependent function. ATP content and damage mitochondrial DNA mass were detected to assess mitochondrial damage and function by ATP Assay Kit and agarose gel electrophoresis, respectively. Soluble protein content was quantified by SDS polyacrylamide gel electrophoresis. Autophagy-related genes transcription levels, autophagy marker proteins lgg-1, and lysosomal content were analyzed to quantify autophagy levels by qRT-PCR, transgenic C. elegans, and lysosomal staining. Autophagy inhibitor chloroquine, daf-16 mutant, and RNA Interference were used to determine the roles of autophagy and DAF-16 in rBTI-mediated effects. RESULTS: In this study, we found that rBTI could decrease the proportions of insoluble protein and impaired mitochondria, finally reduce motility deficits in both models. Further study indicated that rBTI activated the autophagy, and the inhibition of autophagy reduced rBTI-mediated beneficial effects. Genetic analyses showed the transcriptional activity of DAF-16 was increased by rBTI and was required for rBTI-mediated beneficial effects. CONCLUSIONS: These data indicated that rBTI might promote the autophagy to alleviate the age-related functional decline via DAF-16 in C. elegans and suggested a potential role of rBTI as a nutraceutical for the improvement of age-related complications.

Inhibitory site of α-hairpinin peptide from tartary buckwheat has no effect on its antimicrobial activities.

Antimicrobial peptides (AMPs) are known to play important roles in the innate host defense mechanisms of most living organisms. Protease inhibitors from plants potently inhibit the growth of a variety of pathogenic bacteria and fungi. Therefore, there are excellent candidates for the development of novel antimicrobial agents. In this study, an antimicrobial peptide derived from tartary buckwheat seeds (FtAMP) was obtained by gene cloning, expression and purification, which exhibited inhibitory activity toward trypsin. Furthermore, the relationship between the antimicrobial and inhibitory activities of FtAMP was investigated. Two mutants (FtAMP-R21A and FtAMP-R21F) were generated through site-directed mutagenesis. Inhibitory activity analysis showed that both FtAMP-R21A and FtAMP-R21F lost trypsin-inhibitory activity. However, FtAMP-R21A and FtAMP-R21F showed novel inhibitory activities against elastase and α-chymotrypsin, respectively, suggesting that Arg-21 in the inhibitory site loop is specific for the inhibitory activity of FtAMP against trypsin. Antimicrobial assays showed that all three peptides exhibited strong antifungal activity against Trichoderma koningii, Rhizopus sp., and Fusarium oxysporum. These results showed that the changes in FtAMP inhibitory site have no effect on their antifungal properties.

rBTI reduced ß-amyloid-induced toxicity by promoting autophagy-lysosomal degradation via DAF-16 in Caenorhabditis elegans.

Alzheimer's disease (AD) is an age-related neurodegenerative disease, of which ß-amyloid (Aß) induced toxicity was suggested as a main cause. Some substances with prolongevity effects have been shown to be protective against AD. In a previous study we demonstrated that a recombinant buckwheat trypsin inhibitor (rBTI) could prolonge the lifespan in Caenorhabditis elegans (C. elegans). Here, we investigated whether rBTI may benefit to mitigate the AD symptom by feeding the AD model C. elegans CL4176. CL4176 is a transgenic C. elegans expressing human Aß3-42 in muscle tissue. The results showed that rBTI not only could extend lifespan but also could reduce Aß toxicity-triggered body paralysis in AD worms. Further study found the accumulation of Aß was decreased and autophagy-lysosomal degradation pathway was activated in AD worms treated with rBTI. Moreover, the inhibition of autophagy reduced rBTI-mediated paralysis delay. Genetic analyses showed rBTI increased the transcriptional activity of dauer formation abnormal-16 (DAF-16) and the disruption of daf-16 abolished rBTI-mediated protective effect in AD worms. Taken together, these data indicated that rBTI promoted the autophagy-lysosomal degradation pathway to reduce the Aß-induced toxicity via DAF-16 in an AD model C. elegans, implying that BTI has the potential to protect against AD.

Peptides from sesame cake extend healthspan of Caenorhabditis elegans via upregulation of skn-1 and inhibition of intracellular ROS levels.

The peptides from sesame cake (PSC) which are the main by-product of agricultural processing of sesame were prepared. To evaluate benefits of PSC for health and longevity, antioxidant activity and anti-aging effects were studied in vitro and in a Caenorhabditis elegans (C. elegans) model system. PSC exhibited antioxidant activity in vitro, and induced beneficial effects on lifespan and several health parameters of C.elegans, including pharyngeal pumping rate, locomotion and lipofuscin accumulation. In a mev-1 mutant, PSC increased lifespan, and it enhanced oxidative stress tolerance in wild-type nematodes. After treatment with PSC, SOD activity, GSH content, and GSH/GSSG ratio were increased, leading to low intracellular ROS levels in C. elegans. PSC up-regulated skn-1 mRNA, and its target gene gcs-1, and abolished the extension of lifespan in skn-1 mutant, indicating that PSC-mediated longevity is dependent on activation of the skn-1/Nrf-2 transcription factor. Current results warrant research into the use of PSC as nutraceuticals for overall health improvement.

Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

rBTI extends Caenorhabditis elegans lifespan by mimicking calorie restriction.

Buckwheat trypsin inhibitor (BTI) is a low molecular weight polypeptide extracted from buckwheat. This study examined the effects of BTI on the lifespan of Caenorhabditis elegans (C. elegans) and investigated the mechanism involved. Our results showed that recombinant BTI (rBTI) extended life expectancy by mimicking calorie restriction (CR) in C. elegans. rBTI promoted formation of reactive oxygen species (ROS) via increasing respiration, induced activities of ROS defense enzymes by activating DAF-16, and increased oxidative stress resistance and survival rates. The inhibition of ROS signal by antioxidants reduced rBTI-mediated longevity by up to 65%. Moreover, it was shown that the disruption of daf-2 abolished the extension of the lifespan and the increased ROS. Taken together, these data indicate that rBTI-mediated longevity mimics CR by down-regulating insulin/IGF-1 signaling (IIS) pathway, implying that BTI has the potential to be a novel anti-aging drug.

Protective effects of the active fraction from the tuber of Scirpus yagara in mouse endotoxin shock model.

ETHNOPHARMACOLOGICAL RELEVANCE: Scirpus yagara Ohwi is a perennial, aquatic plant, whose dry tubers have long been used as Traditional Chinese Medicine (TCM) "Sanleng" for the treatment of postpartum abdominal pain, hyperemesis gravidarum, amenorrhea, dyspepsia and several inflammatory related diseases. Although it is known to have anti-inflammatory activities, its mechanism of action on lipopolysaccharide (LPS)-induced inflammation has not yet been identified in detail.This study was designed to investigate the anti-inflammatory activity of the active fraction (AF) from the tuber of Scirpusyagara both in vitro and in vivo. MATERIALS AND METHODS: RAW264.7 macrophage was incubated for 16h with 1µg/ml of LPS in absence or presence of AF (0, 10, 50 and 100µg/ml) and the secretions of tumor necrosis-alpha (TNF-α) and interleukin-6 (IL-6) in the medium were determined by using an enzyme-linked immunosorbent assay (ELISA). In the in vivo study, mice were orally administrated with AF (50 and 300mg/kg) for three days consecutively. 1h after the last AF administration, the mice were intraperitoneally injected with LPS (15mg/kg), and the life span of LPS-challenged mice were determined. Furthermore, the levels of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were measured using ELISA kit, and histological change in lungs was examined by light microscopy. Additionally, the components of AF were analyzed by high performance liquid chromatography (HPLC) using a C18 column. RESULTS: AF significantly decreased TNF-α and IL-6 production induced by LPS in RAW264.7 macrophage. In LPS-induced mouse endotoxin shock model, AF pre-treatment significantly improved the survival rate of mice. And LPS-induced increases of pro-inflammatory cytokines TNF-α and IL-6 in the serum, lung and liver were markedly suppressed by AF. Moreover, the histopathological examination indicated that AF could significantly attenuate lung tissues injury in endotoxemic mice. In addition, eight compounds (protocatechuic acid, vanillic acid, p-coumaric acid, ferulic acid, methyl-3,6-dihydroxy-2-[2-(2-hydroxyphenyl)-ethynyl] benzoate, sciryagarol I, sparstolonin B, SanLeng diphenyllactone) of AF were quantified by HPLC analysis. CONCLUSIONS: These results suggested that AF protected mice against LPS-induced lethality by inhibiting the production of multiple cytokines and organ dysfunction. Thus AF may prove beneficial in the prevention and treatment of endotoxin shock.

Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis.

Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells.

Sparstolonin B suppresses lipopolysaccharide-induced inflammation in human umbilical vein endothelial cells.

Sparstolonin B (SsnB) is an isocoumarin compound isolated from the tubers of both Sparganium stoloniferum and Scirpus yagara. We previously demonstrated that SsnB blocked the Toll-like receptor (TLR) 2- and TLR4-triggered inflammatory signaling in macrophages by inhibiting the recruitment of MyD88 to the TIR domains of TLR2 and TLR4. The present study was designed to examine the effects of SsnB on vascular inflammatory responses in human umbilical vein endothelial cells (HUVECs) challenged by lipopolysaccharide (LPS, a TLR4 ligand). We found that SsnB dose-dependently attenuated the LPS-induced expression of interleukin (IL)-1ß and monocyte chemoattractant protein 1 both at the transcription and translation levels in HUVEC. LPS-induced endothelial cell adhesion molecules, intercellular adhesion molecular-1 and vascular cell adhesion molecule-1 expressions were also reduced by treatment with SsnB. In addition, co-incubation with SsnB attenuated THP-1 monocyte adhesion to LPS-activated HUVECs. Furthermore, SsnB efficiently suppressed LPS-induced phosphorylation of extracellular -signal-regulated kinase (Erk1/2) and Akt in HUVECs. These findings show that SsnB can suppress endothelial cell inflammation, suggesting that SsnB might be suitable for development as a therapeutic agent for inflammatory cardiovascular disease.

Bioactive cis-stilbenoids from the tubers of Scirpus yagara.

Two new cis-stilbenoids, sciryagarol I (1) and II (2) were isolated from the EtOAc extract of the tubers of Scirpus yagara, together with four known compounds. The structures of all compounds were determined by comprehensive analyses of their spectroscopic data and comparison with literature information. The compounds 3, 4 and 6 were isolated for the first time from this genus. Some compounds were tested for their cytotoxicity against human tumor cell lines and antimicrobial activity. Compounds 1-4 showed significant cytotoxicity against the Hela cell lines with IC(50) values ranging from 7.21 to 61.21µM. 1 and 2 exhibited some antimicrobial activity against Staphylococcus aureus and Candida albicans with uniform MICs of 79.3µl/ml for 2, and 152µl/ml for 1, respectively.

[Anticancer activity of Crocodylus siamensis blood in vitro and in vivo].

OBJECTIVE: To study the anticancer effects of the blood of Crocodylus siamensis in vitro and in vivo. METHODS: The inhibitory effects of serum and plasma of Crocodylus siamensis on proliferation of HepG2, BGC823, HeLa and SKOV3 cell were measured by MTT assay. The mouse S180 tumor model was used to evaluate the anti-tumor effect in vivo. RESULTS: High dosage serum and plasma of Crocodylus siamensis could inhibit the proliferation of HepG2, BGC823, HeLa and SKOV3 cell. The tumor inhibitory rate of high dosage blood of Crocodylus siamensis on S180 tumor was up to 57.55%. CONCLUSION: The blood of breeding Crocodylus siamensis has anticancer activity.

Preparation and properties of rutin-hydrolyzing enzyme from tartary buckwheat seeds.

A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37°C for 30min in the presence of 20% ethanol, and its Km value for rutin is 1.04×10(-3)M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by (1)H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.

Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb.

Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named F1, F2, F3, and F4, respectively. Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb. According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3). Following purification by Ni(2+) affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient). Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg(139) and Asp(144) might be involved in the allergic activity of TBb. However, R141A had the weakest IgE-binding activity, suggesting that Arg(141) may be the critical amino acid of TBb. This is the first report on the epitope mapping and identification of TBb. Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.

[Preparation and identification of the rat antibody against recombinant buckwheat trypsin inhibitor].

AIM: To prepare the rat antibody against the recombinant buckwheat trypsin inhibitor. METHODS: The recombinant buckwheat trypsin inhibitor was expressed in E.coli. BL21 (DE3) and used as an immunogen to immunize the rat. The titer and specificity of the anti-BTI antibody from the rat were analyzed by ELISA, Western blot and immunohistochemistry, respectively. RESULTS: ELISA detection indicated the titer of the antiserum was about 1:128 000. Western blot analysis showed the antibody reacted specifically with rBTI. The immunohistochemistry analysis proved that rBTI was expressed in the cytoplast and nucleus of EC9706 cells. CONCLUSION: The rat antibody against rBTI has high titer and specificity, which is beneficial to further study on the molecular mechanisms in rBTI-induced apoptosis of tumor cells and also provide an important theory basis for further exploring the relationship between the structure and function of BTI.

Purification and biochemical characterization of a novel allergenic protein from tatary buckwheat seeds.

An allergenic protein from tatary buckwheat seeds, designated TBt, was purified by salting out, anion exchange chromatography and size exclusion chromatography (SEC). Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SEC showed that the allergenic protein was composed of two subunits linked by disulfide bonds, which we named TBa and TBb. TBa, TBb and TBt have estimated molecular weights of 24, 34 and 56 kDa, and isoelectric points of 6.85, 5.85 and 7.00, respectively. The immunological characteristics of the three proteins as analyzed by enzyme-linked immunosorbent assay revealed that TBa and TBb have comporable IgE binding activity, both higher than that of TBt. Determination of sulfhydryl (-SH) and disulfide (-S-S-) groups in TBt showed that most cysteine residues exist as disulfide linkages rather than free -SH groups. We calculated that TBt has an -S-S- content of 1.978 M/M protein, indicating that TBt contains one disulfide bond and could be the precursor of a 24 kDa allergenic protein from tatary buckwheat seeds.