Pepsin Inhibitor Research Articles

Molecular cloning, expression and characterization of aspartyl protease inhibitor from Ancylostoma ceylanicum.

Aspartyl protease inhibitors (APIs) from parasitic intestinal nematodes are highly immunogenic and have been suggested as potential vaccine antigens. Ac-API-1 from Ancylostoma caninum showed strong immunogenicity and its polyclonal antibodies could specifically recognize the excretory/secretory products of adult worms. However, little is known about molecular characteristics and biological function of API from Ancylostoma ceylanicum (Ace-API). In this study, the Ace-API mature peptide coding sequence was cloned and expressed, and molecular characteristics of its full length sequence were analyzed. Ace-API cDNA was 684bp in length, which encoded 228 amino acids. The similarity of the Ace-API amino acid sequence to Ac-API-1 and Adu-API-1 was 96.93% and 96.49%, respectively, and they clustered together in the phylogenetic tree. Escheria coli-expressed recombinant protein was mainly soluble in the supernatant of bacterial cell lysate. Western blot showed that Ace-API protein had good reactivity to the serum of infected dogs. Pepsin inhibition assay revealed that the recombinant protein had inhibitory activity on pepsin. Immunofluorescence results demonstrated that Ace-API was mainly localized to the epidermis, excretory glands, and pseudocoelomic fluid of the adult. Using the quantitative real-time PCR, the expression of Ace-api mRNA in adults was significantly higher than that in the third stage (L3) larvae. Together, these data indicate that Ace-API is secreted extracellularly by the parasite, and might play a role in protecting the parasite against the proteolytic digestion by the host proteases, which stimulate further studies to explore this protein as a potential hookworm vaccine candidate.

Evaluation of the Inhibitory Effect of a Medicinal Herb <i>Phyllanthus amarus</i> on the Activity of <i>α</i>-Amylase, Pepsin and Trypsin

The medical herb Phyllanthus amarus play a crucial role in indigenous medicine. Therapeutically, these plants’ extract acts as potential players that inhibits several digestive enzymes that are relevant to the management of Peptic ulcers and Diabetes Mellitus, which occur due to the overproduction of such enzymes. Evaluation of inhibitory effect of this extract was carried out against Pepsin, α-amylase, Trypsin enzymes along with the effect of thermal stability and ammonium sulphate precipitation on these inhibitory assays. P. amarus leave’s extract with different concentration gradients were used in this research analysis. Results obtained along with the literature analysis revealed photochemical compounds such as polyphenols causes inhibitory nature in the extract. Maximal percentage of inhibition of amylase, pepsin and trypsin were found to be 71% (0.32 mg/ml), 85% (0.08 mg/ml) and 87% (1.28 mg/ml) respectively. In thermal stability assay the maximum percentage of inhibition for amylase, pepsin and trypsin was observed at 30% (80°C), 68% (4°C) and 5% (37°C). Enzymes inhibitory assays on ammonium sulphate precipitation elicited maximum percentage of inhibition for amylase, pepsin and trypsin as 42% (at 45% of (NH4)2SO4), 58% (at 15% of (NH4)2SO4) and 40% (at 30% of (NH4)2SO4) respectively. This research concluded that Phyllanthus amarus leave extracts are potential inhibitors of α-amylase, pepsin and trypsin enzymes. Ammonium sulphate precipitation was helpful to purify the polyphenols the active compounds to a good extend. Also, thermal stability was helpful to check the stability of these active photochemical compounds present in the extract. Thus, P. amarus is an effective inhibitor to be used as supplements in the disease management.

Investigation on the Interaction Behavior Between Oenothein B and Pepsin by Isothermal Titration Calorimetry and Spectral Studies.

Oenothein B (OeB) is a dimeric macrocyclic ellagitannin isolated from Herbs and fruits that have a variety of biological activities. In order to better understand the effect of OeB on the activity of the digestive enzyme pepsin, interactions between OeB and pepsin were investigated in vitro under simulated physiological conditions based on enzyme inhibition studies, fluorescence, isothermal titration calorimetry, CD, and molecular docking. It was found OeB is an effective inhibitor of pepsin, likely acting in a reversible manner through both competitive and noncompetitive inhibition. Fluorescence quenching of pepsin by OeB was a static quenching. CD spectra showed the addition of OeB causes the main chain of pepsin to loosen and expand and the partial β-sheet structure to be converted to a disordered structure. Isothermal titration calorimetry and docking studies revealed the main binding mechanism of OeB and pepsin was through noncovalent interactions, hydrophobic interactions with OeB and the internal hydrophobic group of pepsin, and then hydrogen bonding between OeB and the Val243 and Asp77 residues of pepsin. Noncovalent bonds between OeB and pepsin change the polarity and structure of enzymes, decreasing enzymatic activity. Compared with small molecular polyphenols, OeB has a weaker hydrophobic interaction with pepsin and less effect on the secondary structure of pepsin. These findings are the first direct elucidation of the interactions between the oligomer ellagitannin OeB and pepsin, further contributing to understanding binding between oligomer ellagitannins and digestive enzymes. PRACTICAL APPLICATION: The results of this study indicate that the interaction between OeB and pepsin has a certain inhibitory effect on pepsin. In order to reduce the impact of OeB on human digestion and its own activities, nano-encapsulation technology can be used in the future to protect oligomeric ellagitannin such as OeB.

A randomised study of evaluation of antiulcerogenic activity of quercetin in rats

Background: Peptic ulcer is very common disease. Peptic ulcer results probably due to an imbalance between aggressive factors (acid, pepsin, H. pylori) and defensive factors (gastric mucus, prostaglandins and bicarbonate secretion). Whatever may be the cause of peptic ulcer, it is the gastric acid that prevents ulcer healing and maintain the ulcer. Therefore, most of the drugs available for treatment of peptic ulcer either neutralize the secreted acid or decrease the acid secretion.Methods: The study was conducted on albino rats (Wistar) of 200-250g and maintained under standard conditions (room temperature 24-27oC and humidity 60-65%) with 12h light and dark cycle. The food in the form of dry pellets (Amrut Lab., hyd) and water were available ad libitum. The animal experiments were approved by the ethics committee of the institute.Results: In pyloric ligation model, the rats pre-treated with quercetin showed highly significant protection (p <0.001) when compared to control group and significant protection when compared to ranitidine pre-treated group (p <0.05). In indomethacin model, both quercetin and ranitidine pre-treated groups showed significant protection when compared to control group (p <0.01).Conclusions: In conclusion, it appears that quercetin possess anti-ulcerogenic principles like flavanoids, phenolic compound and caratinoid. These phytoconstituents provides protection against gastric mucosal damage induced by pylorus ligation, aspirin and ethanol, through inhibition of gastric acid, pepsin, histamine and free radical and stimulation of mucus secretion.

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D.immitis. We aimed to fractionate and identify adult D.immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D.immitis secretome. The most immunoreactive proteins were between 10 and 48kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.

Management of acute upper gastrointestinal bleeding

The medical management of patients with acute upper gastrointestinal bleeding includes both diagnostic and therapeutic procedures. Emergency or early endoscopy has greatly improved the diagnosis, but it has not yet changed the therapeutic approach very much. The main emphasis is still on close observation of the patients in order to give blood transfusions when necessary and to identify those who need urgent operation. Most patients stop bleeding spontaneously, but the frequency of rebleeding is high. Patients with continuous bleeding or rebleeding are candidates for emergency operation. Much effort has been made to prevent rebleeding because, if the patients can be operated on electively, the frequency of complications is lower and vagotomy may be preferable to resection. Several medical treatments including antacids, H2-receptor antagonists, pepsin inhibitors and specific antifibrinolytic agents have been tried, but their value is still sub judice. Curiously, methods of treatment that are clearly effective in healing peptic ulcers seem less effective in preventing rebleeding from eroded vessels within such ulcerations.

Recombinant Brugia malayi pepsin inhibitor (rBm33) exploits host signaling events to regulate inflammatory responses associated with lymphatic filarial infections

Prolonged existence of filarial parasites and their molecules within the host modulate the host immune system to instigate their survival and induce inflammatory responses that contribute to disease progression. Recombinant Brugia malayi pepsin inhibitor (rBm33) modulates the host immune responses by skewing towards Th1 responses characterized by secretion of inflammatory molecules such as TNF-α, IL-6, nitric oxide (NO). Here we also specified the molecular signaling events triggered by rBm33 in peripheral blood mononuclear cells (PBMCs) of filarial endemic normals (EN). rBm33 predominantly enhanced the levels of nitric oxide in cultured PBMCs but did not result in oxidative stress to the host cells. Further, rBm33 treatment of human PBMCs resulted in higher GSH/GSSG levels. MYD88 dependent activation was found to be associated with rBm33 specific inflammatory cytokine production. rBm33 triggered intracellular signaling events also involved JNK activation in host PBMCs. In addition, c-Fos and not NF-κB was identified as the transcription factor regulating the expression of inflammatory cytokines in rBm33 stimulated PBMCs. rBm33 marked its role in filarial pathology by altered levels of growth factors but did not have a significant impact on matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) activity of host PBMCs. Thus, the study outlines the signaling network of rBm33 induced inflammatory responses within the host immune cells.

Interaction mechanism of pepsin with a natural inhibitor gastrodin studied by spectroscopic methods and molecular docking

The interaction of gastrodin with pepsin has been investigated by enzyme activity assay, fluorescence, UV–Visible, circular dichroism spectra, and molecular docking. The pepsin activity results suggest that gastrodin is an inhibitor of pepsin. The fluorescence experiments show that gastrodin can quench the fluorescence of pepsin via a static quenching process. The thermodynamic analysis suggests that hydrophobic interaction is the main force between pepsin and gastrodin. UV–Visible and circular dichroism spectra studies suggest that the binding of gastrodin leads to a loosening and unfolding of pepsin backbone with partial α-helix being transformed into β-sheet. All these experimental results have been validated by docking studies, which further show that besides hydrophobic interaction, hydrogen bond also help stabilize the gastrodin–pepsin complex. The results reveal the potential to develop the natural compound gastrodin for the treatment of diseases related to the excessive activity of pepsin.

Heat Stable Protease Inhibitors from Sesbania grandiflora and Terminalia catappa

Aims: Protease inhibitors play a vital role in the regulation of protease activity and display promising therapeutic effects against diseases in humans. The aim of this study was to investigate protease inhibitory activities and their heat stabilities from parts of five medicinal plants. Study Design: Experimental. Place and Duration of Study: Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka between March 2012 and February 2013. Methodology: Water extracts were prepared using fresh plant parts of Mangifera zeylanica (MZ) bark, Sesbania grandiflora (SG) bark, flower, leaves and seeds, Terminalia bellerica bark and seed, Terminalia catappa (TC) bark, fruit and leaves and Terminalia chebula bark and seed. Percentage inhibitory activities of the extracts against pepsin and trypsin were measured. Final concentrations of the extracts used for pepsin and trypsin inhibitory assays were 0.2 and 0.13% respectively. Remaining inhibitory activities were measured after heating the extract at 60, 80 and 100°C up to 1 h. All the experiments were conducted in triplicate for three times. Results: Maximum pepsin inhibitory activity was detected in TC bark (98%) followed by SG bark (67%) and MZ bark (31%). Rest of the extracts showed 10 to 16% pepsin inhibition or no inhibition. Maximum trypsin inhibitory activity was detected in SG bark (95%) followed by TC bark (86%) and MZ bark (39%). Rest of the extracts showed 6 to 18% trypsin inhibition or no inhibition. Inhibitory activities of SG and TC barks remained when heated for 1 h at 60, 80 and 100°C. The maximum loss recorded was with the trypsin inhibitory activity (27% loss) when SG bark was heated at 100°C for 1 h. Conclusion: S. grandiflora and T. catappa barks demonstrated strong protease inhibitory activities which were heat stable. Further studies are necessary to isolate, characterize and elucidate the structures of these protease inhibitors.

Synthesis, characterization, and pepsin inhibition study of Au(III)–3-(2′-thiazolylazo)-2,6-diaminopyridine complex

Complex between Au(III) and 3-(2′-thiazolylazo)-2,6-diaminopyridine (TADAP) was synthesized, characterized and found to inhibit pepsin activities at the concentrations between 0–200μM. The IC50 value of the complex toward pepsin was 9.45μM. Our kinetic study suggested that Au(III)–TADAP complex could act as the uncompetitive inhibitor. These findings have provided the first information of Au(III)–TADAP complex as a protease inhibitor.

Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin

Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase.

Phenylpropanoid glycoside inhibition of pepsin, trypsin and α-chymotrypsin enzyme activity in Kudingcha leaves from Ligustrum purpurascens

Phenylpropanoid glycosides (PPGs) are important ingredients in Kudingcha bitter tea from Ligustrum purpurascens, possessing many beneficial properties. Here, we investigated the effect of PPGs on the activity of the digestive enzymes pepsin, trypsin and α-chymotrypsin. Furthermore, we explored the interactions between PPGs and digestive enzymes by a multi-spectroscopic method and docking studies. PPGs could inhibit digestive enzyme activities with a non-competitive pattern of inhibition. PPGs may have a binding effect on these digestive enzymes, changing the polarity and the structure of enzymes, which results in decreased enzyme activity. This description of the effects of PPG inactivation of pepsin, trypsin and α-chymotrypsin helps reveal the potential anti-nutritional property of PPGs.

Curcumin and Anthocyanin Inhibit Pepsin-Mediated Cell Damage and Carcinogenic Changes in Airway Epithelial Cells

Laryngopharyngeal reflux (LPR) is associated with inflammatory and neoplastic airway diseases. Gastric pepsin internalized by airway epithelial cells during reflux contributes to oxidative stress, inflammation, and carcinogenesis. Several plant extracts and compounds inhibit digestive enzymes and inflammatory or neoplastic changes to the esophagus in models of gastroesophageal reflux. This study examined the potential of chemoprotective phytochemicals to inhibit peptic activity and mitigate pepsin-mediated damage of airway epithelial cells. Cultured human laryngeal and hypopharyngeal epithelial cells were pretreated with curcumin (10 micromol/L), ecabet sodium (125 microg/mL), and anthocyanin-enriched black-raspberry extract (100 microg/mL) 30 minutes before treatment with pepsin (0.1 mg/mL; 1 hour; pH 7). Controls were treated with media pH 7 or pepsin pH 7 without phytochemicals. Cell damage and proliferative changes were assessed by electron microscopy, cell count, thymidine analog incorporation, and real-time polymerase chain reaction array. Pepsin inhibition was determined by in vitro kinetic assay. Micromolar concentrations of curcumin, ecabet sodium, and black-raspberry extract inhibited peptic activity and pepsin-induced mitochondrial damage and hyperproliferation. Curcumin abrogated pepsin-mediated depression of tumor suppressor gene expression and altered the subcellular localization of pepsin following endocytosis. Several phytochemicals inhibit the pepsin-mediated cell damage underlying inflammatory or neoplastic manifestations of LPR. Dietary supplementation or adjunctive therapy with phytochemicals may represent novel preventive or therapeutic strategies for LPR-attributed disease.

The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH2), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure.

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 103 and 6.52 × 103 M−1. The binding reactions were enthalpy driven with ΔHb values between −50.99 and −46.07 kJ mol−1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

Slow-tight binding inhibition of pepsin by an aspartic protease inhibitor from Streptomyces sp. MBR04

The present article reports a low molecular weight aspartic protease inhibitor from a Streptomyces sp. MBR04 exhibiting a two-step inhibition mechanism against pepsin. The kinetic interactions revealed a reversible, competitive, slow-tight binding inhibition with an IC50 and Ki values of 4.5nM and 4nM respectively. The conformational changes induced upon inhibitor binding to pepsin was monitored by far and near UV analysis, demonstrated that the inhibitor binds to the active site and causes inactivation. Chemical modification of the inhibitor with WRK and TNBS abolished the antiproteolytic activity of the inhibitor.

Recombinant Brugia malayi pepsin inhibitor (rBm33) induced monocyte function and absence of apoptotic cell death: An in vitro study

The effect of recombinant Brugia malayi pepsin inhibitor (rBm33) on human monocytes/macrophages has been examined using THP-1 cells. THP-1 cells stimulated with rBm33 showed enhanced levels of expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and diminished levels of IL-12, iNOS and anti-inflammatory cytokine (IL-10) expression suggesting the predominant features of Th1 response. Phorbol-12-myristate-13-acetate (PMA) treated THP-1 cells stimulated with rBm33 and subsequent incubation with GFP expressing Escherichia coli (E. coli) for 2 h enhanced the uptake of E. coli. Nitric oxide (NO) levels measured in the supernatants of these cultures did not show significant changes. Apoptotic studies with Peripheral Blood Mononuclear Cells (PBMCs) from normal individuals stimulated with rBm33 did not induce apoptosis of monocytes or lymphocytes. These observations suggest that rBm33 stimulates macrophages to induce Th1 response and does not promote apoptosis.

Purification of a Novel Pepsin Inhibitor from Coriolus versicolor and its Biochemical Properties

A novel pepsin inhibitor was isolated from Coriolus versicolor. The purification was carried out by a 2-step ultrafiltration followed by DEAE-52 and Mono Q ion-exchange chromatography. SDS-PAGE and gel filtration chromatography analysis showed that the isolated inhibitor was a 22.3 kDa protein with a single subunit. Heat stability of this inhibitor was estimated and only 7% of its inhibitory activity lost after treatment at 98 °C. The inhibitor was more specific against pepsin than several other proteases. The dissociation constant (K(i)) and concentration required for 50% pepsin inhibition (IC50) were 5.84 × 10(-5) M and 26.26 μg/mL, respectively. Apparent decrease of α-helix and increase of random coil were observed in the circular dichroism spectra of pepsin when an equimolar amount of the inhibitor was added. The inhibition mechanism of this inhibitor differs from the reported aspartic protease inhibitors, according to the secondary structure and the kinetic studies of this inhibitor.

Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin1

Phytic acid (IP(6)) and myo-inositol phosphate esters (IP(1-5)), including IP(5) isomers prepared chemically and enzymatically with bacterial and fungal phytases, were examined for their effects on protein aggregation of soy protein and β-casein, interaction with Fe(3+), and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP(1-6)) on the 2 proteins decreased dramatically from IP(6) to IP(5) and became negligible with IP(1-4). Among the IP(5) isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP(3-4) still showed inhibition of pepsin though to a lesser extent than IP(5-6). The in vitro data with IP(1-5) generated with microbial 3- and 6-phytases indicate that, for complete alleviation of pepsin inhibition, IP(6) needs to be broken down to IP(1-2.) In contrast to the aggregation with protein, the reactivity of IP(1-6) toward Fe(3+) decreased proportionally from IP(6) to IP(3.) Based on the radical decrease in turbidity of IP(6) -protein complex observed, as a result of IP(6) dephosphorylation to IP(5), a novel qualitative and semi-quantitative phytase plate assay was established using IP(6)-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP(6) at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe(3+) digestion.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: A human Lymphatic Filarial parasite

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.