Isolation and functional diversity of Bowman-Birk type serine proteinase inhibitors from Hyacinthus orientalis.

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

A stilbene dimer and flavonoids from the aerial parts of Chromolaena odorata with proprotein convertase subtilisin/kexin type 9 expression inhibitory activity.

Investigation of components of the chloroform-soluble and ethyl acetate-soluble extracts of the aerial parts of Chromolaena odorata L. selected by PCSK9 mRNA expression monitoring assay in HepG2 cells led to the isolation of a new stilbene dimer, (+)-8b-epi-ampelopsin A (1), and 30 known compounds (2-31). The structures of the isolates were established by interpretation of NMR spectroscopic data and the stereochemistry of the new stilbene (1) was proposed based on ECD and NMR calculations. Among the isolates, 1, 5,6,7,4'-tetramethoxyflavanone (6), 5,6,7,3',4'-pentamethoxyflavanone (7), acacetin (18), and uridine (21) were found to inhibit PCSK9 mRNA expression with IC50 values of 20.6, 21.4, 31.7, 15.0, and 13.7 µM, respectively. Furthermore, the most abundant isolate among the selected compounds, 6, suppressed PCSK9 and low-density lipoprotein receptor protein expression in addition to downregulating the mRNA expression of HNF-1α.

Neutrophil elastase inhibitor purification strategy from cowpea seeds.

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Identification and pharmaceutical evaluation of novel frog skin-derived serine proteinase inhibitor peptide-PE-BBI (Pelophylax esculentus Bowman-Birk inhibitor) for the potential treatment of cancer.

Amphibian venom-derived peptides have high potential in the field of anticancer drug discovery. We have isolated a novel Bowman-Birk proteinase inhibitor (BBI)-type peptide from the skin secretion of Pelophylax esculentus (PE) named PE-BBI, and evaluated its bio-functions and anti-cancer activity in vitro. PE-BBI is a heptadecapeptide with C-terminal amidation. The mRNA sequence and primary structure of PE-BBI were identified using RT-PCR and LC/MS, respectively. A trypsin inhibitory assay was used to characterize the serine proteinase inhibitory activity of synthetic PE-BBI. PE-BBI's myotropic activity was analyzed using isolated rat bladder and rat-tail artery smooth muscle tissues, and the anti-cancer ability of PE-BBI using human colorectal cancer cells. PE-BBI's mechanism of action was investigated using Discovery studio software. PE-BBI showed trypsin inhibitory activity (Ki = 310 ± 72 nM), strong myotropic activity, and cytotoxicity that were specific to cancer cells, and no side effect to normal epithelial cells. The docking stimulation showed that PE-BBI had high affinity to several members of human kallikrein related peptidase (KLK) family. This finding helps to enrich our understanding of BBI peptides' mode of action. Moreover, the data presented here validates frog secretions as sources of potential novel proteinase inhibitors for cancer treatment.

Identification of a small pacifastin protease inhibitor from Nasonia vitripennis venom that inhibits humoral immunity of host (Musca domestica).

Nasonia vitripennis is an important natural enemy of many flies. Pacifastin protease inhibitors (PPIs) play an important role in development and innate immunity of insects. In this study, cDNA sequences of a small pacifastin protease inhibitor in N. vitripennis (NvSPPI) was characterized and its open reading frame (ORF) contains 243bp. Real-time quantitative PCR (RT-qPCR) results revealed that NvSPPI mRNA were detected specifically in the venom apparatus, while they were expressed at low levels in other tissues tested. In the venom apparatus, NvSPPI transcript was highly expressed on the fourth day post eclosion and then declined gradually. The NvSPPI gene was recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvSPPI to three serine protease inhibitors (trypsin, chymotrypsin, and protease K) were tested and results showed that recombinant NvSPPI could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvSPPI on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by recombinant NvSPPI; however, there was no significant inhibition on the PO activity. Our results suggested that NvSPPI could inhibit PPO activation in host hemolymph and trypsin activity in vitro.

High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.

Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors.

The natural flavone fukugetin as a mixed-type inhibitor for human tissue kallikreins.

The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

Chemical Composition and Inhibitory Effects of Hypericum brasiliense and H. connatum on Prolyl Oligopeptidase and Acetylcholinesterase Activities.

BACKGROUND: The genus Hypericum (family Clusiaceae) comprises various species that are used in traditional medicine, such as wound healing, antidepressant, and anticancer agents. OBJECTIVE: The aim of this study was to evaluate the inhibitory capacity of extracts and fractions from two Hypericum species used in the Brazilian folk medicine (H. brasiliense and H. connatum) against the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase-IV (DPP-IV), and acetylcholinesterase (AChE), as well as to identify their main active constituents. METHODS: Dried aerial parts of H. connatum and H. brasiliense were subjected to extraction with 8:2 methanol-H2O. Each hydroalcoholic extract was fractioned resulting in ethyl acetate and aqueous fractions. The activity of POP, DPP-IV and AChE was determined in vitro in 96-well microplates. RESULTS: The main components identified in the plant extracts were chlorogenic acid (1), quercitrin (2), rutin (3), quercetin (4), and isoquercitrin (5). Hydroalcoholic extracts, ethyl acetate and aqueous fractions showed high POP inhibitory activity with IC50 values of 2.6 to 3.7 µg/mL. AChE and DPP-IV inhibitory effects were very low for all extracts and substances. CONCLUSION: Chlorogenic acid (1) and quercetin (4) were the main constituent responsible for the activity observed against POP. Parallel artificial membrane permeability assay of ethyl acetate fractions of both species showed that the metabolite that can effectively pass through the lipid membrane is 4, the aglycone form of 2, 3 and 5.

Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.

Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly ß sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

Inhibitory effects of the recombinant human serine protease inhibitor Hespintor on the proliferation, migration and invasion of hepatocellular carcinoma cells.

Hespintor is a new Kazal-type serine proteinase inhibitor (Serpin) screened from the HepG2 hepatoblastoma cell line using the suppression subtractive hybridization (SSH) technique. Seprin is closely associated with the progression and remission of malignant tumors, and has certain significance in the diagnosis and treatment of tumors. Investigations on the antitumor activity of Serpin are expected to aid in the development of a new method for tumor treatment based on the serine protease inhibitor. Although the Hespintor prokaryotic expression strain and recombinant Hespintor protein (recombinant fusion protein of Hespintor and rHespintor) have already been obtained, the protein extraction efficiency is low due to the low initial amount of extracted protein and large number of purification steps, which affect the study of the protein function. The aim of the present study was to improve the purification method of rHespintor, increase the protein extraction efficiency, and investigate its effects on the proliferation, migration and invasion of the HepG2 hepatoblastoma cell line. The results demonstrated that the application of urea gradient washing of inclusion body of the protein may effectively remove the majority of impure proteins from the targeted protein. After one-step purification, the target protein rHespintor exhibited a high inhibitory effect of Trypsin Hydrolysis, which was exhibited in a dose-dependent manner. Hoechst 33258 staining was used to determine cell apoptosis. After treating HepG2 hepatoblastoma cells with rHespintor, the cell growth was inhibited, the proliferation ability was reduced, and the number of migrated and invaded cells were significantly decreased. Hoechst 33258 staining and flow cytometry assay results showed clear cell apoptosis. The results reveal showed that rHespintor significantly inhibited proliferation, migration and invasion of the HepG2 hepatoblastoma cell line in vitro, and induced cell apoptosis to a certain extent, indicating that the recombinant protein Hespintor exerts an antitumor effect in vitro, and has the potential and feasibility to become an antitumor drug.

Recovery, viscoelastic and functional properties of Barbel skin gelatine: investigation of anti-DPP-IV and anti-prolyl endopeptidase activities of generated gelatine polypeptides.

The characteristics and functional properties of gelatine from freshwater fish skin (Barbus callensis) were investigated. The gelatine extraction efficiency was improved by an acid-swelling process in the presence of barbel crude acid protease extract. Barbel skin gelatine (BSG) contained 92.15% protein, 0.31% lipid and 0.72% ash. The amino acid profile of BSG showed a high percentage of imino acids. The electrophoretic profile showed that BSG is mainly composed of α- and ß-components. BSG showed an excellent solubility and possessed interfacial properties, which were governed by the protein concentration. Biological activities of the hydrolysates obtained after digestion of BSG with several commercial proteases were evaluated. The results suggested that these hydrolysates are a good source of natural inhibitors of dipeptidyl peptidase-IV and prolyl endopeptidase and could potentially be used as dietary ingredients in the management of type 2-diabetes and/or neuropathological disorders.

Functional characterization of a new non-Kunitz serine protease inhibitor from the scorpion Lychas mucronatus.

Serine protease inhibitors have been widely discovered from different animal venoms, but most of them belong to Kunitz-type toxin subfamily. Here, by screening scorpion venom gland cDNA libraries, we identified four new non-Kunitz serine protease inhibitors with a conserved Ascaris-type structural fold: Ascaris-type toxins Lychas mucronatus Ascaris-type protease inhibitor (LmAPI), Pandinus cavimanus Ascaris-type protease inhibitor (PcAPI), Pandinus cavimanus Ascaris-type protease inhibitor 2 (PcAPI-2), and Hottentotta judaicus Ascaris-type protease inhibitor (HjAPI). The detailed characterization of one Ascaris-type toxin LmAPI was further carried out, which contains 60 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges. Enzyme and inhibitor reaction kinetics experiments showed that recombinant LmAPI inhibits the activity of chymotrypsin potently with a Ki value of 15.5 nM, but has little effect on trypsin and elastase. Bioinformatics analyses suggested that LmAPI contains unique functional residues "TQD" and might be a useful template to produce specific protease inhibitors. Our results indicated that animal venoms are a natural source of new type of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.

PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis.

Development and homeostasis of the vascular system requires integrin-promoting endothelial cell adhesion, migration and survival. Nowadays, integrins represent potential targets for pharmacological agents and open new avenues for the control of metastatic spread in the treatment of tumor malignancies. We have already reported that PIVL, a serine protease inhibitor isolated from Macrovipera lebetina venom, displays an anti-tumor effect through interference with integrin receptor function. Here, we report that PIVL inhibits human vascular endothelial cell adhesion and migration onto fibrinogen and fibronectin in a dose-dependent manner without any cytotoxicity. Furthermore, we show that PIVL increases microtubule dynamic instability in HMEC-1 transfected with EGFP-tagged α-tubulin. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrate that PIVL exhibits a strong anti-angiogenic effect both in vitro and in vivo. Interestingly, results herein reveal that the potent anti-angiogenic properties of PIVL are mediated by its RGD-like motif ((41)RGN(43)).

Bowman-Birk inhibitors from legumes as colorectal chemopreventive agents.

Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract (GIT) has prompted scientists to investigate the potential of serine protease inhibitors, both natural and synthetic, as modulators of their proteolytic activities. Protease inhibitors of the Bowman-Birk type, a major protease inhibitor family in legume seeds, which inhibit potently and specifically trypsin- and chymotrypsin-like proteases, are currently being investigated as colorectal chemopreventive agents. Physiologically relevant amounts of Bowman-Birk inhibitors (BBI) can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT. Studies in animal models have proven that dietary BBI from several legume sources, including soybean, pea, lentil and chickpea, can prevent or suppress carcinogenic and inflammatory processes within the GIT. Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated, the emerging evidence suggests that BBI exert their preventive properties via protease inhibition; in this sense, serine proteases should be considered as primary targets in early stages of carcinogenesis. The validation of candidate serine proteases as therapeutic targets together with the identification, within the wide array of natural BBI variants, of the most potent and specific protease inhibitors, are necessary to better understand the potential of this protein family as colorectal chemopreventive agents.

Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom.

A 7.1 kDa basic peptide (Rusvikunin-II) was purified from a previously described protein complex (Rusvikunin complex, consists of Rusvikunin and Rusvikunin-II) of Daboia russelii russelii venom. The N-terminal sequence of Rusvikunin-II was found to be blocked, but peptide mass fingerprinting analysis indicated its identity as Kunitz-type basic protease inhibitor 2, previously reported from Russell's Viper venom. A tryptic peptide sequence of Rusvikunin-II containing the N-terminal sequence HDRPTFCNLFPESGR demonstrated significant sequence homology to venom basic protease inhibitors, Kunitz-type protease inhibitors and trypsin inhibitors. The secondary structure of Rusvikunin-II was dominated by ß-sheets (60.4%), followed by random coil (38.2%), whereas α-helix (1.4%) contributes the least to its secondary structure. Both Rusvikunin-II and the Rusvikunin complex demonstrated dose-dependent anticoagulant activity; however, the anticoagulant potency of latter was found to be higher. Both inhibited the amidolytic activity of trypsin > plasmin >> FXa, fibrinogen clotting activity of thrombin, and, to a lesser extent, the prothrombin activation property of FXa; however, the inhibitory effect of the Rusvikunin complex was more pronounced. Neither Rusvikunin-II nor Rusvikunin complex inhibited the amidolytic activity of chymotrypsin and thrombin. Rusvikunin-II at 10 µg/ml was not cytotoxic to Colo-205, MCF-7 or 3T3 cancer cells; conversely, Rusvikunin complex showed ∼30% reduction of MCF-7 cells under identical experimental conditions. Rusvikunin-II (5.0 mg/kg body weight, i.p. injection) was not lethal to mice or House Geckos; nevertheless, it showed in vivo anticoagulant action in mice. However, the Rusvikunin complex (at 5.0 mg/kg) was toxic to NSA mice, but not to House Geckos, suggesting it has prey-specific toxicity. Rusvikunin complex-treated mice exhibited dyspnea and hind-limb paresis prior to death. The present study indicates that the Kunitz-type protein complex Rusvikunin from Russell's Viper venom significantly contributes to venom toxicity, and an important biological role in venoms appears to be facilitation of prey subjugation.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.