PCTH: A novel orally active chelator for the treatment of iron overload disease

Our laboratories have prepared a novel class of iron (Fe) chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) class. This article will review the iron chelation efficacy of this series of chelators, both in cell culture and in animal models. Several PCIH analogs were shown to be effective at inducing iron mobilization and preventing iron uptake from the iron-transport protein, transferrin. Moreover, several of these ligands were effective at permeating the mitochondrion and inducing iron release. Studies in mice demonstrated that the PCIH analog, PCTH, was orally active and well tolerated by mice at doses ranging from 50 to 100 mg kg(-1) , twice daily (b.d.). A dose-dependent increase in fecal Fe-59 excretion was observed in the PCTH-treated group. This level of iron excretion was similar to that found for the orally effective chelators, pyridoxal isonicotinoyl hydrazone (PIH) and deferiprone (L1). The PCIH group of ligands clearly has the potential for the treatment of ss-thalassemia (thal) and Friedreich's Ataxia (FA).

Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P3 is considered to be important to complex Fe3+ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P4], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe3+, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca 2+ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe3+ have been determined. The complexes formed between Fe 3+ and 4,6-bispyrenoyl Ins(1,2,3,5)P4 display similar stability to those formed with Ins(1,2,3)P3, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P4, which closely resemble those obtained for Ins(1,2,3)P3. The data presented confirms that Fe3+ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif. © 2010 The Royal Society of Chemistry.

Development of a new analytical approach based on cellulose membrane and chelator for differentiation of labile and inert metal species in aquatic systems

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD-TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM-TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0 mol L-1 of HCl and NaOH solutions. After the MD-TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0-24 h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60 mg L-1) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD-TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD-TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95-75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100 min, and Cu and Fe approached equilibrium after 400 min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS-metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems. (c) 2006 Elsevier B.V. All rights reserved.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

Tungsten(0)-carbonyl complexes of naphthylazoimidazoles

The reaction of W(CO)(6) with 1-alkyl-2-(naphthyl-alpha-azo)imidazole (alpha-NaiR) has synthesized [W(CO)(5)(alpha-NaiR-N)] (alpha-NaiR-N refers to the monodentate imidazole-N donor ligand) at room temperature. The structure of[W(CO)(5)(alpha-NaiMe-N)] shows a monodentate imidazole-N coordination of 1-methyl-2-(naphthyl-alpha-azo)imidazole (alpha-NaiMe). The complexes are characterized by elemental, mass and other spectroscopic data (IR, UV-Vis, NMR). On refluxing in THF at 323 K, [W(CO)(5)(alpha-NaiR-N)] undergoes decarbonylation to give [W(CO)(4)(alpha-NaiR-N,N')] (alpha-NaiR-N,N' refers to the imidazole-N(N), azo-N(N') bidentate chelator). Cyclic voltammetry shows metal oxidation (W-0/W-1) and ligand reductions (azo/azo(-), azo(-)/azo(=)). The redox and electronic properties are explained by theoretical calculations using an optimized geometry. DFT computation of [W(CO)(5)(alpha-NaiMe-N)] suggests that the major contribution to the HOMO/HOMO - 1 come from W cl-orbitals and the orbitals of CO. The LUMOs are occupied by alpha-NaiMe functions. The back bonding interaction thus originates from the W(CO)(n) moiety to the LUMO of alpha-NaiR. A TD-DFT calculation has ascribed that HOMO/HOMO - 1 -> LUMO is a mixture of metal-to-ligand and ligand-to-ligand charge transfer underlying the CO -> azoimine contribution. The complexes show emission spectra at room temperature. [W(CO)(4)(alpha-NaiR-N,N')] shows a higher fluorescence quantum yield (phi = 0.05-0.07) than [W(CO)(5)(alpha-NaiR-N)] (phi = 0.01-0.02). (C) 2008 Elsevier Ltd. All rights reserved.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6•0–6•5, when the pH of the external medium was varied between 2•3–7•0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn2+ ions partially protected the enzyme against this inactivation. Mn2+-dependent glutamine synthetase activity was higher at acidic pH (6•0) compared to Mg2+-supported activity. While the concentration of Mg2+ required to optimally activate glutamine synthetase at pH 6•0 was very high (≥ 50 mM), Mn2+ was effective at 4 mM. Higher concentrations of Mn2+ were inhibitory. The inhibition of both Mn2+ and Mg2+-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn2+ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

Estudo das alterações biológicas causadas pela aderência de cepas de Escherichia coli enteroagregativa (EAEC) com macrófagos humanos ativados da linhagem U-937

Escherichia coli enteroagregativa (EAEC) é um patógeno relacionado ao desenvolvimento de quadros de diarréia aguda ou persistente. A resposta inflamatória induzida por EAEC está relacionada à liberação de interleucina 8, que atua estimulando a transmigração de neutrófilos através do epitélio. Os macrófagos, de forma similar aos neutrófilos, são células fagocíticas que produzem espécies reativas de oxigênio (ERO), como o peróxido de hidrogênio (H2O2). Neste trabalho, avaliamos as consequências da interação de diferentes cepas clínicas com macrófagos humanos ativados da linhagem U-937. Todas as cepas testadas apresentaram filamentos nos testes de aderência aos macrófagos, diferentemente do que ocorre na interação com outras linhagens celulares como HEp-2, T84 e Caco-2. O ferro é um microelemento essencial para bactérias, sendo utilizado como cofator de enzimas e que também pode participar da geração de ERO através da reação de Fenton. Considerando-se a possibilidade de que o H2O2 produzido pelos macrófagos possa gerar radical hidroxil através da reação de Fenton, testes de aderência foram realizados com as amostras cultivadas na presença do captador de ferro 2,2-dipiridil. Tal fato não suprimiu a formação de filamentos, entretanto diminuiu a aderência das cepas EAEC 042 e 17-2. Com o objetivo de produzir uma resposta adaptativa ao H2O2, as culturas bacterianas foram pré-tratadas com uma dose sub-letal de H2O2 por 60 minutos antes de aderirem aos macrófagos. Nossos resultados mostraram que o pré-tratamento também não inibiu o aparecimento de filamentos em relação às culturas não tratadas. Além disto, foi observado que o pré-tratamento com o H2O2 reduziu a aderência das amostras de EAEC ao tapete celular. A filamentação é uma das respostas SOS, induzida pela presença de danos e/ou bloqueio na síntese da molécula de DNA. Com o objetivo de verificar se o H2O2 produzido pelos macrófagos estaria causando danos induzindo o sistema SOS e a filamentação bacteriana, foram realizados testes de viabilidade com mutantes derivados de E. coli K12 deficientes em enzimas do reparo por excisão de bases (BW535) e na resposta SOS (DM49). Nossos resultados mostram que os mutantes apresentaram os níveis de sobrevivência semelhantes ao observado para cepa selvagem isogênica (AB1157). Todos estes resultados em conjunto indicam que o H2O2 não é o indutor da filamentação nos testes de aderência. Macrófagos ativados apresentam ação microbicida através da ação da enzima indolamina dioxigenase (IDO), associada à redução do aminoácido L-triptofano. Desta forma, realizamos testes qualitativos de aderência de EAEC aos macrófagos suplementando o meio de interação com este aminoácido. Nossos resultados mostram que a adição de triptofano ao meio de interação reduz o número de filamentos por campo. Desta forma, aventamos a hipótese de que a depleção do triptofano seja responsável pela indução de resposta SOS, tendo como conseqüência a filamentação das bactérias.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Effects of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator

The action of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation was examined in vitro and a strong anticoagulant effect was observed. This action was abolished after treatment with a specific inhibitor of phospholipase A(2) activity (p-bromophenacyl bromide), revealing a procoagulant action in low concentrations of treated venom (around 1 mu g/ml). The effect of the venom an haemostasis was further characterized by measuring its ability to activate purified blood coagulation factors. It is concluded that A. halys pallas venom contains prothrombin activation activity. A prothrombin activator (aharin) was purified from the venom by Sephadex G-75 gel filtration and ion-exchange chromatography on a Mono-Q column. It consisted of a single polypeptide chain, with a mol. wt of 63,000. Purified aharin possessed no amidolytic activity on chromogenic substrates. It did not act on other blood coagulation factors, such as factor X and plasminogen, nor did it cleave or clot purified fibrinogen. The prothrombin activation activity of aharin was readily inhibited by ethylenediamine tetracetic acid (a metal chelator), but specific serine protease inhibitors such as diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride had no effect on it. These observations suggest that, like those prothrombin activators from Echis carinatus and Bothrops atrox venoms, the prothrombin activator from A. halys pallas venom is a metalloproteinase. (C) 1998 Elsevier Science Ltd. All rights reserved.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

Long-term depression in rat CA1-subicular synapses depends on the G-protein coupled mACh receptors

The subiculum, which is the primary target of CA1 pyramidal neurons and sending efferent fibres to many brain regions, serves as a hippocampal interface in the neural information processes between hippocampal formation and neocortex. Long-term depression (LTD) is extensively studied in the hippocampus, but not at the CA1-subicular synaptic transmission. Using whole-cell EPSC recordings in the brain slices of young rats, we demonstrated that the pairing protocols of low frequency stimulation (LFS) at 3 Hz and postsynaptic depolarization of -50 mVelicited a reliable LTD in the subiculum. The LTD did not cause the changes of the paired-pulse ratio of EPSC. Furthermore, it did not depend on either NMDA receptors or voltage-gated calcium channels (VGCCs). Bath application of the G-protein coupled muscarinic acetylcholine receptors (mAChRs) antagonists, atropine or scopolamine, blocked the LTD, suggesting that mAChRs are involved in the LTD. It was also completely blocked by either the Ca2+ chelator BAPTA or the G-protein inhibitor GDP-beta-S in the intracellular solution. This type of LTD in the subiculum may play a particular role in the neural information processing between the hippocampus and neocortex. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.