Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27–30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds.     

Dose-Response Analysis of Chemotactic Signaling Response in Salmonella typhimurium LT2 upon Exposure to Cysteine / Cystine Redox Pair

The chemotaxis system enables motile bacteria to search for an optimum level of environmental factors. Salmonella typhimurium senses the amino acid cysteine as an attractant and its oxidized dimeric form, cystine, as a repellent. We investigated the dose-response dependence of changes in chemotactic signaling activity upon exposure to cysteine and cystine of S. typhimurium LT2 using in vivo fluorescence resonance energy transfer (FRET) measurements. The dose-response curve of the attractant response to cysteine had a sigmoidal shape, typical for receptor-ligand interactions. However, in a knockout strain of the chemoreceptor genes tsr and tar, we detected a repellent response to cysteine solutions, scaling linearly with the logarithm of the cysteine concentration. Interestingly, the magnitude of the repellent response to cystine also showed linear dependence to the logarithm of the cystine concentration. This linear dependence was observed over more than four orders of magnitude, where detection started at nanomolar concentrations. Notably, low concentrations of another oxidized compound, benzoquinone, triggered similar responses. In contrast to S. typhimurium 14028, where no response to cystine was observed in a knockout strain of chemoreceptor genes mcpB and mcpC, here we showed that McpB / McpC-independent responses to cystine existed in the strain S. typhimurium LT2 even at nanomolar concentrations. Additionally, knocking out mcpB and mcpC did not affect the linear dose-response dependence, whereas enhanced responses were only observed to solutions that where not pH neutral (>100 μM cystine) in the case of McpC overexpression. We discuss that the linear dependence of the response on the logarithm of cystine concentrations could be a result of a McpB / C-independent redox-sensing pathway that exists in S. typhimurium LT2. We supported this hypothesis with experiments with defined cysteine / cystine mixed solutions, where a transition from repellent to attractant response occurred depending on the estimated redox potential.     

l-Cystine inhibits aspartate-β-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-β-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by l- and d-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, β-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK_a of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [³⁵S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [³⁵S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)₂TVSL, thus demonstrating that the essential ¹³⁵Cys is the amino acid residue modified by the treatment with cystine.     

Incorporation of specifically labeled cysteine into Escherichia coli protein

Protein obtained from several strains of Escherichia coli grown in the presence of [3,3′-¹⁴C]cystine contained the radiolabel in nearly all the other amino acids, suggesting catabolism of cysteine to pyruvic acid. Utilization in amino acid synthesis of the pyruvate thus generated can be blocked by growing the bacteria in a medium specifically enriched with most of the naturally occurring amino acids. Cysteine that is incorporated intact is diluted by de novo synthesis at low cystine concentrations; also, it was found that E. coli can use the sulfur of methionine for cysteine biosynthesis. Both of these latter two processes can be prevented by supplying an excess of exogenous cystine. This regiment leads to protein that is highly specifically labeled in the cysteine residues, with a minor amount (20–25%) of the label also appearing in alanine residues. Although this strategy was developed expressly for cysteine, it may be useful for incorporating other labeled amino acids that are also readily catabolized.     

Cystine lyase: β-cystathionase from turnip roots

Cystine lyase (EC 4.4.1.-) was purified 277-fold by a combination of ammonium sulfate precipitation, chromatography on calcium phosphate and DEAE-cellulose with a 6% recovery. The MW as measured by gel filtration on Biogel p-300 was ca 150 000. The enzyme catalysed the pyridoxal phosphate-dependent degradation of cystine to pyruvate, ammonia and cysteine persulfide. Cysteine persulfide normally degraded spontaneously to elemental sulfur and cysteine, that further reacted to yield cystine and H₂S. Pyridoxal phosphate stabilized the enzyme. The K_m value for cystine was 0.94 mM. The enzyme was insensitive to thiol reagents but was inhibited by some thiols (which may have reduced the cystine). Cystine lyase degraded many compounds having the L-α-amino propionic acid group with a thioether or disulfide linkage attached to the β-carbon but was inactive towards D-configuration at the α-carbon or L-homocystine. The cystine lyase was also a β-cystathionase as indicated by (1) a constant ratio of β-cystathionase activity to cystine lyase activity throughout a 277-fold purification, (2) the inhibition of cystine lyase activity by cystathionine and inhibition of β-cystathionase activity by cystine and (3) similarity in sensitivity to heat, cyanide and hydroxylamine. Using DL-cystathionine as substrate, the K_m value was 4 mM.     

Gyrfalcons <Emphasis Type="Italic">Falco rusticolus</Emphasis> adjust <Emphasis Type="Italic">CTNS</Emphasis> expression to food abundance: a possible contribution to cysteine homeostasis

Melanins form the basis of animal pigmentation. When the sulphurated form of melanin, termed pheomelanin, is synthesized, the sulfhydryl group of cysteine is incorporated to the pigment structure. This may constrain physiological performance because it consumes the most important intracellular antioxidant (i.e., glutathione, GSH), of which cysteine is a constitutive amino acid. However, this may also help avoid excess cysteine, which is toxic. Pheomelanin synthesis is regulated by several genes, some of them exerting this regulation by controlling the transport of cysteine in melanocytes. We investigated the possibility that these genes are epigenetically labile regarding protein intake and thus contribute to cysteine homeostasis. We found in the Icelandic population of gyrfalcon Falco rusticolus, a species that pigments its plumage with pheomelanin, that the expression of a gene regulating the export of cystine out of melanosomes (CTNS) in feather melanocytes of developing nestlings increases with food abundance in the breeding territories where they were reared. The expression of other genes regulating pheomelanin synthesis by different mechanisms of influence on cysteine availability (Slc7a11 and Slc45a2) or by other processes (MC1R and AGRP) was not affected by food abundance. As the gyrfalcon is a strict carnivore and variation in food abundance mainly reflects variation in protein intake, we suggest that epigenetic lability in CTNS has evolved in some species because of its potential benefits contributing to cysteine homeostasis. Potential applications of our results should now be investigated in the context of renal failure and other disorders associated with cystinosis caused by CTNS dysfunction.     

Adsorption of cysteine on hematite,magnetite and ferrihydrite: FT-IR,Mössbauer,EPR spectroscopy and X-ray diffractometry studies

In the present paper, the adsorption of cysteine on hematite, magnetite and ferrihydrite was studied using FT-IR, electron paramagnetic resonance (EPR), Mössbauer spectroscopy and X-ray diffractometry. Cysteine was dissolved in artificial seawater (two different pHs) which contains the major constituents. There were two main findings described in this paper. First, after the cysteine adsorption, the FT-IR spectroscopy and X-ray diffractometry data showed the formation of cystine. Second, the Mössbauer spectroscopy did not show any increase in the amount of Fe²⁺ as expected due the oxidation of cysteine to cystine. An explanation could be that Fe²⁺ was oxidized by the oxygen present in the seawater or there occurred a reduction of cystine by Fe²⁺ generating cysteine and Fe³⁺. The specific surface area and pH at point of zero charge of the iron oxides were influenced by adsorption of cysteine. When compared to other iron oxides, ferrihydrite adsorbed significantly (p < 0.05) more cysteine. The pH has a significant (p < 0.05) effect only on cysteine adsorption on hematite. The FT-IR spectroscopy results showed that cystine remains adsorbed on the surface of the iron oxides even after being mixed with KCl and the amine and carboxylic groups are involved in this interaction. X-ray diffractometry showed no changes on iron oxides mineralogy and the following precipitated substances were found along with the iron oxides after drying the samples: cysteine, cystine and seawater salts. The EPR spectroscopy showed that cysteine interacts with iron oxides, changing the relative amounts of iron oxides and hydroxide.     

Role of N-acetylcysteine and cystine in glutathione synthesis in human erythrocytes

Abstract

Glutathione is an intracellular antioxidant that often becomes depleted in pathologies with high oxidative loads. We investigated the provision of cysteine for glutathione synthesis to the human erythrocyte (red blood cell; RBC). Almost all plasma cysteine exists as cystine, its oxidized form. In vitro, extracellular cystine at 1.0 mM sustained glutathione synthesis in glutathione-depleted RBCs, at a rate of 0.206 ± 0.036 μmol (L RBC)^?1min^?1 only 20% of the maximum rate obtained with cysteine or N-acetylcysteine. In plasma-free solutions, N-acetylcysteine provides cysteine by intracellular deacetylation but to achieve maximum rates of glutathione synthesis by this process in vivo, plasma N-acetylcysteine concentrations would have to exceed 1.0 mM, which is therapeutically unattainable. ¹H-NMR experiments demonstrated that redox exchange reactions between NAC and cystine produce NAC-cysteine, NAC-NAC and cysteine. Calculations using a mathematical model based on these results showed that plasma concentrations of N-acetylcysteine as low as 100 μM, that are attainable therapeutically, could potentially react with plasma cystine to produce ～50 μM cysteine, that is sufficient to produce maximal rates of glutathione synthesis. We conclude that the mechanism of action of therapeutically administered N-acetylcysteine is to reduce plasma cystine to cysteine that then enters the RBC and sustains glutathione synthesis.     

Further studies of the transport of amino acids in rat liver slices

Further studies of amino acid transport by the rat liver slice have shown that the transport of α-aminoisobutyric acid is inhibited by glycine as well as dinitrophenol, Na⁺-free medium, and iodoacetate. Glycine itself is actively transported by the rat liver slice, although some metabolism also takes place. Cystine is transported by a single transport system, although reduction to cysteine occurs intracellularly and to some extent in the medium also. Cysteine is transported faster than cystine and to greater concentration gradients. Kinetic studies showed that cystine was transported by a single system that was inhibited by glycine but not by α-amino-isobutyric acid. Two transport systems were involved in cysteine transport, each inhibited to a certain extent by α-aminoisobutyric acid and glycine. Lysine and valine both exist at a higher concentration intracellularly than in the plasma in vivo but no intracellular gradients were obtained after in vitro incubations. It is suggested that the intracellular gradients for these amino acids are maintained by protein catabolism.     

Cystine catabolism in mycelia of Microsporum gypseum,a dermatophytic fungus

The fate of ³⁵S label was studied during cystine degradation by mycelia of the dermatophytic fungus Microsporum gypseum. Excess free cystine in the medium was readily taken up and its sulfur moiety excreted as inorganic sulfate and sulfite. At intervals after 3–60 min of incubation with ³⁵S cystine the products of cystine catabolism were extracted from the mycelia by boiling water and separated by thin layer chromatography and electrophoresis. A total of 10 sulfur-containing compounds were identified, and their relative radioactivity was assessed. After 3 min the mycelia contained, in addition to cystine, labeled cysteine and particularly cysteine sulfinic acid which was accompanied by a smaller amount of cysteic acid. Later on, oxidized and reduced glutathione, inorganic sulfate and taurine appeared consecutively. In all extracts, small amounts of labeled S-sulfocysteine were found, not, however, sulfite.The results suggest that the intermediates of cysteine degradation in the fungal mycelia are cysteine, cysteine sulfinate, unstable sulfinylpyruvate, sulfite and sulfate, i.e., that the catabolic pattern is similar to that of higher organisms.The formation and the role of S-sulfocysteine, cysteic acid, and of taurine is not yet completely understood, although certainly autoxidative processes are involved in the formation of the latter two compounds, and sulfitolysis in that of the former compound.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural and biochemical analyses of Microcystis aeruginosa O-acetylserine sulfhydrylases reveal a negative feedback regulation of cysteine biosynthesis

O-acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis from O-acetylserine (OAS) and inorganic sulfide in plants and bacteria. Bioinformatics analyses combined with activity assays enabled us to annotate the two putative genes of Microcystis aeruginosa PCC 7806 to CysK1 and CysK2, which encode the two 75% sequence-identical OASS paralogs. Moreover, we solved the crystal structures of CysK1 at 2.30 ? and cystine-complexed CysK2 at 1.91 ?, revealing a quite similar overall structure that belongs to the family of fold-type II PLP-dependent enzymes. Structural comparison indicated a significant induced fit upon binding to the cystine, which occupies the binding site for the substrate OAS and blocks the product release tunnel. Subsequent enzymatic assays further confirmed that cystine is a competitive inhibitor of the substrate OAS. Moreover, multiple-sequence alignment revealed that the cystine-binding residues are highly conserved in all OASS proteins, suggesting that this auto-inhibition of cystine might be a universal mechanism of cysteine biosynthesis pathway.     

Sulfate reduction in a cell-free system ofChlorella

Summary During sulfate reduction in a cell-free system ofChlorella activated sulfate of APS is transferred to a thiosulfonate reductase. The sulfate thus bound to the thiosulfonate reductase (i.e. bound sulfite) is reduced to bound sulfide in a ferredoxin dependent reaction. This bound sulfide can be used with O-acetylserine as acceptor for cysteine biosynthesis; serine and O-phosphoserine are not used. An assaysystem for thiosulfonate reductase activity using methylviologen dependent reduction of S₂O₄ ^2– to S^2– is developed and a procedure for isolating thiosulfonate reductase fromChlorella cells is presented. During isolation of thiosulfonate reductase a low weight molecular factor, needed for optimal enzyme activity was lost. The bound sulfite seems to be attached to this factor. Reduction of APS or GS-SO₃H to the level of S^2– is inhibited by cysteine. 50% inhibition of GS-SO₃H reduction was found at a molar cysteine concentration of 6.8×10^–5.Abbreviations APS adenosine-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - GSH reduced glutathion - GS-SO₃H S-sulfoglutathion - fd ferredoxin - Mv methylviologen - DTT dithiothreitol     

The Cysteine Desulfhydrase CdsH Is Conditionally Required for Sulfur Mobilization to the Thiamine Thiazole in Salmonella enterica

Thiamine pyrophosphate is a required coenzyme that contains a mechanistically important sulfur atom. In Salmonella enterica, sulfur is trafficked to both thiamine biosynthesis and 4-thiouridine biosynthesis by the enzyme ThiI using persulfide (R-S-S-H) chemistry. It was previously reported that a thiI mutant strain could grow independent of exogenous thiamine in the presence of cysteine, suggesting there was a second mechanism for sulfur mobilization. Data reported here show that oxidation products of cysteine rescue the growth of a thiI mutant strain by a mechanism that requires the transporter YdjN and the cysteine desulfhydrase CdsH. The data are consistent with a model in which sulfide produced by CdsH reacts with cystine (Cys-S-S-Cys), S-sulfocysteine (Cys-S-SO₃⁻), or another disulfide to form a small-molecule persulfide (R-S-S-H). We suggest that this persulfide replaced ThiI by donating sulfur to the thiamine sulfur carrier protein ThiS. This model describes a potential mechanism used for sulfur trafficking in organisms that lack ThiI but are capable of thiamine biosynthesis.     

The reactions of the sulfur-containing amino acids with phosphoric and sulfuric acids

Both cystine and cysteine react very differently toward 6.0 M phosphoric and sulfuric acids. With phosphoric acid, extensive decomposi tion occurs; with sulfuric acid, very little.Cystine, in phosphoric acid at 100 °C. produces hydrogen sulfide, free sulfur, cysteic acid, ammonia, alanine, serine, and pyruvic acid. Cysteine produces a similar series but with marked quantitative differences.Cystine, in sulfuric acid, is merely racemized.Methionine is largely demethylated by sulfuric acid under these conditions. It is practically inert toward phosphoric acid.The behavior of hair toward phosphoric and sulfuric acids is similar to that of cystine but is modified by reactions of other hydrolytic products.     

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamidetreated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-ME_ox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [³⁵S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [³⁵S]cystine with 2-ME_ox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-ME_ox and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.     

Release of sulfhydryl compounds by the pathogenic fungusHistoplasma capsulatum

The pathogenic fungusHistoplasma capsulatum undergoes a mycelial to yeast transition when the temperature of incubation is switched from 25° to 37°C. The presence of exogenous cysteine or cystine has been reported to be required for this phase transition and for maintenance of yeast form and growth. However, the initial stages of mycelial to yeast transition and yeast growth are associated with the rapid utilization and conversion of exogenous cystine to at least one sulfhydryl compound, which appears in the extracellular milieu as detected by dithiodinitrobenzoic acid. Attempts to identify the extracellular thiol-containing compound with cystine metabolic standards indicate that the released thiols are not cysteine or reduced glutathione. These results indicate that other thiols may be important in supportingH. capsulatum yeast morphogenesis.     

Ion-exchange and potentiometric characterization of Al–cystine and Al–cysteine complexes

The interaction between aluminium and cysteine and cystine was evaluated by means of ion-exchange experiments and potentiometry. Ion-exchange experiments included other ligands with affinity for aluminium and two kinds of resins, either a Na⁺-form or an Al³⁺-form exchanger. The ability of the ligands to keep aluminium in solution in the presence of the Na⁺ exchanger or to withdraw it from the Al³⁺-form resin was evaluated. Aluminium quantification was carried out by either graphite-furnace or flame atomic absorption spectrometry. Aluminium extraction isotherms were linearised using the Scatchard plot, and stability constants were obtained from the curves’ slopes. The experiments showed that the ability of the ligands to withdraw aluminium from the Al³⁺-form resin increased following the order cysteine < oxalate < citrate = cystine < nitrilotriacetic acid < ethylenediaminetetraacetic acid. Potentiometric titrations, carried out in aqueous solution with constant ionic strength and temperature, showed that the predominant species in solution have a metal–ligand proportion of 1:1 for both amino acids. The main species are Al(OH)₃L, with log K of 6.2 for cysteine, and AlL and Al(OH)L, with log K of 10.3 and 1.7, respectively, for cystine. Stability constants obtained from the Scatchard plots showed a linear correlation with the stability constants obtained by potentiometry for cystine and cysteine in this work and those collected from the literature for the other ligands. These results show that cysteine and cystine extract and maintain aluminium in solution, which may explain elevated concentrations of aluminium in parenteral nutrition solutions containing these amino acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The transsulfuration pathway: a source of cysteine for glutathione in astrocytes

Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (γ-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the γ-glutamylcycle leading to formation of glutathione (l-γ-glutamyl-l-cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the x_c⁻ cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from the indispensable amino acid, methionine, has recently been identified as a significant contributor to GSH synthesis in astrocytes. The purpose of this review is to evaluate the importance of the transsulfuration pathway in these cells, particularly in the context of a reserve pathway that channels methionine towards cysteine when the demand for glutathione is high, or under conditions in which the supply of cystine by the x_c⁻ exchanger may be compromised.     

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 ??mol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.