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Ruma Banerjee - One of the best experts on this subject based on the ideXlab platform.

  • Chlorocob(II)alamin Formation Which Enhances the Thiol Oxidase Activity of the B12-Trafficking Protein CblC.
    Inorganic chemistry, 2020
    Co-Authors: Elizabeth D Greenhalgh, Markus Ruetz, Thomas C. Brunold, Umar T. Twahir, Albert Kallon, Kurt Warncke, Ruma Banerjee
    Abstract:

    CblC is a chaperone that catalyzes removal of the β-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the β-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust Thiol Oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the Thiol Oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced Thiol Oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful Thiol Oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.

  • The human B12 trafficking protein CblC processes nitrocobalamin.
    The Journal of biological chemistry, 2020
    Co-Authors: Romila Mascarenhas, Markus Ruetz, Carmen Gherasim, Ruma Banerjee
    Abstract:

    In humans, cobalamin or vitamin B12 is delivered to two target enzymes via a complex intracellular trafficking pathway comprising transporters and chaperones. CblC (or MMACHC) is a processing chaperone that catalyzes an early step in this trafficking pathway. CblC removes the upper axial ligand of cobalamin derivatives, forming an intermediate in the pathway that is subsequently converted to the active cofactor derivatives. Mutations in the cblC gene lead to methylmalonic aciduria and homocystinuria. Here, we report that nitrosylcobalamin (NOCbl), which was developed as an antiproliferative reagent, and is purported to cause cell death by virtue of releasing nitric oxide, is highly unstable in air and is rapidly oxidized to nitrocobalamin (NO2Cbl). We demonstrate that CblC catalyzes the GSH-dependent denitration of NO2Cbl forming 5-coordinate cob(II)alamin, which had one of two fates. It could be oxidized to aquo-cob(III)alamin or enter a futile Thiol Oxidase cycle forming GSH disulfide. Arg-161 in the active site of CblC suppressed the NO2Cbl-dependent Thiol Oxidase activity, whereas the disease-associated R161G variant stabilized cob(II)alamin and promoted futile cycling. We also report that CblC exhibits nitrite reductase activity, converting cob(I)alamin and nitrite to NOCbl. Finally, the denitration activity of CblC supported cell proliferation in the presence of NO2Cbl, which can serve as a cobalamin source. The newly described nitrite reductase and denitration activities of CblC extend its catalytic versatility, adding to its known decyanation and dealkylation activities. In summary, upon exposure to air, NOCbl is rapidly converted to NO2Cbl, which is a substrate for the B12 trafficking enzyme CblC.

  • Coordination chemistry controls the Thiol Oxidase activity of the B12-trafficking protein CblC.
    The Journal of biological chemistry, 2017
    Co-Authors: Aranganathan Shanmuganathan, Markus Ruetz, Kazuhiro Yamada, Nicholas A. Lesniak, Bernhard Kräutler, Thomas C. Brunold, Markos Koutmos, Ruma Banerjee
    Abstract:

    The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust Thiol Oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of Thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its Thiol Oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and β-faces of cobalamin promote the Thiol Oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent Thiol Oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the Thiol Oxidase activity of ceCblC.

  • Unusual Aerobic Stabilization of Cob(I)alamin by a B12-Trafficking Protein Allows Chemoenzymatic Synthesis of Organocobalamins
    2015
    Co-Authors: Nicholas A. Lesniak, Ruma Banerjee
    Abstract:

    CblC, a B12 trafficking protein, exhibits glutathione transferase and reductive decyanase activities for processing alkylcobalamins and cyanocobalamin, respectively, to a common intermediate that is subsequently converted to the biologically active forms of the cofactor. We recently discovered that the Caenorhabditis elegans CblC catalyzes Thiol-dependent decyanation of CNCbl and reduction of OH2Cbl and stabilizes the paramagnetic cob­(II)­alamin product under aerobic conditions. In this study, we report the striking ability of the worm CblC to stabilize the highly reactive cob­(I)­alamin product of the glutathione transferase reaction. The unprecedented stabilization of the supernucleophilic cob­(I)­alamin species under aerobic conditions by the intrinsic Thiol Oxidase activity of CblC, was exploited for the chemoenzymatic synthesis of organocobalamin derivatives under mild conditions

  • Unusual aerobic stabilization of Cob(I)alamin by a B12-trafficking protein allows chemoenzymatic synthesis of organocobalamins.
    Journal of the American Chemical Society, 2014
    Co-Authors: Nicholas A. Lesniak, Ruma Banerjee
    Abstract:

    CblC, a B12 trafficking protein, exhibits glutathione transferase and reductive decyanase activities for processing alkylcobalamins and cyanocobalamin, respectively, to a common intermediate that is subsequently converted to the biologically active forms of the cofactor. We recently discovered that the Caenorhabditis elegans CblC catalyzes Thiol-dependent decyanation of CNCbl and reduction of OH2Cbl and stabilizes the paramagnetic cob(II)alamin product under aerobic conditions. In this study, we report the striking ability of the worm CblC to stabilize the highly reactive cob(I)alamin product of the glutathione transferase reaction. The unprecedented stabilization of the supernucleophilic cob(I)alamin species under aerobic conditions by the intrinsic Thiol Oxidase activity of CblC, was exploited for the chemoenzymatic synthesis of organocobalamin derivatives under mild conditions.

Kai Hell - One of the best experts on this subject based on the ideXlab platform.

  • structural and functional roles of the conserved cysteine residues of the redox regulated import receptor mia40 in the intermembrane space of mitochondria
    Journal of Biological Chemistry, 2009
    Co-Authors: Nadia Terziyska, Barbara Grumbt, Christian Kozany, Kai Hell
    Abstract:

    Abstract Oxidative folding drives the import of proteins containing twin CXnC motifs into the intermembrane space of mitochondria. This import pathway employs a disulfide relay system whose key components are the redox-regulated import receptor Mia40 and the Thiol Oxidase Erv1. Mia40 contains six cysteine residues in a CPC-CX9C-CX9C arrangement in a highly conserved domain. We show that this domain is sufficient for the function of Mia40. By analysis of Mia40 cysteine mutants we demonstrate that the cysteine residues have distinct roles and are not equally important for Mia40 function. The second cysteine residue is essential for viability of yeast cells. It is required for the interaction of Mia40 with Erv1 in a disulfide intermediate and forms a redox-sensitive disulfide bond with the first cysteine residue. Both cysteine residues are required for the oxidation of the substrate, Tim10, in a reconstituted system comprised of Mia40 and Erv1. Mutants with amino acid exchanges in the third and sixth cysteine residues have severe defects in growth and in the import of intermembrane space proteins. These Mia40 variants are not tightly folded. We conclude that the cysteine residues of the twin CX9C motif have a structural role and stabilize Mia40. In particular, the disulfide bond formed by the third and sixth cysteine residues apparently supports a conformation crucial for the function of Mia40. Furthermore, the disulfide bond in the CPC segment mediates the redox reactions with the Thiol Oxidase Erv1 and substrate proteins in mitochondria.

  • The disulfide relay system of mitochondria is required for the biogenesis of mitochondrial Ccs1 and Sod1.
    Journal of molecular biology, 2008
    Co-Authors: Silvia Reddehase, Barbara Grumbt, Walter Neupert, Kai Hell
    Abstract:

    Abstract Cells protect themselves against oxygen stress and reactive oxygen species. An important enzyme in this process is superoxide dismutase, Sod1, which converts superoxide radicals into water and hydrogen peroxide. The biogenesis of functional Sod1 is dependent on its copper chaperone, Ccs1, which introduces a disulfide bond and a copper ion into Sod1. Ccs1 and Sod1 are present in the cytosol but are also found in the mitochondrial intermembrane space (IMS), the compartment between the outer and the inner membrane of mitochondria. Ccs1 mediates mitochondrial localization of Sod1. Here, we report on the biogenesis of the fractions of Ccs1 and Sod1 present in mitochondria of Saccharomyces cerevisiae. The IMS of mitochondria harbors a disulfide relay system consisting of the import receptor Mia40 and the Thiol Oxidase Erv1, which drives the import of substrates with conserved cysteine residues arranged in typical twin Cx3C and twin Cx9C motifs. We show that depletion of Mia40 results in decreased levels of Ccs1 and Sod1. On the other hand, overexpression of Mia40 increased the mitochondrial fraction of both proteins. In addition, the import rates of Ccs1 were enhanced by increased levels of Mia40 and reduced upon depletion of Mia40. Mia40 forms mixed disulfides with Ccs1, suggesting a role of Mia40 for the generation of disulfide bonds in Ccs1. We suggest that the disulfide relay system transfers disulfide bonds via Mia40 to Ccs1, which then shuttles disulfide bonds to Sod1. In conclusion, the disulfide relay system is crucial for the import of Ccs1, thereby affecting the transport of Sod1, and it can control the distribution of Ccs1 and Sod1 between the IMS of mitochondria and the cytosol.

  • functional characterization of mia40p the central component of the disulfide relay system of the mitochondrial intermembrane space
    Journal of Biological Chemistry, 2007
    Co-Authors: Barbara Grumbt, Nadia Terziyska, Vincent Stroobant, Lars Israel, Kai Hell
    Abstract:

    Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX9C motif and bridge the cysteine residues of two CX9C segments. In contrast to the stabilizing disulfide bonds of the twin CX9C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX9C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the Thiol Oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p.

  • The sulfhydryl Oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway.
    FEBS letters, 2007
    Co-Authors: Nadia Terziyska, Barbara Grumbt, Melanie Bien, Walter Neupert, Johannes M. Herrmann, Kai Hell
    Abstract:

    The Thiol Oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin "CX(3)C" or twin "CX(9)C" motifs which are crucial for import of typical substrates of this pathway and it does not need two "CX(2)C" motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.

Akihiko Kondo - One of the best experts on this subject based on the ideXlab platform.

  • Enzymatic improvement of mitochondrial Thiol Oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae
    Microbial Cell Factories, 2017
    Co-Authors: Jyumpei Kobayashi, Daisuke Sasaki, Kiyotaka Y. Hara, Tomohisa Hasunuma, Akihiko Kondo
    Abstract:

    Background Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial Thiol Oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different Thiol Oxidase genes. Results We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 ^ S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. Conclusions This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.

  • Enzymatic improvement of mitochondrial Thiol Oxidase Erv1 for oxidized glutathione fermentation by Saccharomyces cerevisiae
    Microbial cell factories, 2017
    Co-Authors: Jyumpei Kobayashi, Daisuke Sasaki, Kiyotaka Y. Hara, Tomohisa Hasunuma, Akihiko Kondo
    Abstract:

    Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial Thiol Oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different Thiol Oxidase genes. We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.

  • Improvement of oxidized glutathione fermentation by Thiol redox metabolism engineering in Saccharomyces cerevisiae
    Applied Microbiology and Biotechnology, 2015
    Co-Authors: Kiyotaka Y. Hara, Jyumpei Kobayashi, Naoko Aoki, Kentaro Kiriyama, Keiji Nishida, Michihiro Araki, Akihiko Kondo
    Abstract:

    Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae . Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering Thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of Thiol Oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of Thiol Oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the Thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Chandran Karunakaran - One of the best experts on this subject based on the ideXlab platform.

  • Recent trends in electrochemical biosensors of superoxide dismutases
    Biosensors & bioelectronics, 2018
    Co-Authors: Murugesan Balamurugan, Paulraj Santharaman, Thangamuthu Madasamy, Seenivasan Rajesh, Niroj Kumar Sethy, Kalpana Bhargava, Srigiridhar Kotamraju, Chandran Karunakaran
    Abstract:

    Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as perOxidase, nitrite Oxidase, Thiol Oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and Thiols using Thiol Oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

  • Electrochemical cysteine biosensor based on the selective Oxidase–perOxidase activities of copper, zinc superoxide dismutase
    Sensors and Actuators B: Chemical, 2010
    Co-Authors: Pitchaimani Dharmapandian, Seenivasan Rajesh, Sarkkarai Rajasingh, Ayyappan Rajendran, Chandran Karunakaran
    Abstract:

    Abstract A new cysteine (CySH) biosensor was developed based on its Oxidase–perOxidase activities with copper, zinc superoxide dismutase (SOD1). The biosensor comprising of SOD1 coimmobilized with horseradish perOxidase (HRP) on polypyrrole (PPy)-platinum (Pt) electrode was characterized by cyclic voltammetry. The bienzymatic electrode exhibited an electrochemical response with cysteine due to the bicarbonate-dependent perOxidase activity stimulated by Thiol Oxidase activity of SOD1. Among the Thiols, viz. , CySH, HCY and GSH, CySH only showed a remarkable amplification of current. Thus, the biosensor is CySH selective. The electrode linearly responded to cysteine concentrations up to 500 μM with a detection limit of 10 μM. The CySH biosensor is highly stable, selective and reproducible, making it suitable for analytical purposes. Using this biosensor, the levels of CySH present in commercially available l -CySH dietary supplement tablets, serum and urine were estimated.

  • Thiol Oxidase activity of copper, zinc superoxide dismutase stimulates bicarbonate-dependent perOxidase activity via formation of a carbonate radical
    Chemical research in toxicology, 2005
    Co-Authors: Chandran Karunakaran, Hao Zhang, Joy Joseph, William E. Antholine, Balaraman Kalyanaraman
    Abstract:

    Here, we investigated the effect of bicarbonate anion (HCO3-) on the perOxidase activity stimulated by the Thiol Oxidase activity of copper, zinc superoxide dismutase (SOD1) using electron spin res...

José M. C. Ribeiro - One of the best experts on this subject based on the ideXlab platform.

  • RHODNIUS PROLIXUS SALIVARY NITROPHORINS DISPLAY HEME-PEROxidase ACTIVITY
    Insect Biochemistry and Molecular Biology, 1998
    Co-Authors: José M. C. Ribeiro
    Abstract:

    Rhodnius prolixus is a blood feeding triatomine bug that contains salivary nitric oxide bound to hemoproteins previously named nitrophorins. Nitrophorins, in addition to storing and transporting NO, have two other functions such as anti-histaminic and anti-clotting (displayed by nitrophorin 2 only). Additionally, nitrophorins display a Thiol Oxidase reaction, where cysteine is oxidized to cystine with the production of hydrogen peroxide. In this paper the heme-perOxidase reaction of nitrophorins is described. The heme moiety of nitrophorins is destroyed by addition of cysteine or hydrogen peroxide. No biliverdin is produced during this reaction. We have also found that during the Thiol Oxidase reaction, nitrophorins can destroy norepinephrine, conferring an additional vasodilatory competence for this class of salivary molecules.

  • Salivary Thiol Oxidase activity of Rhodnius prolixus
    Insect biochemistry and molecular biology, 1996
    Co-Authors: José M. C. Ribeiro
    Abstract:

    Cysteine and other Thiol compounds can accelerate the unloading of nitric oxide (NO) from salivary nitrosyl-nitrophorins of the blood sucking bug Rhodnius prolixus. The dependence of NO unloading on cysteine concentration is biphasic, showing a maximum between 0.5 and 1 mM cysteine. The proposed mechanism of action for the unloading is a series of reactions where cysteine (at low concentrations) reacts with the heme group of nitrophorins to form cystine and superoxide. The superoxide then reacts with NO to form peroxynitrite, which decays to a mixture of nitrite and nitrate anions. At high cysteine concentrations, cysteine is converted to cystine and H2O and thus no removal of NO from nitrophorins is observed. The Thiol Oxidase activity of Rhodnius nitrophorins is similar to that observed before in plant perOxidases [Pichorner et al., Phytochemistry 31, 3371 (1992)]. The possible physiological significance of this reaction to probing and feeding by R. prolixus is discussed.