- Email: [email protected]
USA - unexpected binding mode implications for development of zinc biosensor
Biosensors & Bioelectronics Vol. 11 No. 8 (1996)
employed for the fn'st time in such a way. By potentiometrically detecting the pH shiRs of the typical reactions of the immobilized enzymes with the corresponding substrates before and after an incubation time, the relative inhibition of eleven pesticides was detenltined by forming the ratio of both the potential differences. The combination of the inhibition results of three enzyme electrodes enables to get more information, both additional and excluding, about these pollutants than with only one enzyme electrode. In this way, a separation scheme of the used substances to be detected could be derived. Contact: lnstitut fur Anorganische Chemie, Technische Universitat Clausthal, Paul-Ernst-Strasse 4, D-38678 Clausthal-Zellerfeld, Germany. USA - Unexpected binding mode implications for development of zinc biosensor In J. BIOL. CHEM. (271/2 (1003-1007) 1996) S.K. Nair, D. Elbaum & D.W. Christianson of the University of Pennsylvania report on 'Unexpected binding mode of the sulfonamide fluorophore 5-dimethylamino-1- naphthalene sulfonamide to human carbonic anhydrase II. Implications for the development of a zinc biosensor'. The three-dimensional structure of human carbonic anhydrase II (CAll) complexed with the sulfonamide fluorophore 5-di methylamino- 1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-A resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAll, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val- 121, Phe- 131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Leu-198 variants (Nail S. K., Krebs, J. F., Christianson, D. W., and Fierke, C. A. (1995) Biochemistry 34, 39813989). Modeling studies indicate that a second binding mode, in which the fused aromatic ring is rotated out of the hydrophobic pocket, is sterically feasible. Both
experimentally observed and modelled binding modes have implications for new leads in the design of avid CAII inhibitors. Finally, the structure of the CAII-dansylamide complex has implications for its exploitation in zinc biosensor applications, and possible routes toward the optimization of fluorophore design are considered on the basis on this structure. Contact: Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA. USA- Quantitating staphylococcal enterotoxin B In ANAL. BIOCHEM. (233/1 (50-57) 1996) L.A. Tempelman, K.D. King, G.P. Anderson & F.S. Ligler of the Naval Research Laboratory report on 'Quantitating staphylococcal enterotoxin B in diverse media using a portable fiber-optic biosensor'. A new, portable fiber-optic biosensor has been used to detect staphylococcal enterotoxin B, a causative agent of food poisoning, at levels as low as 0.5 ng/ml in buffer. The toxin (SEB) can also be detected and quantitated in other relevant media: human serum, urine, and aqueous extract of ham. The level of toxin, from 5 to 200 ng/ml, can be accurately predicted in these media by calibrating each fiber and by comparing results to a single standard curve based on toxin in buffer. The quantitative fluorescent sandwich immunoassay provides results in 45 min; qualitative results are provided in 15-20 min. Using a blender and a benchtop centrifuge, fast, simple aqueous extracts of contaminated ham samples were prepared and tested. Ham spiked with 5 or 40 ttg SEB per 100 g food resulted in biosensor readings indicative of 11 or 69% recovery of the toxin, respectively. Finally, the SEB assay is highly specific; SEA and SED give only 2-3% of the signal at 5000 ng/ml as SEB gives at 1000 ng/ml. This specific, sensitive assay for SEB on the portable fiberoptic biosensor permits easy monitoring of clinical samples or on-site analysis of suspect food samples. Contact: Center for Bio/Molecular Sci./Engg., Naval Research Laboratory, Washington, DC 203 75, USA.
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employed for the fn'st time in such a way. By potentiometrically detecting the pH shiRs of the typical reactions of the immobilized enzymes with the corresponding substrates before and after an incubation time, the relative inhibition of eleven pesticides was detenltined by forming the ratio of both the potential differences. The combination of the inhibition results of three enzyme electrodes enables to get more information, both additional and excluding, about these pollutants than with only one enzyme electrode. In this way, a separation scheme of the used substances to be detected could be derived. Contact: lnstitut fur Anorganische Chemie, Technische Universitat Clausthal, Paul-Ernst-Strasse 4, D-38678 Clausthal-Zellerfeld, Germany. USA - Unexpected binding mode implications for development of zinc biosensor In J. BIOL. CHEM. (271/2 (1003-1007) 1996) S.K. Nair, D. Elbaum & D.W. Christianson of the University of Pennsylvania report on 'Unexpected binding mode of the sulfonamide fluorophore 5-dimethylamino-1- naphthalene sulfonamide to human carbonic anhydrase II. Implications for the development of a zinc biosensor'. The three-dimensional structure of human carbonic anhydrase II (CAll) complexed with the sulfonamide fluorophore 5-di methylamino- 1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-A resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAll, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val- 121, Phe- 131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Leu-198 variants (Nail S. K., Krebs, J. F., Christianson, D. W., and Fierke, C. A. (1995) Biochemistry 34, 39813989). Modeling studies indicate that a second binding mode, in which the fused aromatic ring is rotated out of the hydrophobic pocket, is sterically feasible. Both
experimentally observed and modelled binding modes have implications for new leads in the design of avid CAII inhibitors. Finally, the structure of the CAII-dansylamide complex has implications for its exploitation in zinc biosensor applications, and possible routes toward the optimization of fluorophore design are considered on the basis on this structure. Contact: Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA. USA- Quantitating staphylococcal enterotoxin B In ANAL. BIOCHEM. (233/1 (50-57) 1996) L.A. Tempelman, K.D. King, G.P. Anderson & F.S. Ligler of the Naval Research Laboratory report on 'Quantitating staphylococcal enterotoxin B in diverse media using a portable fiber-optic biosensor'. A new, portable fiber-optic biosensor has been used to detect staphylococcal enterotoxin B, a causative agent of food poisoning, at levels as low as 0.5 ng/ml in buffer. The toxin (SEB) can also be detected and quantitated in other relevant media: human serum, urine, and aqueous extract of ham. The level of toxin, from 5 to 200 ng/ml, can be accurately predicted in these media by calibrating each fiber and by comparing results to a single standard curve based on toxin in buffer. The quantitative fluorescent sandwich immunoassay provides results in 45 min; qualitative results are provided in 15-20 min. Using a blender and a benchtop centrifuge, fast, simple aqueous extracts of contaminated ham samples were prepared and tested. Ham spiked with 5 or 40 ttg SEB per 100 g food resulted in biosensor readings indicative of 11 or 69% recovery of the toxin, respectively. Finally, the SEB assay is highly specific; SEA and SED give only 2-3% of the signal at 5000 ng/ml as SEB gives at 1000 ng/ml. This specific, sensitive assay for SEB on the portable fiberoptic biosensor permits easy monitoring of clinical samples or on-site analysis of suspect food samples. Contact: Center for Bio/Molecular Sci./Engg., Naval Research Laboratory, Washington, DC 203 75, USA.
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