Enzymatic spectrophotometric determination of nitrites in beer,
Girotti, S., Ferri E. N., Fini F., Ruffini F., Budini R., Moura I., Almeida G., Costa C., Moura J. J. G., and Carrea G.
, Analytical Letters, 1999, Volume 32, Number 11, p.2217-2227, (1999)
AbstractA colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10(-8) - 10(-2) M range with a detection limit of 10(-8) M. and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NIR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10(-5) - 10(-2) M) and a higher detection limit (10(-5) M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.
EPR and Mossbauer spectroscopic studies on enoate reductase,
Caldeira, J., Feicht R., White H., Teixeira M., Moura J. J., Simon H., and Moura I.
, J Biol Chem, Aug 2, Volume 271, Number 31, p.18743-8, (1996)
AbstractEnoate reductase (EC 1.3.1.31) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN. The enzyme reduces the alpha,beta carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen. UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm. EPR redox titration shows two widely spread mid-point redox potentials (-190 and -350 mV at pH 7. 0), and a nearly stoichiometric amount of this species is detected. The data suggest the semiquinone radical has an anionic nature. In the reduced form, the [Fe-S] moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4. 2-60 K) with g values at 2.013, 1.943, and 1.860 (-180 mV at pH 7.0). The gmax value is low when compared with what has been reported for other iron-sulfur clusters. Mossbauer studies reveal the presence of a [4Fe-4S]+2/+1 center. One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states. Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mossbauer parameters. The Mossbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.
ESR studies of cytochrome c3 from Desulfovibrio desulfuricans strain Norway 4: Midpoint potentials of the four haems, and interactions with ferredoxin and colloidal sulphur,
Cammack, R., Fauque G., Moura J. J. G., and Legall J.
, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Volume 784, Number 1, p.68-74, (1984)
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