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(19) Hemolysate was permitted to react with [H.sub.2][O.sub.2] along with GSH, then the remainder of GSH was permitted to react with DTNB. Yellow colour yielded was measured at 412 nm.
After 10 mins, the color reagent DTNB was poured on to the plates and the AChE activity was observed.
The effect of substrate concentration on the velocity of the enzyme reaction was investigated by using varying concentrations of NADPH (0.015-0.24 mM) and DTNB (0.19-3 mM) as substrates.
In the 96-well plates, 25 [micro]L of tissue homogenate, followed by 125 [micro]L of 3mM DTNB and 25 [micro]L of buffer, was added.
The levels of sulfhydryl compound, pancreatic T-SH, and NP-SH were determined by Ellman's reaction using 5'5'-dithio-bis-2nitrobenzoic acid (DTNB).
Aliquots of supernatant (50 [micro]L for brain and 100 [micro]L for muscle) were incubated at 30 [degrees]C for 2 min with a solution containing 0.1 M sodium phosphate buffer, pH 7.5, and 1 mM DTNB. After the incubation period, the reaction was initiated by the addition of acetylthiocoline (0.5 mM).
After centrifugation, the supernatants were assayed for glutathione content using DTNB. The activity of GPx is expressed as U/g hemoglobin of erythrocyte lysate.
Foi utilizado acido ditiobisnitrobenzoico (DTNB), iodeto de acetiltiocolina, iodeto de pralidoxima e paraoxon do fabricante Sigma- Aldrich[R] e foi utilizado o leitor de microplacas por espectroscopia do fabricante Molecular Devices[R], disponivel no laboratorio do Instituto de Biologia do Exercito.
All reagents and chemicals used for biochemical analyses [2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), HCl, reduced GSH and oxidized glutathione (GSSG), 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), GSH reductase] were obtained from Sigma-Aldrich Chemie GmbH (Darmstadt, Germany).
Then, 25 [micro]L of acetylthiocholine iodide (Electrophorus electricus, Sigma Aldrich) 1000 U [mL.sup.-1]; the reaction mixture, 125 [micro]L of 5'.5-dithio-bis (2- nitrobenzoate) (DTNB, Sigma D8130) (3 mM) and 50 [micro]L of Tris/HCl (0.1% m/v, pH=8) containing bovine serum albumin was added to each well.
Reagents and Chemicals used in this study were 2 thionitrobenzoic acid (DTNB), ethylenediamine tetracetic acid (EDTA), 2,4,6-tripyridyl-s-triazine (TPTZ) and peroxide hydrogen(H O ).
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