50% bacterial strains and 1-chloro-2,4-dinitrobenzene
(CDNB) compound as a substrate were used in the experiments done to determine GST activity.
CdGSTP1-1 was heterologously expressed in Eschericia coli BL21(DE3) as a ~24 kDa soluble protein and showed to be catalyticly active towards the model substrate 1-chloro-2,4-dinitrobenzene
. The results of the present study provide new information into camelid evolution and give further insights into the diversity and complex enzymatic functions of GST superfamily.
Prabavathi, "Analysis of vibrational spectra of 1-chloro-2,4-dinitrobenzene
based on density functional theory calculations," Spectrochimica Acta--Part A: Molecular and Biomolecular Spectroscopy, vol.
2,2-Azobis(2-methyl-propionamidine) dihydrochloride (AAPH), copper sulphate, Chelex[R] 100 sodium, quercetin, thiobarbituric acid, trichloroacetic acid, Amberlite XAD-7, sodium bicarbonate, methylglyoxal, glutathione (reduced form), 1-chloro-2,4-dinitrobenzene
(CDNB), Lglutamine, and CelLytic[TM] M were purchased from SigmaAldrich (St.
Glutathione and CDNB (1-chloro-2,4-dinitrobenzene
) were served as substrates in this assay.
The enzyme reaction system included 25 uL enzyme solution, 225 uL of 3.3 mmol*L-1 glutathione and 3 mL of 0.1 mol*L-1 phosphate buffer (pH 7.4); 25 uL of 30 mmol L-1 1-chloro-2,4-dinitrobenzene
was used to activate the reaction, and changes in absorbance were measured within 2 min at 340 nm.
For glutathione S-transferase activity, CNDB (1-chloro-2,4-dinitrobenzene
; Sigma, USA; Cat.
GST activity was measured using 1-chloro-2,4-dinitrobenzene
(CDNB) as a substrate by the method of Habig and Jacoby .
Louis, Missouri, USA): nicotinamide adenine dinucleotide phosphate (NADP); glycylglycine; glucose-6-phosphate; l-y-glutamyl-3-carboxyl-4nitronilide; epinephrine; hydrogen peroxide; glutathione (GSH); 5,50-dithio-bis-2-nitrobenzoic acid; thiobarbituric acid; and 1-chloro-2,4-dinitrobenzene
. Acetaminophen was from Panadol, GlaxoSmithKline Consumer Plc.
(CDNB), 5',5'-dithiobis-2-nitrobenzoic acid (DTNB), glutathione (GSH), epinephrine, hydrogen peroxide ([H.sub.2][O.sub.2]), and thiobarbituric acid (TBA) were all purchased from Sigma Chemical Company (London, UK).
Ballatori, "Intrahepatic conversion of a glutathione conjugate to its mercapturic acid: metabolism of 1-chloro-2,4-dinitrobenzene
in isolated perfused rat and guinea pig livers," The Journal of Biological Chemistry, vol.
Quercetin, glutathione, 1-chloro-2,4-dinitrobenzene
(CDNB), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), epinephrine, and hydrogen peroxide ([H.sub.2][O.sub.2]) were all purchased from Sigma Chemical Company (London, UK).