ABSTRACT
Arginase (L-arginine amidinohydrolase, EC.3.5.3.1) from animal tissues such as, liver and kidney has been partially characterized by many researchers. In this study, we purified arginase to homogeneity from buffalo liver with about ~2857 purification fold and a 20% recovery by chromatographic and spectroscopic analysis were obtained. The molecular mass determined by gel filtration and SDS-PAGE was found to be 118 kDa and 47 kDa, respectively. The optimal pH and temperature of the arginase was 9.5 and 40°C, respectively. Kinetic parameters (Km and Vmax) showed activation of arginase in the reaction medium with decrease in Km (7.14, 5.26, 4.0 and control 3.22 mM) and Vmax (0.05, 0.035, 0.027 and control 0.021 mg/mL/min), while co-factor activity of arginase was optimized using metal ions like Mn2+ and Mg2+ at 2 mM, which revealed an increase in Vmax values (0.011, 0.013, 0.015 and control 0.010 mg/mL/min) and a decrease in Km values (2.22, 2.12, 1.88 and control 1.66 mM). The kinetic data suggested that the arginase activity is enhanced in the presence of dihydropyrimidine derivative and metal ions, indicating essential mode of activation.
ABSTRACT
Grape juice and grape skin extracts are important commercial source of polyphenolic compounds which exert different functional properties such as color potential, antimicrobial, antioxidant activity, and health benefits. In this paper we describe a sensitive and specific assay for determination of bioactive polyphenolic compounds in Campbell Early (Vitis labrusca cv. baile). Five polyphenolic components were separated on an Agilent Zorbax Extend C18 Column (250 mm × 4.6 mm × 5 μm) and detected by a diode array detector. The mobile phase was composed of (a) aqueous phosphoric acid (0.2%, v/v); and (b) acetonitrile using a gradient elution. Analytes were performed at 25°C with a flow rate of 0.8 ml/min and UV detection at 280, 360, and 520 nm. All calibration curves showed good linear regression (r2 ≥ 0.9999) within tested ranges. Overall intra- and inter-day variations were less than 1.90%, and the average recoveries were 95.5-105% for analytes. The antioxidant activity determined by DPPH radical assay, ranged from 86-105 for extracts, and 165-252 for studied standards (µM trolox/100 g dry wt.). The proposed method would be sensitive enough and reliable for quality control in functional food and modernization of Campbell Early (Vitis labrusca cv. baile) as potent antioxidant agent.