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Sivelestat mg The isoelectric point of CL ARG was determined
The isoelectric point of CL-ARG was determined by native IEF at a Sivelestat mg gradient in the range (3–10) using standard protein markers with known isoelectric points. Native IEF reveals the presence of only one major band for CL-ARG with a slightly neutral-basic pI value of about 7.7. The calculated pI value for the purified CL-ARG coincides with the behaviour of the enzyme on the anion and cation exchanger columns chromatography. Mammalian liver arginases tend to have basic pI values of about 8.8-9.4 as rat liver arginase (9.4) and human liver arginase (9), while pI values closer to a neutral pH exist in beef liver arginase (5.9), pig liver arginase (6.9 and 8.8), rabbit liver arginase (6.5–7.2) and monkey liver arginase (6.8–7.5) [3]. Türkoglu and Özer [42] separated bovine liver arginase into three distinct peaks by chromatofocusing in the pH range 4–7 since they concluded that the enzyme was homogenous towards subunit size and kinetic behaviour but heterogeneous via its molecular charge. By investigating the enzyme composition, sample of the purified CL-ARG was assayed with anthrone to detect the carbohydrate content. The assays yielded a hexose content of 2.7%. Mammalian liver arginase contains little or no bound carbohydrate. Hexose sugar content for rat and beef liver arginases has been estimated to be 1–3% and 3–5%, respectively [37], [43], while there was no carbohydrate detected in human liver arginase [44]. The effect of substrate concentration on the reaction velocity of purified CL-ARG was examined using arginine concentration in the range (10–50mM). The results reveal the enzymatic hyperbolic dependence of hydrolysis rate on substrate concentration and a linear relationship was obtained when 1/v was plotted against 1/[S]. The Michaelis constant (Km) of CL-ARG was calculated to be 7.1mM. Beef liver arginase has a Km value of 14mM [45], the Km value of buffalo liver arginase was 2mM [25], that of human liver arginase was 5.4mM [46], the Km values recorded for rabbit, monkey and horse were 1.4mM, 6.5mM and 4.6mM, respectively [38]. The major variable in the kinetics of the arginase reaction is the pH determinant, which reflects the amphoteric character of both the substrate (L-arginine) and the enzyme. Another factor is the effect of metal ion cofactor on the dissociation of ionizable groups of the catalytically active center of the enzyme [47]. CL-ARG activity as a function of pH increased giving two optimum pH at 9 and 10.7 using 50mM Tris-HCl buffer and 50mM carbonate-bicarbonate buffer, respectively. The optimum pH of buffalo liver arginase is 9.2 [25], human liver arginase has an optimum pH of 9.3 [36]. The pH optima of rabbit liver arginase is 10, that of monkey is 9.5-10.5 while that of horse is 10.2 [38]. Gasiorowska et al. [48] found four differently charged isozymes of arginase in different rat tissues one of which had a pH optimum of 7.5 also a minor arginase component was reported by Robbins and Shields [49] in beef liver with an optimal pH 7. The variation of activity with pH suggests titration of an ionizable group that may function at the catalytic site. The basic pH-activity profile may reflect ionization of manganese-bound water [3]. Also, our study showed that CL-ARG has an optimum temperature at 70°C. It was found that arginase from buffalo liver showed maximum activity at 42°C [25]. A common feature of arginases, whether of eukaryotic or prokaryotic origin, is a requirement of divalent cations for activity. Mn+2 is the physiologic activator, although the divalent cation requirement for some arginases has been reported to be satisfied by Co+2 and Ni+2 and in some instance by Fe+2 and Cd+2. It was shown that fully Mn+2-activated arginase contains 2 Mn+2/subunit and these Mn+2 ions form electron spin-coupled binuclear centers [50]. In the present study, Fe+3 and Co+2 were found to act as good activators while Sr+3 and Zn+2 act as inhibitors for CL-ARG. Dabir et al. [25] observed the activation of buffalo liver arginase with Mg+2, Ca+2, Co+2, Ni+2, Cd+2 and its inhibition by Zn+2.