Physicochemical and kinetic characteristics of rhodanese from the liver of African catfish Clarias gariepinus Burchell in Asejire lake

Abstract

Two forms of rhodanese were purified from the liver of Clarias gariepinus Burchell, designated catfish rhodanese I (cRHD I) and rhodanese II (cRHD II), by ion-exchange chromatography on a CM-Sepharose CL-6B column and gel filtration through a Sephadex G-75 column. The apparent molecular weight obtained for cRHD I and cRHD II was 34,500 +/- 707 and 36,800 +/- 283 Da, respectively. The subunit molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 33,200 +/- 283 and 35,100 +/- 141 Da for cRHD I and cRHD II, respectively. Atomic absorption spectrophotometric analysis revealed that cRHD II contained a high level of iron (Fe), which presumably was responsible for the brownish colour of the preparation. In contrast, no Fe was identified in cRHD I, and its preparation was colourless. Further characterization of cRHD II gave true Michaelis-Menten constant (K(m)) values of 25.40 +/- 1.70 and 18.60 +/- 1.68 mM for KCN and Na(2)S(2)O(3), respectively, an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The Arrhenius plot of the effects of temperature on the reaction rate consisted of two linear segments with a break occurring at 40 degrees C. The apparent activation energy values from these slopes were 7.3 and 72.9 kcal/mol. Inhibition studies on the cRHD II enzyme showed that the activity of the enzyme was not affected by Mn(2+), Co(2+), Sn(2+), Ni(2+) and NH(4) (+), but Zn(2+) inhibited the enzyme considerably.

Properties of rhodanese from the liver of tilapia, Oreochromis niloticus, in Asejire Lake, Nigeria

“…Agboola and Okonji (2004) reported 35°C for the rhodanese in the cytosolic fraction of fruit bat liver. Also, Akinsiku et al (2010) reported 40°C for the rhodanese in the liver of catfish (C. gariepinus) from Asejire Lake, same source of tilapia for this study. Tayefi-Nasrabadi and Rahmani (2012) reported 25°C for rhodanese from the liver of rainbow trout.…”

“…These values are lower than those of rainbow trout (KCN = 36.81mM; Na 2 SO 4 = 19.84 mM) (Tayefi-Nasrabadi and Rahmani, 2012), african catfish liver (KCN = 25.40 mM; Na 2 SO 4 = 18.60 mM) (Akinsiku et al, 2010), fruit bat liver (KCN = 13.36 mM; Na 2 SO 4 = 19.15 mM) (Agboola and Okonji, 2004), mouse liver (KCN = 12.50 mM; Na 2 SO 4 = 8.30 mM) (Lee et al, 1995), bovine liver (KCN = 19.0 mM; Na 2 SO 4 = 6.7 mM) (Sorbo, 1953), and human liver (KCN = 9.50 mM; Na 2 SO 4 = 4.50 mM) (Jarabak and Westley, 1974), indicating that the affinity of tilapia enzyme for these substrates is more than that of the other enzymes and that it would catalyze the detoxification reaction with high efficiency. As reported by Agboola and Okonji (2004) less effective enzymatic system may be due to a lower exposure to cyanide and high affinity in some mammalian animals is due to continue exposure through their diet.…”

“…Tayefi-Nasrabadi and Rahmani (2012) reported 25°C for rhodanese from the liver of rainbow trout. Akinsiku et al (2010) posited that with the level of pollution in this water, there will be various metabolic activities going on in it which likely results in the release of heat and the ambient temperature of 40°C which might have conditioned the enzyme to function at higher temperature. The heat stability experiment showed that the enzyme was stable up to 60°C for about 30 min which means that the enzyme is thermostable.…”

“…In early report from our laboratory we described the physicochemical and kinetic characterization of rhodanese from the liver of Clarias gariepinus Burchell in Asejire lake (Akinsiku et al, 2010), this paper describes the isolation and characterization of rhodanese from the cytosolic fraction of tilapia liver from the lake.…”

“…thiosulphate) to cyanide, leading to formation of less toxic sulphite and thiocyanate (Gupta et al, 2010). The enzyme has been purified and characterised from a number of animal tissues (Lee et al, 1995;Agboola and Okonji, 2004;Akinsiku et al, 2010) some of which are actively exposed to cyanide contaminated environments or food. The specific activity of rhodanese in many animal tissues is present in Table 1 *Corresponding author.…”