Detoxification of cyanide in insects. I. Purification and some properties of rhodanese from the gut of the variegated grasshopper Zonocerus variegatus (Orthoptera: Pyrgomorphidae)

Abstract

The purification and characterization of rhodanese, an enzyme that catalyses the detoxification of cyanide, from the gut of the variegated grasshopper ( Zonocerus variegatus L.) were carried out to understand the biochemical basis of the survival of this grasshopper living on cyanogenic plants such as cassava. All experiments, including enzyme assay, were carried out at room temperature and all buffers contained 10 m m sodium thiosulphate to stabilize the enzyme. Grasshoppers were caught alive from a cassava farm within the locality and kept frozen until analysis. Each grasshopper was dissected and the gut was removed quickly. Approximately 102 g of the gut were homogenized in three volumes of 0.1 m acetate glycine buffer (pH 7.8) containing ɛ-amino- n -caproic acid. The supernatant was collected by centrifugation at 12,000 rpm, for 30 min at 40 °C. The enzyme was purified to homogeneity by a combination of procedures such as ammonium sulphate precipitation, ion-exchange chromatography (CM-Sephadex and DEAE-Sephadex), gel filtration (Sephadex G-75) and Agarose-Blue affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and non-SDS–PAGE were used to ascertain the purity of the enzyme. The native and subunit molecular weights of the enzyme were determined by gel filtration on a Bio-Gel P-200 column and SDS–PAGE, respectively. Kinetic parameters were determined by using varying concentrations of one of the substrates at a fixed concentration of the other, and vice versa. Furthermore, the effects of temperature, pH and cations on the activity of the enzyme were investigated. The purified enzyme had a specific activity of 51.7 μmol thiocyanate formed/ml/min/mg protein (U/mg protein) with a yield of about 29%. The apparent molecular weight of the enzyme estimated by Sephadex G-75 gel filtration was 35,400 ± 482 Da and its subunit molecular weight determined by SDS–PAGE was 33,000 ± 212 Da. The K m values of KCN and Na 2 S 2 O 3 were found to be 29.63 ± 02.87 and 26 ± 03.04 m m , respectively. An optimum pH and temperature of 7.0 and 35 °C, respectively, were obtained for the enzyme. The results of enzyme inhibition showed that the activity of the enzyme was not affected by NH 4 Cl, MgCl 2 , CoCl 2 , CaCl 2 , MnCl 2 , NiCl 2 and SnCl 2 , but inhibited by ZnCl 2 and BaCl 2 . In conclusion, these results suggest that the survival of Z. variegatus depends on the presence of the enzyme rhodanese, which shows high activity and has suitable kinetic properties in the gut of the grasshopper that feeds mainly on cassava leaves which are cyanogenic.