Browsing by Author "Alalade, Foluso Ruth."

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    Purification and characterisation of 3-mercaptopyruvate sulfurtransferase from the liver of fruit bat (eidolon helvum)
    (Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile Ife, Nigeria., 2024) Alalade, Foluso Ruth.
    This study isolated and purified 3-mercaptopyruvate sulfutransferase (3-MST) from the liver of fruit bat (Eidolon Helvum). It also determined the physicochemical properties of 3-MST from E.helvum. These were with a view to providing information on the physicochemical properties of the enzyme. Eidolon helvum (fruit bat) were collected from the Botanical Garden, Obafemi Awolowo University, Ile-Ife, Nigeria and identified at the department of Zoology, O.A.U, Ile-Ife. The E.helvum were transported to the Enzymology Laboratory, Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria and the liver were immediately excised stored in the refrigerator until use. The liver was allowed to thaw weighed and homogenized using 3 volumes of homogenization buffer (0.1M phosphate buffer, pH 7.4). The homogenate was centrifuged at 10,000 rpm for 15 minutes at 4 ºC to remove cell debris. The supernatant was collected and assayed for 3-MST activity and protein concentration was determined following standard methods. Purification of the enzyme from Eidolon helvum was carried out using ammonium sulphate precipitation, ion exchange chromatography on CM Sephadex C-50 cation exchanger and Sephadex G-100 gel filtration chromatography. The native molecular weight of the purified 3- mercaptopyruvate sulfurtransferase was determined using Sephadex G-100 while the subunits molecular weight of the purified 3-mercaptopyruvate sulfurtransferase was determined using SDS PAGE. The purified enzyme was further characterized by determining the effects of pH and temperature (optimum and stability). The kinetic parameters, substrate specificity (using sulphur compounds), effects of inhibitors and effects of metals was also determined. 3-Mercaptopyruvate sulfurtransferase from the liver of E. helvum was purified with a specific activity of 11.08 U/mg, purification fold of 8.21 and % yield of 46.92. The native molecular weight and subunit molecular weight were 36.24 kda and 35.72 kda respectively. The apparent Km and Vmax values for potassium cyanide and mercaptoethanol were 8.60 ± 0.17 mM, 96.65 ± 0.56 MU/ml/min and 7.77 ± 0.42 mM, 48.02 ± 0.38 MU/ml/min respectively. The optimum temperature and pH were 70 ºC and 7.0 respectively. Substrate specificity study involving the use of other sulphur compounds revealed that 3-MST from the liver of E. helvum showed high preference for mercaptoetanol and sodium thiosulphate. Metal salts (SnCl2, HgCl2, MnCl2, NaCl, MgCl2 and KCl) showed different degrees of inhibition of 3-MST from the liver of E. helvum. At 70 ºC, 3-MST from the liver of Eidolon helvum retained 66.67 % of its activity for 40 minutes. Urea and EDTA inhibited the activity of the 3-MST while DPPD did not affect the activity of the enzyme. The study established the presence of Mercaptopyruvate sulfurtransferase (a cyanide detoxifying enzyme) in the liver of Eidolon helvum. It further showed the physicochemical properties of 3-MST in the liver of Eidolon helvum. This could possibly be responsible for the mechanism of survival of the Eidolon helvum through cyanide detoxification in their environment.