Open AccessPurification of NADP-Isocitrate dehydrogenase from Red Kidney beans (Phaseolus vulgaris rogue)
Author(s) -
Mukaram Shikara,
Hiba Muneer Al-Khafagi,
Wasnaa H. Faris
Publication year - 2010
Publication title -
maǧallaẗ markaz buḥūṯ al-taqniyyaẗ al-aḥyāʾiyyaẗ
Language(s) - English
Resource type - Journals
eISSN - 2708-1370
pISSN - 1815-1140
DOI - 10.24126/jobrc.2010.4.1.96
Subject(s) - phaseolus , isocitrate dehydrogenase , nicotinamide adenine dinucleotide phosphate , enzyme assay , enzyme , dehydrogenase , chromatography , antiserum , biochemistry , chemistry , biology , microbiology and biotechnology , botany , oxidase test , antigen , genetics
Nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) was extracted from red kidney beans (Phaseolus vulgaris.) after the beans were placed into Murashige-Skoog medium and incubated under continuous white light (110 μmol photon m−2 s−1), then filtered, centrifuged and the supernatant was used for purification. The enzyme purified using ammonium sulphate precipitation, DEAE-cellulose and Matrex Bio red A (dye-ligand-chromatography) techniques, and exhibits several bands through electrophoresis, with one band corresponds to the IDH activity. Km values for the enzyme was 55.71± 4.56 x 10-6M. The enzyme has an optimum pH at 8.5, and optimum temperature at 30°C. The enzyme can be stable at RT (about 25°C) for 180min, but the activity disappears at 400min. Enzyme activity appears to be independent of divalent metals in deionized water, but the addition of Mg+2 and Mn+2 by 4.5 and 2-folds respectively. The purified enzyme was injected into white rabbits to raise an antiserum against NADP+-IDH. The specificity of the antiserum was assayed by its ability to decrease the NADP+-IDH activity present in the extract. NADP+-IDH activity decreased when the extract was incubated with increasing volumes of the antiserum obtained.