Purification of turnip peroxidase and its kinetic properties.
Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA).
Singh
Naresh
N
Center for Biochemical Technology CSIR, Delhi University Campus, India. ssnaresh@hotmail.com
Gade
W N
WN
Singh
Jai
J
D029688:Brassica napus / Q000201:enzymology*; D002021:Buffers; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D009195:Peroxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D002021:Buffers; D007501:Iron; D009195:Peroxidase