Purification of L-kynurenine 3-monooxygenase from mitochondrial outer membrane of pig liver

Abstract

The aim of the present work is to obtain the homogenous L-kynurenine 3-monooxygenase (Fpk) enzyme preparation by a simple and rapid immunoaffinity purification method. Fpk was purified by monoclonal antibody (mAb) immunoabsorbent column. The column was prepared using hydrazide-activated agarose beads (Affi-Gel Hz) to which IgG molecules were coupled via carbohydrate moieties located on the Fc region and peroxidized with periodate. Partially purified Fpk was charged on the column and after washing the column with buffer containing 0.5M NaCl and 0.5% Triton-X-100 and then with buffer alone, the enzyme was eluted with acidic elution buffer. Despite the loss of the catalytic activity due to the acidic elution, the immunoaffinity preparation may be useful for the analysis of the chemical structure of Fpk and for the production of the polyclonal antibody toward Fpk.