Purification of L-kynurenine 3-hydroxylase by affinity chromatography

NADP immobilized on agarose is able to adsorb L-kynurenine 3-hydroxylase. The enzyme is released from the adsorbent by passage of a buffer containing 0.5 mM NADP through the column. L-Kynurenine 3-hydroxylase was purified 26-fold with a yield of 12% from mitochondrial outer membrane with a procedure involving DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography and NADP-agarose affinity chromatography. This monooxygenase was a homogeneous protein, giving a monomeric molecular weight of 145,000, which had neither any significant NADPH diaphorase activity nor cytochrome b5-like haem protein. However, the enzyme did not show affinity for a column with L-kynurenine coupled to the gel with a suitable spacer group, and AMP did not serve as an effective ligand in an affinity resin.