Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile

Biliverdin IXalpha was purified from the bile of Atlantic salmon (Salmo salar) using a silica gel (Wakogel C-200) column. The yield was 49.5 mg per 100 ml of fresh bile and purity 95.3%. The biliverdin IXalpha in the bile was quite stable when the bile was frozen at -80 degrees C for a period of 40 days. However, 7.1% of the biliverdin IXalpha was lost when the bile was stored at 4 degrees C for 20 days. The purified biliverdin IXalpha appeared as a single spot with Rf value of 0.25-0.27 on thin layer chromatography (TLC) and one main peak on high performance liquid chromatography (HPLC) at 436 or 650 nm. When the biliverdin IXalpha was subjected to enzymic reduction with highly purified biliverdin reductase, two clear isobestic points were seen, at 384 and 670 nm. When the products of the reaction with biliverdin IXalpha were extracted in butanol after completion of the reaction, one absorbance peak was observed at 468 nm. The time course of the reduction of biliverdin IXalpha to bilirubin IXalpha catalyzed by biliverdin reductase depended on reduced pyridine nucleotide. The time course of the NADPH-dependent reaction is different from that of the reaction with NADH. In the reduction of biliverdin IXalpha, per mole of biliverdin IXalpha reduced or per mole of bilirubin IXalpha formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.