Purification and Characterization of Biliverdin IX_alpha from Atlantic Salmon (Salmo salar) Bile

Z. K. Ding^1,2 and Y. Q. Xu^1*

¹Key Laboratory of Marine Biology, Shantou University, Shantou City, Guangdong Province 515063, People's Republic of China

²Department of Zoology, University of Toronto, Toronto, ON, Canada M5S 3G5; E-mail: ZhaokunD@hotmail.com

^* To whom correspondence should be addressed.

Received November 18, 2001; Revision received January 14, 2002

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Biliverdin IX_alpha 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 IX_alpha in the bile was quite stable when the bile was frozen at -80°C for a period of 40 days. However, 7.1% of the biliverdin IX_alpha was lost when the bile was stored at 4°C for 20 days. The purified biliverdin IX_alpha appeared as a single spot with R_f 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 IX_alpha 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 IX_alpha 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 IX_alpha to bilirubin IX_alpha 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 IX_alpha, per mole of biliverdin IX_alpha reduced or per mole of bilirubin IX_alpha formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.

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KEY WORDS: biliverdin, heme degradation, biliverdin reductase, bilirubin, salmon

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