PURIFICATION OF HEMOCYANIN FROM WHITE SHRIMP (PENAEUS-VANNAMEI BOONE)BY IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY

Hemocyanin (He) was isolated from white shrimp (Penaeus vannamei Boone ) plasma by density gradient ultracentrifugation and immobilized metal affinity chromatography. He was contained in the subnatant high densi ty fraction and was adsorbed by an iminodiacetic acid (Ni-IDA) column that bound He and apohemocyanin. He molecular weight was estimated by pore limiting electrophoresis as 400 kDa. It is composed of two subuni ts of approximately 75 and 82 kDa. This 400-kDa protein contained copp er and was detected by antibodies raised against the purified subunits . Isoelectric points of the affinity purified native protein were 4.8 and 4.9. Presence of covalently bound carbohydrates in both subunits w as detected by biotinylated lectins and avidin-horseradish peroxidase. (C) 1997 Elsevier Science Inc.