Purification of hemocyanin from white shrimp (Penaeus vannamei boone) by immobilized metal affinity chromatography

Hemocyanin (Hc) was isolated from white shrimp (Penaeus vannamei Boone) plasma by density gradient ultracentrifugation and immobilized metal affinity chromatography. Hc was contained in the subnatant high density fraction and was adsorbed by an iminodiacetic acid (Ni-DA) column that bound Hc and apohemocyanin. Hc molecular weight was estimated by pore limiting electrophoresis as 400 kDa. It is composed of two subunits of approximately 75 and 82 kDa. This 400-kDa protein contained copper and was detected by antibodies raised against the purified subunitsl Isoelectric points of the affinity purified native protein were 4.8 and 4.9. Presence of covalently bound carbohydrates in both subunits was detected by biotinylated lectins and avidinhorseradish peroxidase.