Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

• Published in: Comparative biochemistry and physiology. Toxicology & pharmacology 2003, Vol.134 (1), p.101-107
• Main Authors: Ryu, Soo-Kyung, Park, Jin-Sung, Lee, In-Sook
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Animals; Carrier Proteins - analysis; Carrier Proteins - chemistry; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Copper; Copper-binding protein; Electrophoresis, Polyacrylamide Gel; Environmental Monitoring - methods; Littorina brevicula; Marine; metal-binding protein; metallothionein; Metallothionein - analysis; Metallothionein - chemistry; Molecular Weight; Mollusca - chemistry
• ISSN: 1532-0456; 1878-1659
• DOI: 10.1016/S1532-0456(02)00217-X

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 μM CuCl 2) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 μg/l CuCl 2). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C 18 reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.