Cadmium binding proteins induced in exposed freshwater crayfishProcambarus clarkii

Abstract

This work describes results on the characterization of cadmium binding proteins (Cd-BPs) obtained from cadmium exposed freshwater crayfish Procambarus clarkii. After acclimation to laboratory conditions, induction of Cd-BPs was achieved by water exposure at a concentration of 100 micrograms Cd/L during 2, 15, and 30 d. In accordance with the method followed by Engel and Brouwer, in each case two midgut glands were minced and homogenized in Tris-HC1 buffer with PMSF to prevent protease activity and DTT to maintain reducing conditions. The homogenate was centrifuged, heat treated, applied to a column of Sephadex G-75, and eluted with the same buffer (pH 8.6). Absorbances of the fractions collected were measure at 254 and 280 nm. Cadmium concentrations were determined by flame photometry. In midgut glands of two-d treated crayfish, significant levels of cadmium occurred in the void volume, and no cadmium-binding protein peak was resolved. On the contrary, a cadmium peak was clearly resolved in samples of 15 and 30 d. Cadmium was accumulated in the low molecular weight fractions (about 20,000). These fractions had high ultraviolet absorption at 254 nm and a higher 254/280 ratio.