The evolutionary patterns of the metallothionein family of proteins in nonmammalian species is an area of intense interest since these data may illuminate genetic mechanisms for the insertion of cysteine based sequences into this protein and the impact of this process on changes in structure and formation of the SH-mediated binding sites which characterize this protein. The American oyster (Crassostrea virginica) produces a low molecular weight cadmium-binding protein (CdBP) which is similar in size to metallothionein (MT) but which has a lower cysteine content and binds less Cd and no Zn. Studies of the CdBP binding site via kinetic analysis of the titration rate of SH groups by 5,5'-dithiobis-(2-nitrobenzoic) acid or 2,2 dithiobispyridine (DTP) showed a single phase reaction versus biphasis from MT. Circular dichroism studies of CdBP incubated in vitro with excess Cd disclosed a 40-50% reduction in the 259 nm Cd-S bond peak but no marked changes in the UV spectrum suggesting geometric alteration of the site. EDTA (1mM)chelation studies of CdBP showed that, like MT, release of Cd+2 from the protein via this method was an extremely slow process which required days at 20 degrees C as measured by the observed decrease in the 259 nm Cd-S circular dichroism peak. The above data indicate that the metal binding site of CdBP is similar to mammalian MT in that it is SH-mediated and resistant to EDTA chelation of Cd+2 but also different with respect to the monopasic response to SH titration and unusual circular dichroic properties after addition of excess Cd+2. Similar structural studies examining the nature of the SH-Mediated metal binding sites of the 45,000 dalton scallop kidney CdBP in relation to secondary structure are in progress. These data taken in concert with ongoing amino acid sequence and structural studies of oyster CdBP suggest that one evolutionary pathway for MT may involve the insertion of cysteine based sequences into a more ordered protein with concomitant changes in structure and metal-binding site formation or gene cleavage with production of a smaller more efficient molecule from a larger protein.
