Similarly, the reduction in ion intensities for Cd and Zn were quantified using the FIA responses (Figure 2c-d).  The absolute and relative mass of sample injected into the system was calculated by Coomassie Blue analysis of collected fractions and from the integrated areas of the peaks obtained from the diode array detector respectively (Figure 1c-e).  The degree of depletion of copper from hemocyanin was calculated from the protein-normalized samples using the following expression, (1-[(apo-hemocyanin ng Cu/µg protein)/(hemocyanin ng Cu/µg protein)]) x 100%.  The results showed a 58% depletion in Cu content that also corresponded to attenuation in the absorbance of the copper-oxygen bond at 350 nm (Table 2).  These results were confirmed by the relative decrease in absorbance at 350nm in the apo-hemocyanin relative to the native form of the protein (Figure 1a-b).  After normalization for protein content at 280nm, there was a 310% reduction in absorbance at 350nm (Table 1). 

Table 1.       Purified and isolated Helix aspersa hemocyanin/apo-hemocyanin absorbance ratios at 280, 254, and 350 nm.
 

This absorption band is characteristic of the Cu-O couple in hemocyanin.  The reduction of Cd and Zn were also analyzed to determine the ability of CN^- to remove these metals.  The results showed a 1.25% reduction in Cd and a 16% reduction in Zn in the apo-protein (Table 2). 

Table 2.      ng Cu/µg protein associated with Helix aspersa hemocyanin and apo-hemocyanin  obtained by HPLC/ICP-MS (ng metal) and Coomassie blue assay (µg protein). ng Cu/µg protein
 

Figure 2a-2d