Abstract
Introduction
Material & Methods
Results
Discussion
 Literature Cited
Acknowledgements

NSF Student Research

kelly Thrippleton: Purification, Isolation and Molecular Analysis of Apo-Hemocyanin from the Garden Snail, Helix aspersa, Using Potassium Cyanide and Coupled HPLC/ICP-MS
The preliminary experiments described in this poster form part of a larger study to elucidate the cellular mechanisms by which specific metals are donated to apo-proteins for activation.  In the current study, protocols were evaluated to determine the efficacy of a number of agents in sequestering Cu from native hemocyanin.  The formation of apo-hemocyanin is a necessary initial step in the study of Cu insertion and successive re-activation of this class of respiratory protein.   Initial comparative ICP-MS analyses of data generated using the strong Cu-chelating agent tetraethylene-pentamine (TEPA) and potassium cyanide (KCN) showed that, despite a high affinity constant for Cu, log K1 = 24.3, TEPA was ineffective in removing the metal from the protein.  Conversely, the addition of KCN to hemocyanin preparations caused an instantaneous blanching and loss of blue color from the purified protein.  Diode array spectroscopy of the HPLC fractionated protein showed that this chromatic shift resulted in a significant reduction in the characteristic Cu-O absorption band at 350nm.  

Upon normalization for protein content, it was calculated that there was a 58% reduction at this wavelength for the apo-form.  In contrast, spectral comparisons between the apo- and native forms of the protein showed no change in absorption at 280 nm, characteristic for aromatic residues.  There was, however, an approximate 50% increase in absorption at 254nm after removal of the metal indicating that the chelation process may have also resulted in an increase in detectable mercaptide bonds.  Quantitative analyses of the Cu content of the native and apo- forms of the protein by tandemly coupled HPLC/ICP-MS showed a reduction in Cu content from 2.32 ng Cu/µg to 0.97 ng Cu/µg protein.  Good agreement was observed using both of the stable isotopes for Cu indicating that there were no polyatomic interferences during analyses.  In addition to Cu removal, quantitative results showed a significant reduction in both Cd and Zn in the apo-protein preparation.