PURIFICATION, ISOLATION and MOLECULAR ANALYSIS 
of APO-HEMOCYANIN from the GARDEN SNAIL, Helix aspersa, 
USING POTASSIUM CYANIDE and COUPLED HPLC/ICP-MS

K. Thrippleton and A. Z. Mason, Ph.D.  
Department of Biological Sciences, California State University, Long Beach, CA.     

Copper is an essential trace element that is crucial for the biological activity of numerous enzymes and metalloproteins.  Although the requirement for dietary copper is well known, the mechanisms by which this trace metal is transported intracellularly and inserted in apometalloproteins is poorly understood.   The properties that underlie the biological function of copper are also responsible for the toxic properties it exhibits when free in solution.¹  It is therefore necessary for organisms to limit the concentration of excess free copper to a minimum, which raises the question of how copper is transported within an organism and how the metal is made available for incorporation into apometalloproteins. 

Hemocyanin is a copper based oxygen-carrying metalloprotein found in the hemolymph of most mollusks and crustaceans.  It is well established that the addition of CN^- to a hemocyanin solution causes the sequestration of copper and the release of oxygen. This results in blanching the solution of its characteristic blue color and causes a corresponding attenuation in absorbance of the copper-oxygen bond at 350 nm.⁵  It has been shown that the intracellular levels of the metal-binding protein metallothionein, MT, in the blue crab, Callinectes sapidus, are intricately linked to extracellular levels of hemocyanin.²  Evidence previously reported suggest that Cu(I)-MT from the American lobster, Homarus americanus, can restore the oxygen-binding capacity of CN^- prepared, partially copper-depleted hemocyanin, under anaerobic conditions.³  Although no direct measurements of copper transfer were undertaken in these experiments, hemocyanin re-activation in these studies was estimated indirectly from the increase of the copper-oxygen charge transfer band at 350 nm. 

 This preliminary study aims to establish the efficacy of CN^- for the removal of copper from native hemocyanin by directly quantifying the copper content of the apo-protein using HPLC/ICP-MS.  The formation of apo-hemocyanin is necessary to further elucidate the mechanism of copper insertion and consequent re-activation of these respiratory proteins.