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Kelly Thrippleton:
Purification, Isolation and Molecular
Analysis of Apo-Hemocyanin from the Garden Snail, Helix
aspersa, Using Potassium Cyanide and Coupled HPLC/ICP-MS
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Copper is essential to the biological function
and activation of a large number of proteins. The respiratory role
of hemocyanin, a copper based protein found in the hemolymph of mollusks
and crustaceans, has been extensively studied. However, little is
known about the post-translational mechanisms by which Cu is introduced
into the oxygen-binding site of the protein. It is well established that
the addition of CN- to a hemocyanin solution causes the removal
of Cu 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. This preliminary
study aims to establish the efficacy of CN- for the removal
of Cu from native hemocyanin in order to further elucidate the mechanism
of Cu insertion and consequent re-activation of these respiratory proteins.
The displacement of copper by CN- from purified Helix aspersa
hemocyanin was quantified by FIA using coupled HPLC/ICP-MS. The results
showed a 58% reduction in Cu content which also corresponded to a specific
reduction in absorbance at the copper-oxygen charge transfer band at 350
nm.
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