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
Hemocyanin Extraction
  • Hemolymph was collected from the tentacular sinus of the garden snail, Helix aspersa
  • Hemolymph was centrifuged for 10 min at 10,000 x g at 4°C
  • Supernatant was then centrifuged for 60 min at 100,000 x g at 4°C
  • Hemocyanin pellet was reconstituted in 50mM Tris HCl, 10mM CaCl2 buffer solution, pH 8, to form a stock solution of protein
Apo-Hemocyanin Purification and Isolation
  • Equal volumes of stock hemocyanin and 50mM Tris, 10mM CaCl2, 20mM KCN buffer solution, pH 8, were added together and incubated at room temperature for 10 minutes
  • An Amicon Pressure Dialysis system with an XM50 Diaflo Ultrafiltration membrane ( > 50,000 MW) was used to remove CuCN and excess KCN buffer from the apo-protein
  • Following pressure dialysis, the proteins were washed with KCN buffer (2x), then 50mM Tris HCl, 10mM CaCl2 buffer (2x)
  • The apo-protein was then re-suspended in 50mM Tris HCl, 10mM CaCl2, pH 8
  • This process was repeated
Molecular Analysis of Apo-Hemocyanin
  • Hemocyanin and apo-hemocyanin samples were fractionated by size exclusion HPLC.  Individual fractions containing the protein peak were assayed for protein content by Coomassie Blue assays using a standard curve generated using Bovine Serum Albumin (Pierce, US)
  • Stock hemocyanin and apo-hemocyanin were then analyzed by tandemly coupled HPLC/ICP-MS. 
  • The copper content of the sample was quantified by injecting known masses of Cu and other elements into the eluent stream post chromatographically.  The absorbance profiles of the chromatographed proteins were monitored automatically from 220-600nm using a diode array UV-Vis detector placed upstream of the ICP-MS