Lobster carapace was obtained fresh from commercial fisheries. Preparation and extraction of the crustacyanin was based on the method described by Zagalsky (1985). The shell was removed immediately, cleaned of any attached tissue, washed in iced water and allowed to dry overnight at 0–2 °C. The dry shell was ground to a fine powder at low temperature and then soaked for 18 h, with agitation, in a solution of 0.3 M boric acid neutralised to pH 6.8 with solid Tris. Soaked shell was removed by filtration, washed with fresh borate–Tris solution and resuspended in pre-cooled 10%(w/v) EDTA, pH 7.0 using 25 g shell/1000 g solution. The mixture was stirred continuously overnight, after which shell was removed and the purple coloured filtrate containing the crustacyanin brought to 55% saturation with ammonium sulphate. The mixture was allowed to stand at 0–2 °C for around 18 h and the resultant precipitate, the crustacyanin, was collected by centrifugation at 10 000 g for 30 min at 5 °C. The precipitate was redissolved in 50 mM potassium phosphate buffer pH 7.0, and interfering protein removed by fractional precipitation with ammonium sulphate to 15% or 20% saturation. The desired crustacyanin was then precipitated by increasing the saturation level to 55%. This material was stored at 0 ± 2 °C. A total of 13 extractions were carried out over a six month period and pooled into four batches for further use for characterisation or in food preparations.
2.2. Purification