Two-step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4-9.

Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.