Purification and Characterisation of Two Novel Pigment Proteins from the Carapace of Red Swamp Crayfish (Procambarus clarkii)

Pigment proteins play a vital role in the red colour change of the red swamp crayfish (Procambarus clarkii) shell after cooking. In this study, two red-change-related pigment proteins with molecular weights of approximately 170 and 43 kDa—denoted as F1 and F2, respectively—were purified by ammonium sulphate salting-out and size exclusion chromatography. F1 and F2 entirely comprised homomultimeric protein complexes composed of 21 kDa subunits. LC-MS/MS analysis showed that the 21 kDa protein subunit belonged to the crustacyanin family, named P. clarkii crustacyanin A2 (PcCRA2). The full-length cDNA of PcCRA2 was cloned, which encoded 190 amino acid residues and was highly homologous (91.58%) with Cherax quadricarinatus crustacyanin A. The predicted 3D structure showed that PcCRA2 had a β-barrel structure for pigment encapsulation. The colour change of F1 was first detected at 40 °C, and the red change occurred upon heating above 60 °C. Additionally, with increasing temperature, its β-sheet content increased, and its α-helix content reduced. Correlation analysis showed that the redness value of F1 was significantly related to the heating temperature and the β-sheet content.