One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobac teria widely used as a fluorescent probe. In this study, phycoerythrin of t he red macroalga Palmaria palmata was extracted by grinding the algal sampl e in liquid nitrogen, homogenisation in phosphate buffer and centrifugation . Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous elution system and detected by its pink colour and fluorescence. The pigment presented a typical spectrum of R-phycoerythrin, with three absorbance maxima at 499, 5 45 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorban ce ratio A(565)/A(280), a criterion for purity, was 3.2. A single protein o f relative molecular mass 240 000 was detected on native-PAGE with silver s taining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two majo r subunits with M-r 20 000 and 21 000, respectively, and a very minor subun it of M-r 30 000. These observations are consistent with the (alpha beta)(6 )gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autu mn and showed a good stability up to 60 degrees C and between pH 3.5 and 9. 5. In conclusion, phycoerythrin of Palmaria palmata was purified in a singl e-step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria p almata will allow one to evaluate its fluorescence properties for future ap plications ih biochemical techniques. (C) 2000 Elsevier Science B.V. All ri ghts reserved.