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 cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample 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, 545 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A565/A280, a criterion for purity, was 3.2. A single protein of relative molecular mass 240,000 was detected on native-PAGE with silver staining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two major subunits with Mr 20,000 and 21,000, respectively, and a very minor subunit of Mr 30,000. These observations are consistent with the (alphabeta)6gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autumn 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 single-step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria palmata will allow one to evaluate its fluorescence properties for future applications in biochemical techniques.
Galland-Irmouli
A V
AV
Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, EP CNRS 0616, Faculté de Médecine, BP 184, Vandoeuvre lès Nancy, France.
Pons
L
L
Luçon
M
M
Villaume
C
C
Mrabet
N T
NT
Guéant
J L
JL
Fleurence
J
J
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010799:Phycoerythrin / Q000302:isolation & purification* / Q000378:metabolism; D000461:Rhodophyta / Q000737:chemistry*; D013054:Spectrophotometry, Atomic / Q000379:methods; D013696:Temperature
D010799:Phycoerythrin