Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus.
Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.
Institut für Biophysik und Strahlenbiologie, Freiburg i. Br., Germany.
S
S
Ritter
J
J
Komenda
E
E
Setlíkova
I
I
Setlik
W
W
Welte
D002498:Centrifugation; D002846:Chromatography, Affinity / Q000379:methods*; D000458:Cyanobacteria / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008670:Metals; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000302:isolation & purification*
D008670:Metals; D045322:Photosynthetic Reaction Center Complex Proteins