Purification of multiple pea ferredoxins by chromatography at high ionic strength on unsubstituted sepharose 4B

Abstract

At high concentrations of ammonium sulfate (3–4 M) pea ferredoxin (which is soluble under these conditions) can be adsorbed to Sepharose 4B, either by column chromatography or by batchwise treatment. A reverse (3 M to 1 M) ammonium sulfate gradient results in the elution of three peaks of ferredoxin. The spectral ratio A 420/A 280 of 0.47–0.54 indicates that each peak of ferredoxin is highly purified by this single step. A further gel filtration removes residual high molecular weight contaminants from the ferredoxin. The spectrum of the purified pea ferredoxin is typical of other plant ferredoxins in having absorbance peaks at 276 nm, 330 nm, 422 nm and 465 nm. Other chromatographic matrices are capable of adsorbing ferredoxin. Sepharose and Sephacryl were the best adsorbents while Sephadex and cellulose adsorbed ferredoxin less tenaciously. The polyacrylamide-based resins Biogel P-4 and P-200 did not adsorb ferredoxin at high ionic strength.