Entamoeba histolytica: Isolation of ferredoxin

Abstract

A significant portion of the nonheme iron and acid-labile sulfide of axenically grown Entamoeba histolytica was obtained in a colored, low molecular weight protein fraction from centrifuged amoeba homogenates following gel filtration. The colored component was further purified by adsorption on and elution from DEAE-cellulose. Preparations so purified had 390 280 absorbance ratios as great as 0.87. The oxidized form had absorbance peaks at 390, 315, and 280 nm; the enzymatically reduced form, at 350 nm. The difference spectra showed a major peak at 420 nm, and a minor one at 310 nm. The substance was converted to apoprotein and reconstituted with iron and H 2S by methods developed for clostridial ferredoxin. When chromatographed on a Sephadex G-50 column calibrated with spinach and clostridial ferredoxins its molecular weight appeared to be about 6000, i.e., close to that of the clostridial protein. The substance is considered to be amoebal ferredoxin, but there remains some uncertainty concerning the number of atoms of iron and acid-labile sulfide per molecule. Its physiological role in the organism is not yet understood.