Soluble maize root NADH ferric-chelate reductase

A combination of ammonium sulphate fractionation, Cibacron Blue 3 GA affinity chromatography and size exclusion chromatography was employed to partially purify a 28 kDa NADH Fe3+-chelate reductase from maize root 110,000 g supernatants. This form was similar to a previously purified 28 kDa microsomal ferric reductase, showing the same chromatographic behavior, ratio of ferricyanide to ferric-chelate reduction (3:1 to 2:1, depending on the iron chelator) and high sensitivity to PHMB. The two forms were subjected to IEF in nondenaturing PAGE and the gel was activity-stained. Two bands of pI 8.5 and 7.6 were detected in both cases. A fainter pI 6.9 band was also present in the reductase from the supernatant. The supernatant form seems to be derived from the microsomal form, which was previously shown to be loosely bound to membranes and mainly hydrophilic.