A major and a minor ascorbate free radical (AFR) reductase were separa
ted from the soluble fraction in the human lens cortex by DEAE-cellulo
se ion-exchange column chromatography. These AFR reductases also exhib
ited diaphorase activity using dichlorophenolindopbenol and ferricyani
de as electron accepters. The major AFR reductase was partially purifi
ed by 5'AMP-Sepharose 4B affinity column chromatography. This partiall
y purified AFR reductase showed a single band of diaphorase activity i
n native polyacrylamide disc gel electrophoresis. This activity band c
orresponded to the major protein observed in protein staining by Cooma
ssie Brilliant Blue. However, the protein staining by Coomassie Brilli
ant Blue showed this activity band surrounded by diffused staining. Mo
lecular weight of the partially purified AFR reductase was determined
to be 32 kDa by gel filtration, and the apparent K-m value for AFR was
about 15 mu M. This major lens AFB reductase could be distinguished f
rom soluble Neurospora, Euglena and cucumber AFR reductases, and from
two ubiquitous enzymes with reduction activity of AFR and/or foreign c
ompounds, ie, NADH-cytochrome b(5) reductase and DT-diaphorase, by the
ir molecular weights, K-m values and/or ion-exchange chromatogaphic be
haviors.