SOLUBLE ASCORBATE FREE-RADICAL REDUCTASE IN THE HUMAN LENS

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.