Isolation of ascorbate free radical reductase from rabbit lens soluble fraction

Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.