The purification and properties of human lens glutathione reductase

A very simple and rapid method for the purification of human lens glutathione reductase has been developed. The method involves only two steps--affinity chromatography on 2',5'-ADP-Sepharose 4B and gel filtration on Sephacryl S-200. With whole lenses, the purification achieved is over 18000-fold and 80% of the activity in the tissue homogenate is recovered as an enzyme with a specific activity of 218 IU/mg-1. Glutathione reductase was purified from the nucleus and cortex of type I cataractous human lenses and the properties of the two enzymes were compared. No differences could be detected between these enzymes in their specific activities, molecular weights, pH-activity profiles, heat labilities, reactivity towards various substrates and kinetic parameters (Vmax and Km) for glutathione, NADPH2 and NADH2. Therefore, it was concluded that specific alterations in the properties of nuclear glutathione reductase were not responsible for the decreased ability of the lens nucleus to protect itself against oxidative insults. Several different glutathione reductase preparations were examined for their ability to cleave mixed disulphides of lens proteins and glutathione. Only crude tissue extracts (wheat germ and whole lens) were able to cleave the mixed disulphides: no activity was observed with purified lens or yeast glutathione reductases. Therefore, it was concluded that glutathione reductase does not cleave mixed disulphides.