Spontaneous Generation of Superoxide Anion by Human Lens Proteins and by Calf Lens Proteins Ascorbylated In Vitro

Abstract

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process. To assess whether the glycation-related modifications in human lens proteins spontaneously generate ROS, superoxide anion formation was measured using both cataractous lens proteins and calf lens proteins glycated in vitro with ascorbic acid (ascorbylated).The water-insoluble fraction from aged normal human lenses generated 0.3–0.6 nmol superoxide h−1mg protein−1, whereas the activity increased to 0.5–1.8 nmol h−1mg protein−1with the WI fraction from brunescent cataracts, and 2.3 nmol h−1mg protein−1with calf lens proteins ascorbylated for 4 weeks in vitro. The activity in the human lens proteins was observed in both the water-soluble and water-insoluble fractions, and was completely dependent upon the presence of oxygen. The pH optimum curve for superoxide formation increased from pH 6.5 to 10 with both the cataract and ascorbylated proteins. The superoxide-generating activity in human lens was completely bound to a boronate affinity column, but only partially bound with the ascorbylated proteins.The superoxide anion produced by a 5 m m solution of purifiedNϵ -fructosyl–lysine was barely detectable, and therefore, could not account for the superoxide formed by any of the lens protein preparations. Also, superoxide formation increased 10-fold at pH 8.8 with fructosyl–lysine, but only 1.3–1.8-fold with human lens proteins. The addition of copper-stimulated superoxide formation with glycated bovine serum albumin, but no stimulation was seen with cataractous proteins. Assays of specific compounds showed that catechol, hydroquinone, 3-OH kynurenine and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short halflife. 2,3-Dihydroxypyridine and 4,5 dihydroxynaphthalene were one and two orders of magnitude less reactive.In long-term incubations at 37°, cataractous proteins retained the potential to produce superoxide anion, losing only half of the initial activity after 6–7 days. Therefore, the water-insoluble fraction from aged human lenses and dark brown cataracts are potentially capable of generating >100 nmol mg protein−1and >170 nmol mg protein−1of superoxide anion respectively, likely due to the presence of advanced glycation endproducts in human lens proteins. This spontaneous generation of superoxide anion in vivo could account for a major portion of the oxidation of sulfur amino acids seen during aging and cataract formation.