Human lenses were obtained from the Heartland Lions Eye Tissue Bank of Missouri after removal of the cornea. Procedures adhered to the provisions of the Declaration of Helsinki for research in human tissue. Newborn calf lenses were obtained from Pel-Freeze Biologicals (Rogers, AR), shipped in dry ice, and stored frozen at −70°C until used. A pool of 10 to 20 lenses were homogenized, and the dialyzed water-soluble (WS) proteins and the water-insoluble sonication-solubilized (WISS) proteins were prepared as described previously,
14 15 and also dialyzed before use.
l-Ascorbic acid was obtained as an ultrapure reagent from Sigma-Aldrich (St. Louis, MO).
l-Dehydroascorbic acid (DHA) and
l-diketogulonic acid (DKG) were prepared from
l-ascorbic acid, according to published procedures.
16 DHA was also purchased commercially from several suppliers. Solutions of this reagent were yellow, but one pass through a charcoal filter removed the colored impurities, and the nuclear magnetic resonance (NMR) spectrum showed only DHA resonances.
l-Erythrulose was obtained from Sigma-Aldrich. The chromatography standard, [U-
14C]oxalate (OA), was purchased from DuPont (Wilmington, DE), whereas [1-
14C]erythrulose was synthesized from [1-
14C]erythrose by reduction and selective oxidation by
Gluconobacter oxidans.
17 Similarly, [U-
14C]
d-glucose was reduced to glucitol and converted to [U-
14C]
l-sorbose by cultures of
G. oxidans, and then converted into [U-
14C]
l-ascorbic acid by the method of Reichstein and Grüssner as described in Crawford and Crawford
17 and as we have described previously.
18 Two preparations of [U-
14C]ascorbate were synthesized in this manner, with specific activities of 1.6 and 7.8 mCi/millimole. Aliquots of the [U-
14C]ascorbate preparations were chromatographed over a Resex RNM-carbohydrate column in the sodium form (Phenomenex, Torrance, CA), to remove any remaining residual impurities and were used as such for the protein incorporation studies. When a sample of the [U-
14C]ascorbate was purified in this manner and treated with ascorbate oxidase, no radioactivity eluted at the original position of ascorbate, indicating no impurities were present in the pooled peak. All phosphate buffers were pretreated with 10 g/L resin (200–400 mesh; Chelex; Bio-Rad Laboratories, Richmond, CA), to remove heavy metal ion contaminants, and were filtered through a 0.2-μm nitrocellulose filter before use.