Timolol Binding to Bovine Ocular Melanin In Vitro

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Abstract
The binding characteristics of 3H-timolol to bovine iris melanin were determined by using a ligand binding assay. The association and dissociation kinetics were performed using 100 nM 3H-timolol and 0.5 mg/ml melanin at 37 °C. The effects of pH, ethanol, 2.5 μM isoxuprine and 1 μM d- and 1-propranolol on 100 nM 3H-timolol binding were also determined. In saturation experiments 3H-timolol (from 1.25 nM to 5 μM) was equilibrated with 0.5 mg/ml melanin. The binding was saturable with the binding maximum of about 1 μM timolol/g melanin. The binding of 100 nM 3H-timolol to melanin increased up to 5 hours and amounted 24 % of the added radioactivity. The best fit of the association constants was obtained by using a two fit model. The association rate constants were 4.92 × 105 M−1min−1 and 5.95 × 106 M−1min−1. The dissociation was rapid in vitro and uniphasic with a dissociation rate constant of 5.08 × 10−3 min−1. The pH, ethanol and both enantiomers of propranolol did not appreciably alter the timolol binding, while isoxuprine diminished it.