Abstract
Purpose: :
IRBP, the major protein component of the interphotoreceptor matrix can preserve the isomeric and oxidative state of retinol by an unknown mechanism (Crouch et al., Photochem. Photobiol. 1992). While attempting to concentrate IRBP for crystallography trials, we found that molar excesses of thiol-based reducing agent, DTT is required to concentrate bovine IRBP (bIRBP) above ~3 mg/ml in a soluble, stable and functional form. IRBP is known to have 10 cysteines, 8 of which are free thiols. Our hypothesis is that IRBP is a thiol-dependent antioxidant.
Methods: :
We examined the effect of modifying IRBP’s free thiols on its antioxidant activity, and ability to bind all-trans retinol. Native bIRBP was extracted from the soluble interphotoreceptor matrix, and purified by Con-A, ion exchange and S-300 size exclusion chromatography in the presence of 0.5 mM DTT. The concentration of purified bIRBP was determined by absorbance spectroscopy and amino acid analysis. bIRBP was incubated with N-ethylamalimide (NEM), which covalently modifies thiols, at a molar ratio of 10:2, 10:5 and 10:10 cysteines:NEM. LC-MS/MS with coverage of 80-90% was used to identify modified thiols. Retinol binding was evaluated by monitoring its fluorescence enhancement, and tryptophan quenching. Antioxidant activity was assayed by monitoring the formation of radical cation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS°+). Thioredoxin, trolox and ovalbumin were used as controls. Oxidation of retinol was monitored by absorbance at 330 nm.
Results: :
bIRBP showed a concentration-dependent reduction of ABTS°+. This activity was greater than that of thioredoxin or trolox. Ovalbumin, NEM, and saline had no antioxidant activity. Cysteine modified bIRBP (10:5, 10:10 cysteines:NEM) was less active in reducing ABTS°+ compared to bIRBP. Cysteine modification also inhibited IRBPs ability to protect retinol from oxidation. However, it had no effect on its retinol binding ability by fluorescence spectroscopy. LC-MS/MS showed the modified residues were Cys40, Cys304, Cys1051 and Cys1173.
Conclusions: :
Our data indicates that IRBP has robust antioxidant activity. This activity appears to rely on a thiol dependent mechanism. Preserving the oxidative state of visual cycle retinoids may be an important role of IRBP. As the most abundant soluble protein in the interphotoreceptor matrix, IRBP may have a more general role in protecting the outer retina from oxidative stress.
Keywords: antioxidants • retinoids/retinoid binding proteins • retina: distal (photoreceptors, horizontal cells, bipolar cells)