Abstract
Purpose :
Interphotoreceptor retinoid-binding protein (IRBP, RBP3) is the most abundant protein in the subretinal space. Decreased [IRBP] is associated with DR and increased [IRBP] is protective. IRBP has free radical scavenging activity and protects visual cycle retinoids from photodegradation (Gonzalez-Fernandez et al. Photochem Photobiol 2015; Exp Eye Res, 2014). However, little is known regarding the mechanism(s). Assaying photodecomposition is challenging because light must be used to simultaneously initiate and monitor. Furthermore, the low sensitivity of spectrophotometry with 1 cm pathlength cuvettes and light scatter are obstacles.
Methods :
We exposed 10 µM all-trans retinol and retinal within an integrating cavity cuvette, containing 3 right angle ports for: 1) actinic light delivery via LEDs firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec via an OLIS RSM 1000 UV/Vis rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector PMT. The RSM spectral intermediate slit was partially covered to allow for a “dark” period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the PMT blocked by a perforated spinning shutter disk (see Gonzalez-Fernandez Mol. Vis. 2016). IRBP was purified by Con-A affinity, anion exchange and size exclusion chromatography and glycated by nonenzymatic exposure to glucose.
Results :
Using the RSM to trigger the LED flash and PMT shutter closure during the “dark” period allowed actinic flashes to be placed between scans. Exposure of the retinoid to 366 nm flashes markedly reduced absorbance through 3 spectral intermediates. IRBP significantly retarded the rate of photodecomposition and glycation inhibited this function.
Conclusions :
The pump-probe system allowed retinoid photodecomposition to be followed in real time while increasing sensitivity ~20 fold. The system, which is immune to light scatter, allowed us to show that IRBP significantly reduces retinoid photodecomposition and that IRBP glycation inhibits this biochemical activity.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.