A sensitive method to investigate the interaction of alpha-crystallin with cortical and nuclear lens lipid membranes derived from a single lens.

Abstract

The levels of α-crystallin in eye lens cytoplasm decrease with a proportional increase in membrane-bound α-crystallin with age and cataract formation. The influence of differences in lipids composition between cortical membrane (CM) and nuclear membrane (NM) derived from a single lens on α-crystallin binding to these membranes is still unclear. This study aims to develop a sensitive electron paramagnetic resonance (EPR) spin-labeling method to investigate the binding of α-crystallin to the CM and NM derived from total lipids extracted from a single lens of a bovine. No binding of α-crystallin below the surface of CM and small binding of α-crystallin below the surface of NM was observed using cholesterol analogue spin-label (CSL) within these membranes. However, significant binding of α-crystallin on the surface of CM and NM was observed using 4-palmitamido-TEMPO (4PT) spin-label within these membranes. The percentage of membrane surface occupied and the binding affinity for NM are higher than for CM, correlating with less mobility and more order below and on the surface of NM than CM. The mobility decreases with no significant change in order and hydrophobicity below and on the surface of CM and NM with α-crystallin binding. Our results imply that α-crystallin primarily binds on the surface of bovine CM and NM, and such surface binding of α-crystallin to membranes in young and clear lenses may be helpful for membrane stability. However, α-crystallin binding increases hydrophobicity below the surface of the cortical membrane with low cholesterol (CMLC), mimicking the decreased cholesterol content in cataractous lenses, suggesting that α-crystallin binding below the surface of the CMLC forms a hydrophobic barrier for the passage of polar molecules supporting the barrier hypothesis in cataract formation.