Analysis of Crystallin Species Present in Water–Insoluble–Urea–Insoluble Protein Fractions in Normal and Age–Matched Cataractous Human Lenses

Abstract

Abstract: : Purpose: The aim of this study was to identify the components that form species present in water–insoluble–urea–insoluble (WI–UI) protein fractions of normal and age–matched cataractous human lenses. Methods: Proteins from normal and age–matched cataractous (removed intracapsularly) human lenses were separated into water–soluble (WS) and water–insoluble (WI) protein fractions. The WI–protein fraction was further separated into water–insoluble–urea–soluble (WI–US) and water–insoluble–urea–insoluble (WI–UI)–protein fractions. The WI–UI–protein fractions from normal and age–matched cataractous lenses were subjected to two–dimensional (2D) gel electrophoresis (IEF in the first dimension and SDS–PAGE in the second dimension). The protein spots from each gel were excised, digested with trypsin, and analyzed by MALDI–TOF and ES–MS/MS mass spectrometric methods. Results: On a 2D–gel, the WI–UI–protein fraction from a normal lens contained 22 protein spots between M_r 14–29 kDa and 2 spots with M_r of 55 kDa. Similarly, the WI–UI–protein fraction from a cataractous lens contained 14 protein spots between M_r 18–29 kDa. The MALDI–TOF analyses showed that protein spots (M_r 14–29) from normal and cataractous human lenses were covalent multimers of αA–, αB–, ßA3/A1–, ßB1–, and ßB2–crystallin fragments. Conclusions: The WI–UI–protein fractions from normal and cataractous human lenses contained covalent multimers composed of fragments of α– and ß–crystallins.

Keywords: aging • cataract • crystallins