Capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection as a tool to monitor reactions of proteins.

Abstract

Capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) has the characteristics of high resolution, high speed and high sensitivity for separation of amphoteric biomolecules. These features enable a CIEF-LIF-WCID system to monitor the dynamic process of a protein reaction. The reaction can be a physical change or a chemical reaction, provided that the kinetics of the reaction is slower than the focusing speed or that the intermediates involved have long enough life-span compared to the analysis time. The processes of denaturation (a physical reaction), reduction and carbamylation (both chemical reactions) were dynamically monitored. The CIEF profiles at successive reaction times clearly displayed the formation of different products at different stages. At incomplete denaturation, intermediates with higher apparent pI values relative to the products at complete denaturation were detected. Carbamylation products of a protein were detected when the protein reacted with a urea solution that had prepared three months earlier, exhibiting gradually decreased pI values. Mechanisms involved in these reactions were rationalized. A combined mechanism of denaturation and reduction was suggested to explain the denaturing process under high concentrations of urea. Potential applications and critical factors to manipulate these reactions were also discussed.