Kinetics of formation of fluorescent products from hexanal and L-lysine in a two-phase system

Kinetics of formation of fluorescent condensation products from hexanal and L-lysine (or its N-acetylated forms) including mass-transfer has been studied in a two-phase system consisting of lysine (or lysine derivative) in an aqueous phosphate buffer and a 1-octanol solution of hexanal as model for formation of fluorophores between protein and carbonyl compounds in peroxidizing biological systems. The initial rate of formation of fluorescent products in the aqueous phase was found to be proportional to the concentration of hexanal and lysine and to increase in both phases with increasing pH in the aqueous phase, in contrast to a higher-order dependence on hexanal in the octanol phase. At pH = 6.8, the temperature dependence of the appearance of fluorescent products corresponds to apparent energies of activation of 63 kj.mol-1 and 87 kj.mol-1 in the aqueous phase and the octanol phase, respectively. Fluorescent condensation products appeared faster in the octanol phase. However, by a kinetic analysis, the fluorescent products were shown to be formed in the aqueous phase, corresponding to the lower energy of activation and to the simple second-order kinetics, and subsequently distributed between the aqueous phase and the octanol phase. L-Lysine reacted faster than N alpha-acetyl-L-lysine which reacted faster than N epsilon-acetyl-L-lysine. Using fluorescence quantum yields, determined to be 1.4.10(-2) in octanol and 8.10(-3) in water at pH 6.8, an apparent partition coefficient of 17 (octanol/water) was determined for the condensation product of L-lysine. The steady-state fluorescence in the octanol phase was attributed to two components with fluorescence lifetimes at 25 degrees C of 0.7 +/- 0.05 ns and 5.1 +/- 0.2 ns, assigned to hexanal and the condensation product, respectively. The emission spectra were resolved in the two components using phase-sensitive detection, and the condensation product had emission maximum at 405 nm.