Separation of glutathione and its novel analogues and determination of their dissociation constants by capillary electrophoresis

Abstract

A CE protocol was developed to separate reduced glutathione and its four novel analogues UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), UPF17 (Tyr(Me)-α-Glu-Cys-Gly), UPF50 (β-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), and UPF51 (β-Ala-His-Tyr(Me)-α-Glu-Cys-Gly), and their homo- and heterodimers by varying the ionic strength and/or pH of different BGEs. For the determination of dissociation constants (pK(a)) of the above-mentioned peptides the CE method was used. Effective electrophoretic mobilities of analytes were measured in the pH range 5.50-10.00 using optimized BGE with an ionic strength of 50 mM at 25°C. pK(a) values were calculated by fitting the experimental points to a suitable model with correlation coefficients higher than 0.99. The pK(a) values for imidazolyl, amino and thiol moieties of the analyzed peptides were in the range 5.94-6.29, 8.81-9.10, and 7.86-8.13, respectively.