Development of an analytical HPLC methodology to study the effects of thymosin ?4 on actin in sputum of cystic fibrosis patients
A high-performance liquid chromatography (HPLC) methodology is presented. Using bovine and rabbit F- and G-actin, this methodology results in both fractions as being well-resolved peaks, which were confirmed by dot blot immunoassay and fluorescence microscopy. F- and G-actin were incubated with thymosin ?4 (T?4) and DNase and then analyzed by HPLC, which indicated that T?4 and DNase inhibit G-actin polymerization and that T?4 depolymerizes F-actin in a dose- and time-dependent manner. The F- and G-actin content in sputum from healthy controls and cystic fibrosis (CF) patients were measured by HPLC before and after incubation with T?4, DNase, and gelsolin. These data demonstrate higher quantities of F-actin in the sputum of CF patients compared to healthy individuals, and also demonstrate a significantly increased F/G-actin ratio in CF sputum. Further, T?4, DNase, and gelsolin each increase the depolymerization of F-actin in CF sputum in a dose-dependent fashion that is additive when these agents are combined.
hplc thymosin ?4 actin sputum cystic fibrosis