HPLC and post-column derivatization with fluorescamine: isolation of actin-sequestering β-thymosins by reversed-phase HPLC

Three different procedures are summarized that minimize the possibility of artificial proteolysis during the extraction of small peptides like β-thymosins from tissues or cells. The extracts are desalted and concentrated by solid phase extraction. The peptides can be further purified by isoelectric focusing in a granulated gel, chromatofocusing or conventional ion-exchange chromatography. The final step for purification as well as for quantitative analysis of extracts from tissues or cells is reversephase HPLC. We describe the set-up of a post-column derivatization system with fluorescamine in connection with reversephase or cation-exchange columns. The fluorescence detection offers high sensitivity and permits the use of organic buffering substances, which are volatile but exclude UV detection due to their strong UV absorbance. The isolated β-thymosins are in a saltfree form and thus suitable for studies on biological activities and amino acid sequencing. The procedures summarized here are also applicable to other small peptides.