HPLC AND POSTCOLUMN DERIVATIZATION WITH FLUORESCAMINE - ISOLATION OF ACTIN-SEQUESTERING BETA-THYMOSINS BY REVERSED-PHASE HPLC

Three different procedures are summarized that minimize the possibilit y of artificial proteolysis during the extraction of small peptides li ke beta-thymosins from tissues or cells. The extracts are desalted and concentrated by solid phase extraction. The peptides can be further p urified by isoelectric focusing in a granulated gel, chromatofocusing or conventional ion-exchange chromatography. The final step for purifi cation as well as for quantitative analysis of extracts from tissues o r cells is reverse-phase HPLC. We describe the set-up of a post-column derivatization system with fluorescamine in connection with reverse-p hase or cation-exchange columns. The fluorescence detection offers hig h sensitivity and permits the use of organic buffering substances, whi ch are volatile but exclude UV detection due to their strong UV absorb ance. The isolated beta-thymosins are in a saltfree form and thus suit able for studies on biological activities and amino acid sequencing. T he procedures summarized here are also applicable to other small pepti des. (C) 1997 Elsevier Science B.V.