Isolation of an Immunoreactive Thymosin Fraction by Chromatofocusing and High-Performance Liquid Chromatography

Abstract Thymosin fraction 5 (TSN-5), a partially purified thymic preparation, was active in enhancing a murine mixed lymphocyte reaction (MLR) at a concentration of 10-100 μg/ml. To isolate the polypeptide(s) responsible for the MLR activity, TSN-5 was fractionated by column chromatography on CM-cellulose. Further purification was achieved by chromatofocusing on polybuffer exchanger PBE 94 and by high-performance liquid chromatography (HPLC) on a μBondapak C 18 reversed phase column. The active fraction consists of two main polypeptide components (β 2 and β 2a ) separable by HPLC. These two polypeptides are indistinguishable by amino acid analysis and HPLC tryptic peptide mapping. Each of them contains all tryptic peptides expected to be derived from polypeptide β 1 except for the absence of the C-terminal dipeptide-Leu-Arg. Polypeptide β 2 and β 2a are not active in the MLR assay individually. Whether the activity is attributed to a yet undefined minor component or to the binding of β 2 and/or β 2a to such minor component remains to be investigated.