Separation of tryptic peptides of normal and abnormal alpha, beta, gamma, and delta hemoglobin chains by high-performance liquid chromatography

Abstract

High-performance liquid chromatography (HPLC) was used to separate tryptic peptides of the normal alpha, beta, gamma, and delta chains of human hemoglobins A, F, and A2 and of the abnormal chains of 25 hemoglobin variants. In addition, the separation of chymotryptic peptides of the oxidized core of the normal alpha chain by HPLC was evaluated. HPLC has several advantages over conventional methods used for the separation of proteolytic fragments of hemoglobin chains. The method is fast, and reproducible, and requires only small quantities of material. Several peptides are eluted as single zones, thus eliminating the need of rechromatography for further purification. Characteristic changes in the elution pattern of the peptides often indicate specific modifications.