Fast preparative separation of human hemoglobins A, A2, E, F, Bart’s and Portland I

Abstract

Background Preparative separation of hemoglobins is useful for preparing purified hemoglobins used as standard and control materials, as antigen for immunization or for immunological tests for hemoglobins.

Objectives: This study was aimed to develop the fast procedures for preparative separation of hemoglobins in

hemolysate.

Methods: For fast preparative separation of Hbs A, A2, E (with A2) and F, three stepwise pH gradient liquid

chromatography was developed namely Protocol A, Protocol A2/E and Protocol F using the medium pressure liquid chromatography (MPLC) system and Tris-HCl-KCN (THK) buffer. The techniques of CAE with band elution was also developed to quickly separate and purify Hb Bart’s and Hb Portland I.

Results: Compared to the conventional continuous pH gradient anion-exchange liquid chromatography run on the MPLC system which lasted 240-340 minutes, this newly established protocols took considerably short separating time. The Protocol A, Protocol A2/E and the Protocol F required only 50, 50 and 70 minutes, respectively, to complete run. The outcomes of separation of conventional and the new methods were comparable. Hbs Bart’s and Portland were successfully separated within 15 minutes in CAE, followed by band elution.

Conclusion: The new stepwise pH gradient anion-exchange liquid chromatography protocols run in the MPLC

system and CAE with band elution techniques were successfully developed for fast preparative separation of

hemoglobins. These protocols should be useful in preparation of purified hemoglobins that might be used as

antigen, standard and control materials for hemoglobin study after specific types and separating patterns of purified hemoglobins were correctly confirmed by other standard techniques.

Bull Chiang Mai Assoc Med Sci 2015; 48(2): 100-106. Doi: 10.14456/jams.2015.13