Rapid HPLC Techniques for Globin Chain Synthesis Studies

Abstract

Globin chain synthesis and α/β ratios were determined in a number of normal subjects, α-thalassemia-2 homozygotes, and β-thalassemia trait using three different techniques. Cation exchange high performance liquid chromatography on a Pharmacia Mono-S, HR 5/5 and reversed phase high performance liquid chromatography on a semi-preparative Vydac C⁴ column were compared. with the traditional carboxymethylcellulose chromatography.

Both high performance liquid chromatography columns give excellent results when 2 mg of hemoglobin was chromatographed in each analysis. By modifying the protocols for column equilibration and gradient shape for preparative Vydac C⁴ columns, conditions were found yielding excellent resolutions of the labeled globin chains in less than an hour without the need for substantial increase of the flowrate. This method was found to be superior to other methods and may be a suitable alternative for the classical carboxymethylcellulose chromatography. Up to five specimens could easily be analyzed in a single day with this system.