Rapid analysis of labeled globin chains without acetone precipitation or dialysis by high-pressure liquid chromatography and ion-exchange chromatography

Abstract

Human red blood cells were incubated with tritiated leucine. The cells were washed several times, lysed, and aliquots of the lysates were acidified with trifluoroacetic acid and applied on reversed-phase columns to separate the α-, β-, G γ-, and A γ- globin chains. The ratios of the labeled non-α- to α-globin chains separated by this method were practically identical to those obtained using the standard method of globin chain precipitation with acidified acetone. Hemoglobins present in very small amounts or containing chains which are not readily separated in the reversed-phase system can be purified first using a semi-preparative column of carboxymethyl-Sepharose. The hemoglobins of cell lysates are eluted using linear gradients of NaCl in bis-Tris buffer, and the fractions containing the desired hemoglobin can be applied directly on reversed-phase columns without sample concentration or acetone precipitation. Both chromatographic systems utilize solutions which are very stable at room temperature, and the columns can be easily regenerated and used many times. This results in a considerable reduction of the time and the cost of analysis of labeled globin chains. This system can be easily applied to the common hemoglobins A, F, C, and S.