Fractionation by Countercurrent Distribution (CCD) of Rat Erythrocytes During Animal Development

Abstract

Preliminary studies have shown that red blood cells from new-born and adult rats may be fractionated in aqueous two-phase systems on the basis of differences in cell size and surface properties [1]. Our aim is to extend this work and to use CCD as an appropriate fractionation procedure for biochemical studies of red cell switching (from fetal to definitive erythrocytes) during animal development. Erythrocytes from 19 days-fetuses, new-born young animals (6,8,12 and 24 days of age) and adult Wistar rats were fractionated by CCD. Dextran T-500 (Pharmacia) and poly(ethylene glycol) (PEG)-6000 (Serva) were used for the preparation of 5% dextran/4% PEG two-phase systems, containing 0.09 M NaPB/0.03 M NaCl, pH 6.8 (charged systems) [1,2]. A CCD apparatus with 60-cavity rotors [1,2] was employed. Experimental CCD conditions were those previously used for the fractionation of erythrocytes from adult rats [1,3,4]: top/bottom phase volume ratio of 1.3 (0.9 ml or 0.75 ml of top phase and 0.7 ml or 0.55 ml of bottom phase, respectively); settling time, t=5 min. Specific activities (U/mgHb) of bisphosphoglycerate mutase (BPGM) (EC 5.4.2.4), the enzyme responsible for 2,3-bisphosphoglycerate (BPG) synthesis, and pyruvate kinase (PK) (EC 2.7.1.40), the glycolytic enzyme involved in the regulation of BPG levels in erythrocytes, have been determined in adjacent pooled CCD fractions from all of the above red cell populations.