Separation of the valence intermediates of human haemoglobin by high-performance chromatofocusing

Abstract

Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (α o) 2β +) 2 and (α + β o 2) 2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 μm) in an HR5/20 column at various temperatures (10–25°C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (α o 2β +) 2, (α + β o 2) 2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.