Behaviour in a silica-based high-performance liquid gel permeation chromatographic column of the apo- and holo-forms of the haem-binding proteins haemopexin, histidine-rich glycoprotein, globin and albumin.

The elution of the apo- and holo-forms of four haem-binding proteins was studied using a TSK G 3000 SW HPLC column. Apo-haemopexin had a higher apparent molecular size [68,000 daltons (d)] than histidine-rich glycoprotein (HRG) (66,000 d) in gel chromatography, contrasting with the values in sodium dodecyl sulphate electrophoresis, 84,000 d for HRG and 69,000 d for haemopexin. The elution of the haem complexes of both proteins correlated better with their true molecular weights. Saturation of albumin with haem did not significantly influence its elution. The peaks were more symmetrical for the holo- than the apo-proteins, except for globin/haemoglobin. The results indicated that the apo-forms of haemopexin and HRG had affinity for the column matrix. HRG, which has several haem binding sites, was retained more than haemopexin, which binds only one haem. Free haem itself was bound to the silica column but could be released by globin. HRG had a tendency to polymerize after haem binding, in contrast to haemopexin, which remained monomeric. Globin was eluted from the column with an apparent molecular size of 16,000 d and after saturation with haem with a molecular size of 31,000 d.