Reduction of Methaemoglobin in Haemoglobin Samples using Gel Filtration for Continuous Removal of Reaction Products

EVEN a small degree of oxidation of haemoglobin to methaemoglobin, such as occurs in the transport and storage of frozen red cells, interferes with the study of its oxygen dissociating properties. When abnormal are separated from normal haemoglobins, even more methaemoglobin accumulates during the manipulations involved. The methaemoglobin may be efficiently reduced by passing a haemolysate of such a partly oxidized sample by means of gel nitration through a band of reducing agent. Advantages of this method are that the methaemoglobin constantly encounters fresh reducing agent and that the continued elution quickly removes the haemoglobin formed from excess reagent. These factors are important when reduction is by dithionite, as side reactions can then destroy the haemoglobin. A similar principle can be applied to the oxidation of cytochromes 1 , and to the conversion of the aldimine form of aspartate aminotransferase ( E.C. 2.6.1.1) into the amino form by transamination with an amino-acid 2 . The continuous removal of the α-oxoacid formed is particularly important in the latter case because the position of equilibrium is unfavourable to formation of the amino form.