Effect of glutaraldehyde on haemoglobin: oxidation-reduction potentials and stability.

Glutaraldehyde is a reagent widely used for the cross-linking of haemoglobin for use as a blood substitute. Most of the previous studies were limited to oxygen binding equilibria of the glutaraldehyde-modified haemoglobin. This paper concerns the impact of glutaraldehyde on oxidation-reduction equilibria, autoxidation kinetics and stability towards heat and urea of haemoglobin cross-linked in the oxy, deoxy and ferri states. The oxidation-reduction potentials and homotropic effects were reduced; however, the oxidation Bohr effect was not significantly different when compared with native haemoglobin. Haemoglobin immobilized in the oxy or ferri state exhibited a lower redox potential than when immobilized in the deoxy state. The autoxidation rates were increased after cross-linking, particularly at basic pH. Cross-linking stabilizes ferrihaemoglobin better than oxy or deoxyhaemoglobin against thermal- and urea-induced denaturation. Glutaraldehyde cross-linking does not stabilize haemoglobin against urea-denaturation. The experimental results were interpreted as indicating a chemical modification of the protein without 'conformation freezing' and by an opening of the haem pocket to the aqueous solvent.