Covalent fixation of polymer-linked benzene hexacarboxylate onto human haemoglobin

Abstract

The reaction of human deoxy and oxyhaemoglobin with a macromolecular effector, monomethoxypolyoxyethylene-linked benzene hexacarboxylate, in the presence of a water soluble carbodiimide, produces under defined conditions, thesame conjugates preferentially acylated at the two valines β1. The oxygen affinity of both these conjugates is decreased by approximately 5-fold compared with that of native Hb (at pH 7.2, in 0.05 M Tris buffer. 25°C, P 50:20.1 and 20.7 Torr versus about 4 Torr for Hb). This difference appears to be due to an overstabilization of the T state probably together with a decreased of the oxygen affinity of the R state. Addition of the IHP to the conjugate solutions does not influence the P 50 but addition of IHP to the reaction mixtures before the coupling limits the substitution of Hb by the macromolecular effector, to 20% (instead of 100% in absence of IHP). The cooperativity curve is shifted to the right with an n max of 3 at about 90% oxygen saturation, which corresponds to a potential release of 48% of oxygen at pH 7.2,25°C, between 100 and 40 Torr, compared with 40% for blood. Such kinds of conjugates especially those obtained from oxyhaemoglobin which are easily prepared, could be of a great interest as non-diffusing oxygen carriers in transfusional and perfusional fluids.