Influence of the Polymerization Step Alone on Oxygen Affinity and Cooperative During Production of Hyperpolymers from Native Hemoglobins with Crosslinkers

Abstract

The aim of this study was to find out how the polymerization per se changes oxygen affinity (P50) and cooperativity (n50) of various soluble huge hyperpolymers prepared from native hemoglobins by crosslinking. Increase of cooperativity would be expected considering natural hemoglobin networks. Those hyperpolymers with molecular weights of some 10⁶ g/mol are candidates for artificial oxygen-carrying blood additives rather than volume substitutes. Human and bovine hemoglobin reacted with several crosslinkers (2, 5-diisothiocyanatobenzenesulfonate (DIBS); 4,4′-diisothiocyana-tostilbene-2,2′-disulfonate (DIDS); 1,3-butadiene diepoxide (BUDE); glutaraldehyde (GDA)) in concentrated (case 1) and diluted (case 2) hemoglobin solutions. With high concentration hyperpolymer and with low concentration only monomer products were obtained. P50 and n50 of the products were determined at pH = 7.4, PCO₂ = 40 mmHg, temp. = 37 °C. The difference of properties in both cases are regarded as the influence of polymerization per se. Considering this difference we found with almost all combinations of hemoglobin and crosslinker an increase of O₂ affinity, with DIBS and DIDS cooperativity was not changed and with BUDE and GDA it was decreased. As compared with native hemoglobin loss of cooperativity is considerable in any combination and condition, but comparing human and bovine hemoglobin the first seems to maintain better cooperativity. In contrast bovine hemoglobin as compared with human hemoglobin maintains better or even decreases its O₂ affinity upon reaction with the crosslinkers forming both, monomer and hyperpolymer products, especially in the deoxy state. DIBS and DIDS react very similarly. As a general conclusion only deoxy state reactions led to appropriate products regarding an artificial oxygen carrier. A differentiated analysis of some samples clearly indicates crosslinking with increased homotropic cooperativity on going from monomer to hyperpolymer reaction products.