CROSS-LINKING HEMOGLOBIN BY DESIGN - LESSONS FROM USING MOLECULAR CLAMPS

The development of a red cell substitute by chemical modification of h emoglobin has been approached as a systematic, iterative process. Acyl phosphate methyl esters were designed as anionic electrophiles to per mit selective acylation of amino groups in the cationic site of hemogl obin which binds polyanions. Kinetic studies with systematically subst ituted acyl phosphates and amines show that the reaction is controlled by a reversible addition step followed by an irreversible elimination step. Acyl phosphate methyl esters which are derivatives of rigid dic arboxylic acids introduce cross-links in human hemoglobin between amin o groups in the beta subunits (epsilon-NH2-Lys-82, alpha-NH2-Val-1) an d permit correlation of oxygen binding properties with cross-link stru cture. The data suggest that the cross-link maintains cooperativity wh ile reducing overall oxygen affinity by lowering the affinity of the R form for oxygen rather than by perturbing the R,T equilibrium of nati ve hemoglobin. Materials produced from deoxyhemoglobin with a cross-li nk between positions 1 and 82 of the two beta units have appropriate o xygen affinity for red cell substitutes. The use of a trifunctional cr oss-linker, trimesyl tris(methyl phosphate) selectively produces hemog lobin with the desired 1-82 connection in good yield. The reagent is r eadily prepared and the properties of this chemically modified hemoglo bin are suitable for trial as a red cell substitute, closely resemblin g those of optimized materials produced by recombinant technology. Fur ther work is producing new chemicals and providing structural informat ion.