Molecular engineering of a polymer of tetrameric hemoglobins

We have engineered a recombinant mutant human hemoglobin, Hb Prisca beta (S 9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular S-S bonds between cysteine residues introduced at position beta9, on the model of Hb Porto Alegre (be ta 9Ser --> Cys) (Bonaventura and Riggs, Science 1967;155:800-802). C beta 93 and C beta 112 were replaced in order to prevent formation of spurious S -S bonds during the expression, assembly, and polymerization events. Dynami c light scattering measurements indicate that the final polymerization prod uct is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersity Q = 0.07 +/- 0.02, is similar to that of purified human hemo globin (Q = 0.02 +/- 0.02), consistent with a good degree of homogeneity. I n the presence of strong reducing agents, the polymer reverts to its tetram eric form. During the depolymerization process, a direct correlation is obs erved between the hydrodynamic radius and the light scattering of the syste m, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the p olymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tet rameric hemoglobin molecules is presented. The conformational and functiona l characteristics of this polymer, such as heme pocket conformation, stabil ity to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin. (C) 2001 Wiley-Lis s, Inc.