Molecular engineering of a polymer of tetrameric hemoglobins.
We have engineered a recombinant mutant human hemoglobin, Hb Prisca beta(S9C+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 (beta9Ser --> Cys) (Bonaventura and Riggs, Science 1967;155:800-802). Cbeta93 and Cbeta112 were replaced in order to prevent formation of spurious S&bond;S bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product 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 hemoglobin (Q = 0.02 +/- 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, 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 polymer 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 tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin.
Fronticelli
C
C
Department of Biochemistry and Molecular Biology, University of Maryland, Medical School, Baltimore, Maryland, USA. cfrontic@jhmi.edu
Arosio
D
D
Bobofchak
K M
KM
Vasquez
G B
GB
D002942:Circular Dichroism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D006454:Hemoglobins; D011108:Polymers; D011994:Recombinant Proteins; D010100:Oxygen