Systematically Cross-Linked Human Hemoglobin: Functional Effects of 10 Å Spans between Beta Subunits at Lysine-82

The structure and properties of hemoglobin are altered by the introduction of cross-links of defined structure between specific residues. The bis methyl phosphates and bis 3,5-dibromosalicylates of 4-carboxy- trans -cinnamic acid as well as the bis methyl phosphate of 2,6-naphthalenedicarboxylic acid produce a 10 Å cross-link between the ε-amino groups of each β-lys-82 of human hemoglobin. The oxygen affinity of the modified proteins fits the correlation interpolated from those for shorter and longer cross-links. The oxygen binding curve shows a high degree of cooperativity. These results support the idea that the length of the semirigid cross-link in a structurally homogeneous series constrains the relaxation of the protein upon oxygen binding by a mechanism that is specifically reflected in the oxygen affinity, while interactions between hemes that affect cooperativity are not diminished.