Bis-tetramers of hemoglobin containing disulfide linkages

There is a considerable interest in developing hemoglobin bioconjugates for therapeutic applications. For example, conjugates of hemoglobin and the enzyme superoxide dismutase might be useful as red cell substitutes with antioxidant properties, while conjugates of hemoglobin and insulin might serve as hepatoselective and long-acting insulin analogues. Currently, conjugation of proteins to hemoglobin relies on gene fusion using a recombinant DNA technology or on oligomerization using polymerizing agents. We developed an alternative approach, which relies on chemical modification and conjugation of proteins in a site-specific manner. Two novel reagents for chemical modification of proteins were synthesized and used to generate hemoglobin-superoxide dismutase and hemoglobin-insulin conjugates. The tetrafunctional reagent N,N'-Bis[bis(sodium methyl phosphate)-5-aminoisophthalyl)]-4,4'-disulfanlydibenzamide, (1) reacts with human hemoglobin A in a site-specific manner at the Val-1 and Lys-82 of the ß globin chain. The reagent simultaneously cross-links hemoglobin tetramers intramolecularly and bridges them with a disulfide bond. The reduction of the disulfide bond generates cross-linked hemoglobins that have an exogenous thiol group, which is available for conjugate formation. Superoxide dismutase is chemically modified at the interface of two subunits using the reagent N,N'-[N-(4-maleimidobenzoyl)-5-aminoisophthalyl]-5-aminoisophthalyl bis(sodium methyl phosphate) (2). This cross-links two sub-units of the enzyme by modifying the [epsilon]-amino groups of the Lys3 and Lys151 on one subunit and the c-amino groups of the Lys68' and Lys134' on the other subunit. Modification with 2 also introduces a maleimide group. Maleimidobenzoyl-N-hydroxy succinimide was used to incorporate a maleimide group into insulin at the Ã-amino group of the Phe1 residue of B chain. The chemoselective reaction of the thiol group of hemoglobin with the maleimide group on the cross-link of a second protein produces conjugates in a controlled manner. Evaluation of the properties of the resulting hemoglobin-superoxide dismutase and hemoglobin-insulin conjugates showed that the conjugated proteins retain their function. This establishes that designed conjugates of hemoglobin can be produced and are functional. These should be useful for applications requiring enhanced stability and circulation time., Hemoglobin tetramers cross-linked and connected to a second hemoglobin molecule (bis-tetramers) are a new class of oxygen-carrying proteins with interesting properties. Hemoglobin bis-tetramers formed from the reaction of hemoglobin with tetrafunctional reagents bind oxygen without cooperative features found in tetramers. This indicates that the dynamics of individual hemoglobins within bis-tetramers are different than those of native or cross-linked hemoglobin mono-tetramers. This thesis reports the synthesis of a new tetrafunctional cross-linking reagent 1 and its reaction with human hemoglobin A. The reagent contains a disulfide bond that allows for the cleavage and reformation of the link between tetramers. 1 modifies hemoglobin in a site-specific manner, cross-linking the amino groups of Val-1 and Lys-82 residues of the ß globin chains, resulting in the formation of hemoglobin bis-tetramers that can be cleaved at the disulfide bond by reduction. The measurement of the physical properties of these unique bis-tetramers and of the resulting cross-linked hemoglobin tetramers, which are formed by cleavage of the hemoglobin bis-tetramers, will provide a comparison of the bis- and mono-states. These measurements are expected to give insight into the dynamics of hemoglobin molecules within the bis-tetramers and to correlate biochemical properties and structure. Potential uses of 1 also include formation of hemoglobin-enzyme conjugates with novel interesting properties.