Conjugation of proteins to hemoglobin.
Published: 2004
Language: eng
Number of pages: 126
Description: 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 beta 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.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.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 alpha-amino group of the Phe1 residue of B chain.
Notes: Adviser:  Ronald H. Kluger.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2414.