Molecular necklaces. Cross-linking hemoglobin with reagents containing covalently attached ligands

Hemoglobin can be cross-linked and converted to a bioconjugate in one step by a "molecular necklace", a reagent that contains two reacting sites and a pendant ligand. The compound to be conjugated is activated as an electroph ile. The activated material is then combined with a reagent (3-aminoisophth alic acid) that contains a nucleophilic (amino) site and two latent (carbox yl) sites. The latent sites of the product are activated as 3,5-dibromosali cylates to produce the cross-linker. Illustrative examples of cross-linking are presented with pendant biotin [bis(3,5-dibromosalicyl) N-biotinyl-5-am inoisophthalate] and pendant N-trifluoroacetyl-L-isoleucylglycine [bis(3,5- dibromosalicyl) N-(N-trifluoroacetyl-L-isoleucylglycyl)-5-aminoisophthalate )]. The resulting modified hemoglobins contain two principal types of cross -link: (beta-Lys-82-beta'-Lys-82) and (alpha-Lys-99-alpha'-Lys-99). The fun ctional properties of the modified hemoglobin containing biotin in a (beta- Lys-82-beta'-Lys-82) cross-link are (pH 7.4, 55 mu M heme, 25 degrees C, 0. 1 M chloride, and 50 mM Bis-Tris) P-50 = 4.9 Torr, n(50) = 3.0, values whic h are approximately the same as for native hemoglobin. The results of affin ity chromatography of the biotinylated crosslinked hemoglobin using a colum n of immobilized avidin indicate that the pendant biotin is much less acces sible than free biotin. We suggest that the results are consistent with the pendant species being strongly attracted into the hemoglobin environment.