title: Molecular necklaces. Cross-linking hemoglobin with reagents containing covalently attached ligands.
abstract: 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 electrophile. The activated material is then combined with a reagent (3-aminoisophthalic acid) that contains a nucleophilic (amino) site and two latent (carboxyl) sites. The latent sites of the product are activated as 3,5-dibromosalicylates to produce the cross-linker. Illustrative examples of cross-linking are presented with pendant biotin [bis(3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate] 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 functional properties of the modified hemoglobin containing biotin in a (beta-Lys-82-beta'-Lys-82) cross-link are (pH 7.4, 55 microM heme, 25 degrees C, 0.1 M chloride, and 50 mM Bis-Tris) P(50) = 4.9 Torr, n(50) = 3.0, values which are approximately the same as for native hemoglobin. The results of affinity chromatography of the biotinylated cross-linked hemoglobin using a column of immobilized avidin indicate that the pendant biotin is much less accessible than free biotin. We suggest that the results are consistent with the pendant species being strongly attracted into the hemoglobin environment.
authors.affiliation: Lash Miller Laboratories, Department of Chemistry, University of Toronto, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Crapatureanu
authors.forename: R
authors.initials: R
authors.lastname: Serbanescu
authors.forename: S B
authors.initials: SB
authors.lastname: Brevitt
authors.forename: R
authors.initials: R
authors.lastname: Kluger
mesh_terms: D001665:Binding Sites; D001970:Bromobenzoates; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D019281:Dimerization; D004151:Dipeptides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010795:Phthalic Acids / Q000737:chemistry; D012459:Salicylates; D020156:Salicylic Acid / Q000737:chemistry
chemical_list: D001970:Bromobenzoates; D003432:Cross-Linking Reagents; D004151:Dipeptides; D006454:Hemoglobins; D008024:Ligands; D010446:Peptide Fragments; D010795:Phthalic Acids; D012459:Salicylates; D005914:Globins; C102688:5-aminoisophthalic acid; D020156:Salicylic Acid; C402306:3,5-dibromosalicylic acid