Ether-linked tetrafunctional acylating reagent and its cross-linking reactions with hemoglobin

A new type of tetrafunctional reagent for cross-linking proteins has been prepared and used to modify human hemoglobin A. DPEE (1,2-bis{2-[3,5-bis(3,5-dibromosalicyloxycarbonyl) phenoxy]ethoxy}ethane)) has two separate pairs of reacting sites connected by a flexible tetraether chain. DPEE is capable of connecting a cross-link within a protein to another cross-link, either within the same protein molecule or between molecules. DPEE was readily prepared by esterification of a tetraether-linked bisphthalate (prepared by coupling of 1,2-bis(2-iodoethoxy)ethane and 5-hydroxyisophthalic acid). DPEE reacts with deoxy hemoglobin to produce a mixture of modified proteins. Ion-exchange HPLC was used to separate the modified proteins in the mixture. The most abundant products were selected for structural analysis, which used data from reverse-phase chromatography and tryptic peptide mapping. To prevent dissociation of the modified proteins during analysis, the products were further reacted with the bifunctional reagent, bis(3,5-dibromosalicyl) fumarate, which produces fumaryl cross-links between α-subunits. From peptide analysis of the separated products, the major modified protein from DPEE was identified as a novel species with four links within the same α2β2 tetramer. In addition, a minor product that involves cross-links in two different proteins was observed. These results imply that the reagent reacts primarily in a folded state within the protein.0