A novel tetrafunctional reagent for simultaneous intramolecular and intermolecular cross-linking of bovine hemoglobin

A novel tetrafunctional reagent, α,γ,α′,γ′-tetra-succinimidyl-hexanediamide-di-glutamate ester (HDG(OSu)4), was successfully synthesized, and a well-defined cross-linked bovine hemoglobin (mainly 128 kDa) was prepared with this reagent. Due to the spatial structure of this cross-linking reagent, the intramolecular and intermolecular cross-linking of bovine hemoglobin was formed simultaneously in one reaction. Although the cross-linked bovine hemoglobin showed a slight decrease in half-saturated O2 pressure value (P50, from 28.1 mm Hg to 21.7 mm Hg) and Hill coefficient (from 2.5 to 2), due to the cross-linkage, it still performed well for O2 delivery.