A novel tetrafunctional reagent, alpha,gamma,alpha',gamma'-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 O(2) pressure value (P(50), 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 O(2) delivery.