A new type of multifunctional reagent creates a specific connection within and between two hemoglobin tetramers, resulting in a cross-linked bis-tetramer ("CLBT"). The tetrafunctional reagent (N,N'-5,5'-bis[bis(3,5-dibromosalicyl)isophthalyl]terephthalamide, DBIT) was prepared by conversion of the corresponding tetraacid to the tetrakis(3,5-dibromosalicylate). Deoxy hemoglobin reacts with DBIT to give a cross linked bis-tetramer as the major product. Patterns in tryptic digests reveal that there are modifications of amino groups at beta -Lys-82 and beta-Val-1, indicative of a structure in which each tetramer is cross-linked between these positions. The cross-linked bis-tetramer has a decreased affinity for oxygen and very low cooperativity in oxygen binding. The properties of the material provide insights into the nature of protein-protein interactions.