STABILIZATION OF THE TETRAMERIC STRUCTURE OF HUMAN AND BOVINE HEMOGLOBINS BY PSEUDOCROSSLINKING WITH MUCONIC ACID

In previous studies mono-3,5-dibromosalicyl-fumarate ate was used to i ntroduce an intramolecular crosslink (pseudo-crosslink) in the beta cl eft between hemoglobin beta subunits. Sedimentation velocity analysis indicated that the product had a mean molecular weight indicating a te tramer with low dissociability. The product had a P-50 higher than tha t of native hemoglobin and a plasma retention time in the rat of about 3 h, i.e., four times longer than untreated hemoglobin. However, the product contained a fraction which was rapidly eliminated in the urine and which had a short plasma halftime of about 20 min, indicating the presence of a dissociable fraction. We have attempted to further enha nce the tetrameric stability of hemoglobin and prevent urine eliminati on by positioning a longer chain carboxylic acid than fumaric acid int o the beta cleft. We reason that a longer molecule would allow for gre ater stabilizing interactions across the beta cleft. In the present st udy human and bovine hemoglobins were reacted with mono-3-5-dibromosal icyl muconate. Muconic acid is two carbons longer than fumaric acid. T he products were acylated at the beta 82 (human) and beta 81 (bovine) lysines of the beta-cleft and had a low degree of dissociability. For reasons not presently understood, urine excretion was high and plasma half-time was not increased above that of untreated hemoglobin. In con clusion, it appears that only covalently crosslinked hemoglobins which are completely nondissociable tetramers escape filtration; tetramers with any degree of dissociability into dimers are filterable. (C) 1996 Academic Press, Inc.