Stabilization of the tetrameric structure of human and bovine hemoglobins by pseudocrosslinking with muconic acid.
In previous studies mono-3,5-dibromosalicyl-fumarate was used to introduce an intramolecular crosslink (pseudo-crosslink) in the beta cleft between hemoglobin beta subunits. Sedimentation velocity analysis indicated that the product had a mean molecular weight indicating a tetramer with low dissociability. The product had a P50 higher than that 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 half-time of about 20 min, indicating the presence of a dissociable fraction. We have attempted to further enhance the tetrameric stability of hemoglobin and prevent urine elimination by positioning a longer chain carboxylic acid than fumaric acid into the beta cleft. We reason that a longer molecule would allow for greater stabilizing interactions across the beta cleft. In the present study human and bovine hemoglobins were reacted with mono-3-5-dibromosalicyl muconate. Muconic acid is two carbons longer than fumaric acid. The 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 conclusion, it appears that only covalently crosslinked hemoglobins which are completely nondissociable tetramers escape filtration; tetramers with any degree of dissociability into dimers are filterable.
Razynska
A
A
Department of Biochemistry, School of Medicine, University of Maryland at Baltimore 21201, USA.
Matheson-Urbaitis
B
B
Fronticelli
C
C
Collins
J H
JH
Bucci
E
E
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D013011:Sorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D003432:Cross-Linking Reagents; D006454:Hemoglobins; C005939:muconic acid; D013011:Sorbic Acid