Stabilization of the tetrameric structure of human and bovine hemoglobins by pseudocrosslinking with muconic acid

Arch Biochem Biophys. 1996 Feb 1;326(1):119-25. doi: 10.1006/abbi.1996.0055.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cattle
  • Cross-Linking Reagents / metabolism
  • Hemoglobins / metabolism*
  • Humans
  • Molecular Sequence Data
  • Protein Conformation
  • Rats
  • Sorbic Acid / analogs & derivatives*
  • Sorbic Acid / metabolism

Substances

  • Cross-Linking Reagents
  • Hemoglobins
  • muconic acid
  • Sorbic Acid