Effects of crosslinking on the thermal stability of hemoglobin. I. The use of bis(3,5-dibromosalicyl) fumarate.
The double-headed aspirin, bis(3,5-dibromosalicyl) fumarate, has been used to crosslink hemoglobin A between Lys 82 beta 1 and Lys 82 beta 2 (J. A. Walder et al. (1979) Biochemistry 18,4265). Denaturation experiments were used to compare the stability of this crosslinked protein to that of hemoglobin A. Thermal denaturations, done in 0.01 M 4-morpholine-propanesulfonic acid, pH 7, containing 0.9 M guanidine to prevent precipitation at high temperatures, were monitored by changes in absorbance between 190 and 650 nm using a diode array spectrophotometer. The sample was heated from 25 to 70 degrees C at 0.3 degrees C/min. The data were analyzed by using both a two-state model and a novel first derivative method. As expected, methemoglobin A had a single, broad transition with a midpoint of 40.7 degrees C. The crosslinked methemoglobin showed a transition at 57.1 degrees C. Two minor transitions, one of which was apparently due to residual unmodified hemoglobin, were also observed in the crosslinked sample. Thus, a single crosslink between only two of the four subunits can lead to a significantly more stable molecule. These results can be explained by Le Chatelier's principle, since crosslinking prevents dissociation of the beta-subunits and, thereby, holds the entire tetramer together.
White
F L
FL
Department of Chemistry, Loyola University of Chicago, Illinois 60626.
Olsen
K W
KW
D001241:Aspirin / Q000031:analogs & derivatives* / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006441:Hemoglobin A; D006358:Hot Temperature; D006801:Humans; D046911:Macromolecular Substances; D008706:Methemoglobin; D011489:Protein Denaturation / Q000187:drug effects; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C021630:bis(3,5-dibromosalicyl)fumarate; D008706:Methemoglobin; D006441:Hemoglobin A; D001241:Aspirin