Effects of crosslinking on the thermal stability of hemoglobins. II. The stabilization of met-, cyanomet-, and carbonmonoxyhemoglobins A and S with bis(3,5-dibromosalicyl) fumarate.
Hemoglobins A and S were crosslinked between Lys 82 beta 1 and Lys 82 beta 2 using bis (3,5-dibromosalicyl) fumarate (J. A. Walder et al. (1979) Biochemistry 18, 4265). Thermal denaturation experiments were used to compare the stabilities of the met, cyanomet, and carbonmonoxy forms of these crosslinked hemoglobins to the corresponding uncrosslinked proteins. Uncrosslinked carbonmonoxy- and cyanomethemoglobins had transition temperatures about 11 degrees C higher than the corresponding met samples. The increase in denaturation temperature (Tm) due to crosslinking was 15 degrees C for the methemoglobins, 10 degrees C for the cyanomethemoglobins, and 4 degrees C for the carbonmonoxy ones. There was no significant difference in stability between the met and carbonmonoxy crosslinked proteins. In order of increasing stability the samples were: met Hb S less than met Hb A less than CO Hb S less than CO Hb A = CN-met Hb A less than met XL-Hb S = CO XL-Hb S less than met XL-Hb A = CO XL-Hb A less than CN-met XL-Hb A. The slight decrease in the stability of Hb S (beta 6 Glu----Val) compared to Hb A can be explained by the replacement of an external ionic group by a hydrophobic residue in Hb S. In mixtures of crosslinked and normal Hb A, the Tm of the uncrosslinked material was slightly increased by the presence of the more stable crosslinked hemoglobin. The effects of both crosslinking and cyanide or carbon monoxide binding can be explained by Le Chatelier's principle since both would favor the native form of the protein.
Yang
T
T
Department of Chemistry, Loyola University of Chicago, Illinois 60626.
Olsen
K W
KW
D001241:Aspirin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002263:Carboxyhemoglobin / Q000032:analysis; D003432:Cross-Linking Reagents; D004586:Electrophoresis; D006451:Hemoglobin, Sickle / Q000032:analysis; D008706:Methemoglobin / Q000031:analogs & derivatives* / Q000032:analysis*; D011485:Protein Binding; D011489:Protein Denaturation; D013696:Temperature
D003432:Cross-Linking Reagents; D006451:Hemoglobin, Sickle; C038874:carboxyhemoglobin, sickle; C007169:cyanomethemoglobin; C021630:bis(3,5-dibromosalicyl)fumarate; D008706:Methemoglobin; D002263:Carboxyhemoglobin; D001241:Aspirin