Control of the allosteric equilibrium of hemoglobin by cross-linking agents.
The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with alpha Val 1 or involve a cross-link between alpha Val 1 and alpha Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 microM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange.
Marden
Michael C
MC
INSERM U473, Hôpital de Bicêtre, 94276 Le Kremlin Bicêtre Cedex, France. marden@kb.inserm.fr
Cabanes-Macheteau
Marion
M
Babes
Alexandru
A
Kiger
Laurent
L
Griffon
Nathalie
N
Poyart
Claude
C
Boyiri
Telih
T
Safo
Martin K
MK
Abraham
Donald J
DJ
D000447:Aldehydes / Q000494:pharmacology*; D000494:Allosteric Regulation / Q000187:drug effects*; D002248:Carbon Monoxide / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D019281:Dimerization; D005456:Fluorescent Dyes; D006441:Hemoglobin A / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D013329:Structure-Activity Relationship
D000447:Aldehydes; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D002248:Carbon Monoxide; D006441:Hemoglobin A; D010100:Oxygen