The anion-transport inhibitor H2DIDS cross-links hemoglobin interdimerically and enhances oxygen unloading.
Human hemoglobin treated with equal concentrations of the anion-transport inhibitor H2DIDS produces a right shift in the oxygen dissociation curve. concomitantly, the Hill coefficient is reduced from n = 2.7 to 2.1. When higher concentrations of H2DIDS are applied (H2DIDS: hemoglobin = 5:0.5 mM), the Hill coefficient decreases further to 1.5 and the oxygen dissociation curve of hemoglobin is shifted slightly to the left of the control. Similar results were also obtained with DIDS instead of H2DIDS. SDS-PAGE shows that H2DIDS cross-links hemoglobin monomers mainly into dimers. Cross-linking is more effective under anaerobic conditions. With tritiated H2DIDS the larger part of the radioactivity is found in the dimer position of hemoglobin. Separation of the alpha and beta units of hemoglobin reacted with tritiated H2DIDS demonstrated a stoichiometry of 2.2 and 2.4 molecules H2DIDS per molecule alpha and beta unit hemoglobin, leading to about 8-9 H2DIDS molecules per native hemoglobin. The right shift produced in the hemoglobin oxygen dissociation curve and the cross-linking of monomers into dimers, especially under anaerobic condition, suggest that H2DIDS can also react with those amino groups of hemoglobin which are involved in 2,3-DPG binding. A comparison of H2DIDS, DIDS and 2,3-DPG at three different concentrations close to the hemoglobin concentration revealed a concentration dependent right shift in the oxygen dissociation curve with the order of potency 2,3-DPG greater than H2DIDS greater than DIDS. The Hill coefficients (n) at the three concentrations of 2,3-DPG demonstrated no changes, but H2DIDS and DIDS reduced in a concentration-dependent manner the cooperativity of hemoglobin. Again, H2DIDS is more potent than DIDS, especially at the low concentration. These anion-transport inhibitors provide novel approaches to the exploration of hemoglobin function.
Fuhrmann
G F
GF
Institut für Pharmakologie und Toxikologie der Philipps-Universität, Marburg, F.R.G.
Kreutzfeldt
C
C
Rudolphi
K
K
Fasold
H
H
D019794:2,3-Diphosphoglycerate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D010100:Oxygen / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology*
D004163:Diphosphoglyceric Acids; D006454:Hemoglobins; D013267:Stilbenes; D019794:2,3-Diphosphoglycerate; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D010100:Oxygen