Affinity labeling of hemoglobin with 4,4'-diisothiocyanostilbene-2,2'-disulfonate: covalent cross-linking in the 2,3-diphosphoglycerate binding site.
The bifunctional reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) reacts with hemoglobin to give various products whose properties are dependent on the ligation state of the protein during the reaction. A major product obtained after reaction of (carbonmonoxy)hemoglobin with DIDS was a high oxygen affinity derivative [P50 = 1.4 mmHg, control P50 = 6 mmHg; 50 mM [bis(2-hydroxyethyl)-amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.4, 0.1 M Cl-, 25 degrees C] which contained two molecules of DIDS per tetramer resulting from adduct formation at each beta-chain amino terminus. In contrast, a major product of the reaction of deoxyhemoglobin with DIDS consisted of hemoglobin which had incorporated one molecule of DIDS per tetramer and was cross-linked between the beta-chain amino termini. This cross-linked hemoglobin was found to have a greatly decreased O2 affinity (P50 = 28 mmHg). Inhibition of the T to R transition due to the structural constraint produced by cross-linking the beta amino termini is likely to be a major factor in the decreased O2 affinity of this product. The structural and functional properties of this molecule make it a potential candidate for a cell-free blood substitute.
Kavanaugh
M P
MP
Department of Biochemistry, Oregon Health Sciences University, Portland 97201.
Shih
D T
DT
Jones
R T
RT
D019794:2,3-Diphosphoglycerate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D001665:Binding Sites; D004163:Diphosphoglyceric Acids / Q000378:metabolism*; D006441:Hemoglobin A / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010108:Oxyhemoglobins / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D013267:Stilbenes / Q000378:metabolism*
D000345:Affinity Labels; D004163:Diphosphoglyceric Acids; D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D010108:Oxyhemoglobins; D013267:Stilbenes; D019794:2,3-Diphosphoglycerate; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006441:Hemoglobin A; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid