Binding of Hemoglobin to Red Cell Membranes with Eosin-5-maleimide-Labeled Band 3: Analysis of Centrifugation and Fluorescence Lifetime Data
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- 作者：Afolorunso Andrew Demehin ; Omoefe O. Abugo ; Rajadas Jayakumar ; Joseph R. Lakowicz ; and Joseph M. Rifkind
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 9, 2002
- 年：2002
- 卷：41
- 期：27
- 页码：8630 - 8637
- 全文大小：77K
- 年卷期：v.41,no.27(July 9, 2002)
- ISSN：1520-4995
文摘
We have studied the binding of hemoglobin to the red cell membrane by centrifugation andfluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specificallyreacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobinbound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding wasquantitatively compared with the binding determined by centrifugation, which measures binding to band3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 associationconstants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpretedin terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interactionassociated with the conformational change produced when hemoglobin binds results in a 2.8-fold decreasein the intrinsic constant of (1.54 ± 0.25) × 107 M-1 for the binding of the second hemoglobin molecule.From the changes in lifetime produced by binding the first and second hemoglobin molecules, it waspossible to show that the conformational change associated with binding the second hemoglobin moleculeresults in a decrease of the heme-eosin distance from 47.90 to 44.78 Å. Reaction of cyanate with the
-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding ofhemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobinmolecule to band 3 decreases by a factor of 29 to (5.34 ± 0.15) × 105 M-1. The anticooperative interactionis greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobintetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobinis very different with the second HbOCN increasing the heme-eosin distance to 55.99 Å. The utilizationof eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directlystudy the binding to band 3. At the same time a sensitive probe of the conformational changes, whichoccur when hemoglobin binds to band 3, is provided.
-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding ofhemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobinmolecule to band 3 decreases by a factor of 29 to (5.34 ± 0.15) × 105 M-1. The anticooperative interactionis greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobintetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobinis very different with the second HbOCN increasing the heme-eosin distance to 55.99 Å. The utilizationof eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directlystudy the binding to band 3. At the same time a sensitive probe of the conformational changes, whichoccur when hemoglobin binds to band 3, is provided.