We have studied the binding of he
moglobin to the red cell
me
mbrane by centrifugation andfluorescence
methods. The intact red cell was labeled with eosin-5-
malei
mide (EM), which specificallyreacts with lysine 430 of band 3. Even though this residue is not part of the cytoplas
mic do
main of band3 (cdb3) associated with he
moglobin binding, fluorescence quenching was observed when he
moglobinbound to inside-out vesicles (IOVs). The use of fluorescence quenching to
measure band 3 binding wasquantitatively co
mpared with the binding deter
mined 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 deter
mined fro
m chy
motrypsin-treated IOVs. The binding of he
moglobin to band 3 was interpretedin ter
ms of the binding of two he
moglobin tetra
mers to each band 3 di
mer. An anticooperative interactionassociated with the confor
mational change produced when he
moglobin binds results in a 2.8-fold decreasein the intrinsic constant of (1.54 ± 0.25) × 10
7 M
-1 for the binding of the second he
moglobin
molecule.Fro
m the changes in lifeti
me produced by binding the first and second he
moglobin
molecules, it waspossible to show that the confor
mational change associated with binding the second he
moglobin
moleculeresults in a decrease of the he
me-eosin distance fro
m 47.90 to 44.78 Å. Reaction of cyanate with the
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mino group of he
moglobin (HbOCN) is shown to produce a very dra
matic decrease in the binding ofhe
moglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first he
moglobin
molecule to band 3 decreases by a factor of 29 to (5.34 ± 0.15) × 10
5 M
-1. The anticooperative interactionis greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second he
moglobintetra
mer to band 3. In addition, the nature of the confor
mational change produced by binding he
moglobinis very different with the second HbOCN increasing the he
me-eosin distance to 55.99 Å. The utilizationof eosin-5-
malei
mide-reacted red cell
me
mbrane to study he
moglobin binding
makes it possible to directlystudy the binding to band 3. At the sa
me ti
me a sensitive probe of the confor
mational changes, whichoccur when he
moglobin binds to band 3, is provided.