Identification of Chloride‐Binding Sites in Hemoglobin by Nuclear‐Magnetic‐Resonance Quadrupole‐Relaxation Studies of Hemoglobin Digests

Open Access
Publisher Website
Google Scholar
Abstract
35Cl−-nuclear magnetic resonance (NMR) studies indicate that various digests of human hemoglobin with carboxypeptidase A and B, or a combination of the two, may be used for the identification of chloride binding sites. All the digestion products contain, like hemoglobin itself, at least two classes of binding sites, one of high, the others of low affinity. The pH dependence of the excess linewidth of the 35Cl−-NMR signal indicates that in the simple digests with either carboxypeptidase A or B, chloride is bound with high affinity at or near His-β146–Asp-β94 and at or near Val-α1–Arg-α141. The high-affinity sites show, in the case of the simple digests, a strong oxygen linkage which is lost in the forms digested with both carboxypeptidase A and B; this linkage may thus be correlated to the presence of quaternary conformational changes. Organic phosphates, like inositol hexaphosphate, show competition for some of the high-affinity chloride binding sites in hemoglobin and in the simple digests. This competition is likewise lost in the doubly digested hemoglobins.