Specifically Carboxymethylated Hemoglobin as an Analogue of Carbamino Hemoglobin: Solution and X-Ray Studies of Carboxymethylated Hemoglobin and X-Ray studies of Carbamino Hemoglobin

Hemoglobin can be specifically carboxymethylated at itsNH2-terminal amino groups to form three type of derivatives alfa2Cm-beta2, alfa2Cm-beta2Cm, and alfa2-beta2Cm, where Cm represents carboxymethyl. Previous studies suggested that these derivatives could be used as stable analogues of the corresponding carbamino forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alfa-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons toVal- lalfa. Difference electron density analysis of deoxy alfa2Cm-beta2Cm versus the unmodified deoxyhemoglobin tetramer shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alfa-amino group of Val-18, the epsilon-amino group of Lys-82beta, and backbone NH groups at the NH2-terminal end of the F’-helix. In the case of the alfa-subunits, the carboxymethyl group replaces an anion that is normally bound between the alfa-amino group of Val-l alfa and the beta-OH group of Ser-13l alfa. A corresponding difference electron map of carbamino deoxyhemoglobin in low salt crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The kinetics of carboxymethylation provide a sensitive probe for examining the conformational state of hemoglobin as well as the sites of interaction of the various allosteric effectors. Thus, the rates of carboxymethylation of alfa2-beta2, alfa2Cm-beta2, and alfa2-beta2Cm are 2-4-fold higher in the deoxy state versus the corresponding rates in the oxygenated state. In the deoxygenated form, 2,3-diphosphoglycerate and chloride were effective inhibitors of carboxymethylation in the hybrid alfa2Cm-beta2 where Val-18 is free. Likewise,CO2 and chloride effectively inhibited the carboxymethylation of alfa2-beta2Cm where Val-l alfa is free. Taken together, these studies further characterize the oxygen-linked anion binding sites of hemoglobin and the way in which they interact with each other as well as with oxygen-linked proton binding sites.