Properties of Carboxymethylated Cross-Linked Hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% . In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of M, 64000 species and 15% of M, 128 000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide. Carboxymethylated hemoglobin after being cross-linked with glycolaldehyde for 5 h to yield about 20% of two cross-linked subunits had an average Ps0 of 14 mmHg (average n = 2.0) compared with an average P50 of 7 mmHg for cross-linked, unmodified hemoglobin A (n = 1.6). Thus, carboxymethylated cross-linked hemoglobin A releases its O2 more readily than unmodified hemoglobin. This property forms the basis for further studies on the possible use of this derivative as a blood substitute

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