Modification of human hemoglobin with methyl acyl phosphates derived from dicarboxylic acids. Systematic relationships between cross-linked structure and oxygen-binding properties

Abstract

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta 1Val, beta 82Lys, and, to a minor extent, beta 144Lys. In the case of the smaller reagents, the amino groups of alpha 1Val, alpha 99Lys, and, to a minor extent, alpha 139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta 1Val of one chain and the beta 82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta 1Val of one chain and the beta 82Lys of the other chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation of Human Hemoglobin by Methyl Acetylphosphate

“…Detection utilizing the relatively weak polarization transfer between the carbonyl 13 C and methyl protons of the acetyl group was facilitated by the high mobility of the acetyllysine side chains, and the carbonyl resonances were found to yield greater shift dispersion than the methyl resonances observed using [2-13 C]acetyl-labeled precursors. The 1 H- 13 C HMQC spectrum of COHbA acetylated using a 10-fold molar excess of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]MAP is shown in Fig. 1A.…”

“…Based on the reported selectivity of methyl acetylphosphate for residues in the 2,3-DPG binding pocket, Jones et al (8) have evaluated a series of bis(methyl phosphate) cross-linking agents that stabilize the hemoglobin tetramer. These agents were reported to be highly selective in cross-linking the aminocontaining residues of the 2,3-DPG binding cleft (␤Val-1 and ␤Lys-82) as well as acylating ␣Val-1 and ␣Lys-99.…”

Cys-93-ββ-Succinimidophenyl Polyethylene Glycol 2000 Hemoglobin A

“…It is the first ␤ 93 ␤ 93 intramolecularly cross-bridged species reported to date. The previously described bisdibromosalicyl derivatives of aliphatic carboxylic acids and the bispyridoxal phosphates stabilize the tetrameric structure of HbA by cross-linking the amino groups within its central cavity (1,(5)(6)(7)(8)(9)(10). In contrast, included in the atomic model used to calculate the F calc structure factor magnitudes and phases.…”

“…Several intramolecular cross-bridging approaches have been developed over the years for the stabilization of Hb tetramer against dissociation into ␣␤ dimers (1,(5)(6)(7)(8)(9)(10). By and large, the currently available intramolecular cross-bridging reagents are targeted to the reactive functional groups within the central cavity of Hb in domains that interact with allosteric effectors.…”

“…In fact, some of the cross-linkers have been designed to mimic the interactions of the allosteric effectors. Their design strategy has focused on the complementarity between the stereochemistry of the tagging ends of the proposed bi-or tri-functional reagents and the reactive functional groups within the central cavity (1,(5)(6)(7)(8)(9)(10). In view of their location within the central cavity in the modified Hbs, these cross-links offer steric hindrance for interactions with the allosteric effectors compared with unmodified HbA.…”