Library

Notes: Abstract The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(β), Val-1(α), Lys-66(β), Lys-61(α), and Lys-16(α). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(α), Val-1(β), Lys-66(β), Lys-82(β), Lys-61(α), and Val-1(α). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16(α) is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(α), Lys-82(β), Lys-66(β), Lys-61(α), Val-1(β), and Val-1(α). The propensity of the microenvironment of Lys-16(α) to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1(α) and is about 50 times higher than that of Val-1(β). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.

Notes: Abstract Interspecies hybrids of HbA and Hb from mouse C57BL/10 [α 2 M Β 2 H and↠ 2 H Β 2 M (H=human, M=mouse)], representing 19 and 27 sequence differences perαΒ dimers (as compared with humanαΒ dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybridα 2 M Β 2 H has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybridα 2 H Β 2 M exhibits a very low O2 affinity. These results suggest that the mouseΒ chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouseα andΒ chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouseΒ chain. DPG binds the tetramer in the central cavity formed by the twoΒ subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of theα chain present in the tetramer. The mouseα chain reduces considerably the DPG right shift of the O2 affinity of the humanΒ-chain containing hybrid. Sequence analysis suggest that perturbations of theα 1 Β 1 (not theα 1 Β 2) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouseα chains in transgenic mouse expressing humanΒ S chains.