O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity:: Structural and functional consequences of β93Cys modification

Reacting human deoxyHbA(0) with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA(0) heterogeneous polymer (O-R-PolyHbA(0)) into six distinct fractions with a molecular weight distribution ranging from 64 to similar to 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA(0), species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA(0), O-R-PolyHbA(0), and O-R-PolyHbA(0) fractions. Proposed sites of intramolecular crosslinking (i.e., beta 1Lys82, beta 2Lys82, and beta 1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta 93Cys and alpha 104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA(0) slight conformational. changes are required to allow for the S on alpha 104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA(0) in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA(0) may provide some basis for the reported toxicity of this oxygen carrier. Published 2005 Wiley-Liss, Inc.*