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O ‐raffinose crosslinked hemoglobin lacks site‐specific chemistry in the central cavity: Structural and functional consequences of β93Cys modification
Author(s) -
Boykins Robert A.,
Buehler Paul W.,
Jia Yiping,
Venable Richard,
Alayash Abdu I.
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20453
Subject(s) - raffinose , chemistry , potentiometric titration , size exclusion chromatography , heme , hemoglobin , kinetics , crystallography , stereochemistry , chromatography , biochemistry , organic chemistry , enzyme , ion , physics , quantum mechanics , sucrose
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 ∼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., β1Lys82, β2Lys82, and β1Val1) 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 β93Cys and α104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA 0 , slight conformational changes are required to allow for the S on α104Cys 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. Proteins 2005. Published 2005 Wiley‐Liss, Inc.