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Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site
Author(s) -
Whitfield Dennis M.,
Choay Jean,
Sarkar Bibudhendra
Publication year - 1992
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360320603
Subject(s) - chemistry , iduronic acid , binding site , heparin , metal , biochemistry , stereochemistry , organic chemistry , heparan sulfate
As model compounds for Ni(II)‐binding heparin‐like compounds isolated from human kidneys (Templeton, D. M. & Sarkar, B. (1985) Biochem. J. 230 35–42.), we investigated two disaccharides—4‐ O ‐(2‐ O ‐sulfo‐α‐ L ‐idopyranosyluronic acid)‐2,5‐anhydro‐ D ‐mannitol, disodium salt ( 1a ), and 4‐ O ‐(2‐ O ‐sulfo‐α‐ L ‐idopyranosyluronic acid)‐6‐ O ‐sulfo‐2,5‐an‐hydro‐ D ‐mannitol, trisodium salt ( 1b )—that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate ( 1a ) was active whereas the disulfate ( 1b ) was inactive in a high‐performance liquid chromatography (HPLC) binding assay with the tracer ions 63 Ni(II) 54 Mn(II), 65 Zn(II), and 109 Cd(II). This result is in accord with the isolation of two 67 Cu(II) and 63 Ni(II) binding fractions from a complete pool of nitrous‐acid‐derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound ( 1a ) and the other as a tetrasulfated trisaccharide by high resolution FAB‐MS, NMR and HPLC‐PAD. Similarly, two synthetic disaccharides—methyl, 2‐ O ‐sulfo‐4‐ O ‐(α‐ L ‐idopyranosyluronic acid)‐2‐deoxy‐2‐sulfamido‐α‐ D ‐glucosamine, trisodium salt [IdopA2S(α1,4)GlcNSαMe, 2a ], and 2‐ O ‐sulfo‐4‐ O ‐ (α‐ L ‐idopyranosyluronic acid)‐2‐deoxy‐2‐sulfamide‐6‐ O ‐sulfo‐α‐ D ‐glucosamine, tetrasodium salt [IdopA2S(α1,4) GlcNS6SαMe, 2b ]—were shown to bind tracer amounts of 63 Ni and 67 Cu using chromatographic assays. Subsequently, 1 H NMR titrations of 1a , 1b , 2a , and 2b with Zn(OAc) 2 were analyzed to yield 1:1 Zn(II)‐binding constants of 472 ± 59, 698 ± 120, 8,758 ± 2,237 and 20,100 ± 5,598 M −1 , respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1 H and 13 C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.