重金属与肝素二糖的结合。I. 艾杜糖醛酸是主要结合位点。

Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

作者信息

Whitfield D M, Choay J, Sarkar B

机构信息

Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.

出版信息

Biopolymers. 1992 Jun;32(6):585-96. doi: 10.1002/bip.360320603.

DOI:10.1002/bip.360320603

PMID:1643264

Abstract

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-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-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 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(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-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H 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 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.

摘要

作为从人肾中分离出的镍（II）结合类肝素化合物的模型化合物（Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35 - 42.），我们研究了两种二糖——4 - O -（2 - O - 磺基 - α - L - 艾杜糖醛酸）- 2,5 - 脱水 - D - 甘露醇二钠盐（1a）和4 - O -（2 - O - 磺基 - α - L - 艾杜糖醛酸）- 6 - O - 磺基 - 2,5 - 脱水 - D - 甘露醇三钠盐（1b）——它们是在亚硝酸水解和还原后从肝素中分离得到的。在使用示踪离子63Ni(II)、54Mn(II)、65Zn(II)和109Cd(II)的高效液相色谱（HPLC）结合试验中，单硫酸盐（1a）具有活性，而双硫酸盐（1b）无活性。该结果与使用硫酸梯度和FPLC系统上的MonoQ阴离子交换柱从亚硝酸衍生的肝素二糖的完整库中分离出两种67Cu(II)和63Ni(II)结合级分一致。通过高分辨率FAB - MS、NMR和HPLC - PAD，其中一种被鉴定为化合物（1a），另一种为四硫酸化三糖。同样，两种合成二糖——甲基，2 - O - 磺基 - 4 - O -（α - L - 艾杜糖醛酸）- 2 - 脱氧 - 2 - 磺酰胺 - α - D - 葡萄糖胺三钠盐[IdopA2S(α1,4)GlcNSαMe, 2a]和2 - O - 磺基 - 4 - O -（α - L - 艾杜糖醛酸）- 2 - 脱氧 - 2 - 磺酰胺 - 6 - O - 磺基 - α - D - 葡萄糖胺四钠盐[IdopA2S(α1,4)GlcNS6SαMe, 2b]——通过色谱分析显示能结合痕量的63Ni和67Cu。随后，分析了用Zn(OAc)2对1a、1b、2a和2b进行的1H NMR滴定，以得到1:1的Zn(II)结合常数，分别为472±59、698±120、8758±2237和20100±5598 M-1。2a和2b的值表明存在螯合作用。有人认为艾杜糖醛酸残基是主要的金属结合位点。这一假设的NMR证据来自于加入抗磁性Zn(II)离子后艾杜糖醛酸共振峰明显的1H和13C化学位移变化。