Development of fine micro analytical method of drug recognition function of sugar chains

建立了一种毛细管电泳质谱(CE-MS)分析系统，用于研究糖蛋白的结构对药物结合亲和力的影响。以α1-酸性糖蛋白(AGP)为模型糖蛋白，研究了AGP糖型和遗传变异体与基础药物的结合亲和力.结果表明：(1)AGP分子上靠近药物结合部位的双天线糖链对AGP与基础药物(异丙基吡胺和维拉帕米)的对映选择性结合没有显著影响.(2)AGP有三个主要的遗传变异体，即A变异体、F1变异体和S变异体.在与模型药物对映体的结合中，A变异体表现出比F1^*S变异体更高的对映体选择性，而在与维拉帕米对映体的结合中，这些遗传变异体表现出几乎相同的对映体选择性。(C)采用无鞘电喷雾电喷雾质谱方法的CE/MS系统在分析agp变异体…的胰酶解酶时的检测率提高了一级为了研究糖蛋白的微观异质性对蛋白质功能和二级结构的影响，用核磁共振、分子轨道方法和毛细管电泳法研究了几种人工合成的钠通道蛋白和病毒蛋白的结构化学。合成的片段对应于负责蛋白质二级结构和/或功能的主要多肽部分。研究发现：(A)由于溶剂的性质和组成，模型肽片段的二级结构随环境的疏水性而变化。(B)同时含有疏水部分和疏水部分的葡聚糖形成局部疏水空间，其微介电常数取决于多糖的性质、序列和分支结构。提示疏水局部环境的形成会引起蛋白质二级结构的改变，从而调节蛋白质的功能和淀粉样原纤维的形成。较少

A novel analytical system using capillary electrophoresis (CE) and mass spectroscopy (MS) was developed to investigate the structural effect of glycoproteins upon drug binding affinity. α1-acid glycoprotein (AGP) was used as a model glycoprotein, and the binding affinities of AGP glycoforms and genetic variants to model basic drugs were evaluated.(a) The biantennary glycochains which locate only near the drug binding site on AGP molecule does not give any significant effect upon the enantioselective binding of AGP to model basic drugs (disopyramide and verapamil).(b) AGP has three major genetic variants, A-variant, F1-variant and S-variant. A-variant shows higher enantioselectivity in the binding with the enantiomers of model drugs than F1^*S variants, while these genetic variants show almost the same enantioselectivity in the binding to verapamil enantiomers.(c) CE/MS system using sheathless ESI method allows one-order higher detectability in the analyses of tryptic digests of AGP var … More iants than conventional CE method using UV detection.In order to investigate the effect of microheterogeneity of glycoproteins upon the function and the secondary structure of the proteins, the structural chemistry of several synthesized peptide fragments of Na-channel protein and prion protein were studied by using NMR, molecular orbital method and CE spectroscopy. The synthesized fragments correspond to the major peptide portion responsible for the secondary structure and/or the function of the proteins. The followings were found.(a) The secondary structure of model peptide fragments changes depending on the hydrophobicity of environment due to the nature and the component of solvents.(b) Glycans containing both hydrophobic moiety and hydrophobic moiety create local hydrophobic space with different micro-permittivity depending on nature, sequence and the branching structure of glycans. It is suggested that the creation of hydrophobic local environment would cause the change of secondary structure of protein, resulting in the regulation of function and amyloid fibril formation. Less