Development of novel system to analyze microheterogeneity of alpha_1-acid glycoprotein

采用毛细管等电聚焦（CIEF）技术分析了α - 1-酸性糖蛋白（AGP）的糖型。由于它们的pI值（pI 3.1-3.5）不同，AGP被分解成8个峰。这些峰的重现性（CV%）小于5% （n=5）。5个主要峰的相对峰面积CV%（各峰面积与总峰面积之比）均小于7.3%，总峰面积CV%为4.99% （n=5）。校准线在AGP浓度0.35 ~ 1.5g/L范围内线性良好（RSQ>0.99），覆盖了AGP生理血药浓度水平。此外，从对映体选择性和结合能力方面考察了AGP的支链聚糖结构在与基本药物相互作用中的作用。用魔芋豆蛋白A凝集素亲和层析法将AGP分离为不含双触角聚糖链的未保留AGP （UR-AGP）和含有双触角寡糖链的保留AGP （R-AGP）两个亚段。采用高效液相色谱法（HPFA）联合毛细管电泳（HPCE/FA）测定UR-AGP溶液和R-AGP溶液中普萘洛尔（PRO）对映体和维拉帕米（VER）对映体的未结合浓度。发现(S)-PRO与UR-AGP和R- agp的结合比(R)-PRO更强，而对VER对映体的结合则相反，并且这种对映体选择性是这些蛋白质所共有的。这说明AGP聚糖链的分支类型在结合这些基础药物的手性识别中没有发挥重要作用。

Capillary isoelectric focusing (CIEF) was applied to analyze the glycoforms of alpha1-acid glycoprotein (AGP). AGP was resolved into eight peaks due to the difference in their pI values (pI 3.1-3.5). And the reproducibility of those peaks (CV%) was less than 5% (n=5). The CV% of relative peak area (ratio of each peak area to total peak area) was less than 7.3% for five major peaks, and that of the total peak area was 4.99% (n=5). The calibration line shows good linearity (RSQ>0.99) within the AGP concentration range of 0.35-1.5g/L, which covers the physiological plasma concentration level of AGP.In addition, The role of the branching glycan structure of AGP in the interaction with basic drugs was investigated in terms of enantioselectivity in binding ability. AGP was separated by concanavalin A lectin affinity chromatography into two subfractions, the unretained AGP (UR-AGP) which has no biantennary glycan chain and the retained AGP (R-AGP) which possesses biantennary oligosaccharide chain(s). The unbound concentrations of propranolol (PRO) enantiomers and verapamil (VER) enantiomers in UR-AGP solution and R-AGP solution were determined by high-performance frontal analysis (HPFA) combined with capillary electrophoresis (HPCE/FA). It was found that (S)-PRO is bound to UR-AGP and R-AGP more strongly than (R)-PRO, whereas the reverse applies to VER enantiomers, and that such enantioselectivity is common to these proteins. This suggests that the branching type of glycan chains of AGP does not play significant role in the chiral recognition in binding these basic drugs.