半夏刺激性蛋白分子量的确定及氨基酸组成解析

Analysis of molecular weight and amino acid composition of irritant protein from Pinelliae Rhizoma

摘要

目的 确定半夏刺激性蛋白的分子量及氨基酸组成。方法 采用Tris-HCl缓冲溶液法提取半夏总蛋白,利用硫酸铵沉淀法、SP-Sepharose HP离子交换柱色谱法与Sephadex G-100葡聚糖凝胶柱法分离、纯化半夏蛋白。然后采用小鼠腹膜炎症模型评估半夏蛋白的刺激性。进一步利用SDS-PAGE和高效凝胶渗透色谱法(HPGPC)及凝胶色谱-示差-多角度激光光散射(GPC-RI-MALS)法测定蛋白的分子量。最后,采用高效液相色谱法与Nano-UPLC-Q-Exactive HF对蛋白进行氨基酸组成分析和鉴定。结果 40%饱和度硫酸铵沉淀半夏蛋白(PT-40)能显著增加小鼠腹腔内液中的蛋白渗出量和PGE₂含量,进一步分离得到的刺激性蛋白(PTP-1)被鉴定为分子量约为56 kD的均质四聚体蛋白,由4个14 kD亚基组成。氨基酸组成分析和Nano-UPLC-Q-Exactive HF鉴定确认PTP-1为半夏凝集素蛋白,与已知具有刺激性的凝集素蛋白的氨基酸组成具有高度相似性。结论 成功确定半夏刺激性蛋白PTP-1的分子量及氨基酸组成,为半夏刺激性凝集素蛋白分子量的确定提供了依据,同时为半夏的临床应用和新药开发提供了科学参考。

Abstract

Objective To determine the molecular weight and amino acid composition of the irritant protein from Pinelliae Rhizoma. Methods Total protein was extracted from Pinelliae Rhizoma using Tris-HCl buffer solution. The protein was isolated and purified through ammonium sulfate precipitation, SP-Sepharose HP ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The irritancy of proteins was evaluated using a murine peritoneal inflammation model. Molecular weight was determined by SDS-PAGE, High-Performance Gel Permeation Chromatography (HPGPC), and Gel Permeation Chromatography coupled with Refractive Index and Multi-Angle Laser light scattering (GPC-RI-MALS). Finally, HPLC and Nano-UPLC-Q-Exactive HF were used to analyze and identify the amino acid composition of the irritant protein. Results Proteins in Pinelliae Rhizoma precipitated by 40% ammonium sulfate (PT-40) can significantly increase the amount of protein exudation and PGE₂ content in the peritoneal fluid of mice. The stimulatory protein (PTP-1) purified from PT-40 using ion exchange column chromatography was identified as a homogeneous tetrameric protein with a molecular weight of 56 kD, consisting of four 14 kD subunits. The results of amino acid composition analysis and Nano-UPLC-Q-Exactive HF identification confirmed that PTP-1 was a lectin protein possess in Pinelliae Rhizoma, which was highly similar to the amino acid composition of reported stimulatory lectin proteins. Conclusion This study successfully determines the molecular weight and amino acid composition of the irritant protein PTP-1 derived from Pinelliae Rhizoma. It revises the molecular weight of pungent lectin protein of Pinelliae Rhizoma previously reported in the literatures, thereby providing a scientific basis for clinical application and the development of new drugs of Pinelliae Rhizoma.

关键词

半夏 / 凝集素蛋白 / 分子量 / 氨基酸组成

Key words

Pinellia ternata / lectin protein / molecular weight / amino acid composition

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