当归生药中两种PR-10蛋白亚型的纯化与表征

Purification and characterization of two PR-10 protein isoforms from the crude drug of Angelica sinensis

摘要:

为了进一步研究当归 (Angelica sinensis) 生药中的蛋白质及其功能，通过80%硫酸铵沉淀、Sephadex G-50凝胶过滤层析、DEAE-Sepharose阴离子交换层析，首次从当归生药中纯化出两种分子量相近的蛋白 (命名为ASPR-C-1和ASPR-C-2)。ASPR-C-1和ASPR-C-2在SDS-PAGE上的分子量分别为17.33 kDa和17.18 kDa，在溶液中主要以单体形式存在，但会部分形成二聚体，二者均为糖蛋白，糖基含量分别为2.6%和8.2%。经基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF-TOF?) 鉴定发现ASPR-C-1和ASPR-C-2均为病程相关 (Pathogenesis-related 10，PR-10) 家族蛋白，且具有核糖核酸酶活性，比活分别为73.60 U/mg和146.76 U/mg。两种蛋白的最适pH相近，均为5.6左右，但最适温度不同，ASPR-C-1的为50 ℃，ASPR-C-2的为60 ℃。二者虽然在60 ℃下都表现出最大的酶活力稳定性，但在更高的处理温度 (80–100 ℃) 下，ASPR-C-1迅速失活，最终仅余20%左右活力，ASPR-C-2则表现出良好的热稳定性，最终仍有80%左右活力。此外，Fe2+对二者的酶活性具有激活作用，而Ca2+、Mg2+、Zn2+、Mn2+、Ag+、Cu2+、EDTA、DTT和SDS则会不同程度地抑制二者的酶活性。研究结果为深入研究来自当归生药的PR-10蛋白的生物学功能奠定了基础。

Abstract:

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe2+ had an activating effect on the ribonuclease activities of two isoforms while Ca2+, Mg2+, Zn2+, Mn2+, Ag+, Cu2+, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.