当归生药中两种PR-10蛋白亚型的纯化与表征

为了进一步研究当归(Angelicasinensis)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。

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

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,Fe^2+ had an activating effect on the ribonuclease activities of two isoforms while Ca^2+,Mg^2+,Zn^2+,Mn^2+,Ag^+,Cu^2+,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.