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

Xiangling Wang; Xian LI; Huocong HE; Lingling LI; Di LÜ; Cuihuang Chen; Xiaoqiang YE; Shutao Liu; Jianru Pan

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

Author: Xiangling WANG ¹ ; Xian LI ¹ ; Huocong HE ² ; Lingling LI ¹ ; Di LÜ ¹ ; Cuihuang CHEN ¹ ; Xiaoqiang YE ¹ ; Shutao LIU ¹ ; Jianru PAN ¹
Author Information

1. College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, Fujian, China.
2. Laboratory of Radiation Oncology and Radiobiology, Fujian Medical University Cancer Hospital & Fujian Cancer Hospital, Fuzhou 350014, Fujian, China.
Publication Type:Journal Article
Keywords: PR-10; crude drug of Angelica sinensis; glycoprotein; purify; ribonuclease
MeSH: Angelica sinensis; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Protein Isoforms; Temperature
From: Chinese Journal of Biotechnology 2019;35(1):159-168
CountryChina
Language:Chinese
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.