title: Covalent Immobilization of Human Placental 17β-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications.
abstract: Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), a potential target in breast cancer prevention and therapy, was extracted from human placenta and immobilized on nonporous silica (∼5 μm) with a covalent method for the first time. The optimum initial enzyme concentration and immobilization time during the immobilization process were 0.42 mg mL-1and 12 h, repectively. The binding was confirmed by scanning electron microscope (SEM) and infrared spectroscopy (FT-IR). It could improve the pH, thermal and storage stability compared to free enzyme. Moreover, the immobilized enzyme could be reused at least four times. A screening method based on it coupled with liquid chromatography-time-of-flight mass spectrometer (LC-TOF/MS) was established, and the half-maximal inhibitory concentration (IC 50) of apigenin for the immobilized enzyme was 291 nM. Subsequently, 10 natural products were evaluated leading to inhibition of the activity of 17β-HSD1 at the concentration of 25 μM, and six of them inhibit the activity over 50%.
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
authors.forename: Wen-Di
authors.initials: WD
authors.lastname: Zhou
authors.affiliation: The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Xian-Min
authors.initials: XM
authors.lastname: Mu
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Yu
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China. dibin@cpu.edu.cn.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China. dibin@cpu.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Di
authors.affiliation: Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, People's Republic of China. sumengxiang@cpu.edu.cn.|Key Laboratory on Protein Chemistry and Structural Biology, China Pharmaceutical University, Nanjing, 210009, People's Republic of China. sumengxiang@cpu.edu.cn.
authors.forename: Meng-Xiang
authors.initials: MX
authors.lastname: Su
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry*; D047310:Apigenin / Q000737:chemistry*; D002853:Chromatography, Liquid; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005260:Female; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D011257:Pregnancy Proteins / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004800:Enzymes, Immobilized; D011257:Pregnancy Proteins; D012822:Silicon Dioxide; D047310:Apigenin; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D005976:Glutaral
keywords: Covalent immobilization; Enzyme stability; Human 17β-hydroxysteroid dehydrogenase type 1; Inhibitor screening; LC-TOF/MS