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An improved HPLC method for the quantitation of 3 ′-phosphoadenosine 5 ′-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells
作者: Xu, Jiaojiao1; Chen, Ye1; Li, Liang1; Li, Zaiquan3; Wang, Chenai1; Zhou, Tianyan1,2; Lu, Wei1,2
关键词: 3 &prime ; -Phosphoadenosine 5 &prime ; -phosphate (PAP) ; HPLC ; Sulfotransferases ; Enzymatic kinetics
刊名: JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
发表日期: 2012-03-25
DOI: 10.1016/j.jpba.2011.12.015
卷: 62, 页:182-186
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology
类目[WOS]: Chemistry, Analytical ; Pharmacology & Pharmacy
研究领域[WOS]: Chemistry ; Pharmacology & Pharmacy
关键词[WOS]: THERMOSTABLE PHENOL SULFOTRANSFERASE ; HUMAN ESTROGEN SULFOTRANSFERASE ; KINETIC-ANALYSIS ; L-SELECTIN ; SULFATION ; LIVER ; 5-PHOSPHOSULFATE ; RAT
英文摘要:

Sulfotransferase-catalyzed (SULT-catalyzed) sulfation is responsible for hormone regulation and xenobiotic detoxification. In the current study, a sensitive and widely applicable method of reversed-phase HPLC-UV was developed for the determination of 3′-phosphoadenosine 5′-phosphate (PAP), a common product of different subtypes of sulfation reactions, in HepG2 cells. The analyte was separated on a ZORBAX Extend-C18 column with the mobile phase of methanol and water containing 75 mM KH2PO4, 100 mM NH4Cl, and 1 mM 1-octylamine (pH 4.55), at a flow rate of 1.0 ml/min. The assay exhibited linearity over the range of 0.1-20 mu M for PAP with a correlation coefficient of 0.9995. The total time per run was under 10 min. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 82.6-102.0%. The method was applied to the determination of kinetic parameters K-m, V-m, and K-cat, of three different SULTs (SULT1A1, SULT2A1, and SULT1E1) in HepG2. This universal HPLC-UV method was easy to perform, economically feasible, and suitably efficient for the investigation of the enzyme kinetics of the SULT family using multiplex substrates. (C) 2011 Elsevier B.V. All rights reserved.

语种: 英语
所属项目编号: 81072699
项目资助者: National Natural Science Foundation of China (NSFC) ; School of Pharmaceutical Sciences, Peking University
WOS记录号: WOS:000301032200025
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/60132
Appears in Collections:北京大学药学院_药剂学系_期刊论文

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作者单位: 1.Peking Univ, Hlth Sci Ctr, Sch Pharmaceut Sci, Dept Pharmaceut, Beijing 100191, Peoples R China
2.Peking Univ, State Key Lab Nat & Biomimet Drugs, Beijing 100191, Peoples R China
3.Peking Univ, Hlth Sci Ctr, Sch Basic Med Sci, Dept Pathol, Beijing 100191, Peoples R China

Recommended Citation:
Xu, Jiaojiao,Chen, Ye,Li, Liang,et al. An improved HPLC method for the quantitation of 3 ′-phosphoadenosine 5 ′-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells[J]. JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS,2012,62:182-186.
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