Immobilization of Malarial (Plasmodium falciparum) Dihydrofolate Reductase for the Selection of Tight-Binding Inhibitors from Combinatorial Library

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• 作者：Chawanee Thongpanchang ; Supannee Taweechai ; Sumalee Kamchonwongpaisan ; Yongyuth Yuthavong ; Yodhathai Thebtaranonth
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：July 1, 2007
• 年：2007
• 卷：79
• 期：13
• 页码：5006 - 5012
• 全文大小：145K
• 年卷期：v.79,no.13(July 1, 2007)
• ISSN：1520-6882

文摘

A simple procedure for selection of tight-binding inhibitorsof mutant dihydrofolate reductases from Plasmodiumfalciparum (PfDHFRs) based on preferential binding tothe enzyme immobilized on a Sepharose column has beendescribed. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to athiopropyl-Sepharose gel via S-S linkage. The amount ofimmobilized DHFRs was estimated to be 4-5 mg/g ofdried gel, and the activities of bound DHFRs werecomparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based onthe affinities of each ligand with the enzyme. Free ligandswere then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry,and the components with high binding affinity of thelibrary could thus be realized. Results could be confirmedby quantitative analysis of the bound ligands released fromthe enzyme by guanidine hydrochloride treatment.