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Immobilization of Malarial (Plasmodium falciparum) Dihydrofolate Reductase for the Selection of Tight-Binding Inhibitors from Combinatorial Library
journal contribution
posted on 2007-07-01, 00:00 authored by Chawanee Thongpanchang, Supannee Taweechai, Sumalee Kamchonwongpaisan, Yongyuth Yuthavong, Yodhathai ThebtaranonthA simple procedure for selection of tight-binding inhibitors
of mutant dihydrofolate reductases from Plasmodium
falciparum (PfDHFRs) based on preferential binding to
the enzyme immobilized on a Sepharose column has been
described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a
thiopropyl-Sepharose gel via S−S linkage. The amount of
immobilized DHFRs was estimated to be 4−5 mg/g of
dried gel, and the activities of bound DHFRs were
comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on
the affinities of each ligand with the enzyme. Free ligands
were then identified and analyzed quantitatively by high-performance liquid chromatography−mass spectrometry,
and the components with high binding affinity of the
library could thus be realized. Results could be confirmed
by quantitative analysis of the bound ligands released from
the enzyme by guanidine hydrochloride treatment.