Immobilization of Malarial (<i>Plasmodium
falciparum</i>) Dihydrofolate Reductase for the
Selection of Tight-Binding Inhibitors from
Combinatorial Library
Chawanee Thongpanchang
Supannee Taweechai
Sumalee Kamchonwongpaisan
Yongyuth Yuthavong
Yodhathai Thebtaranonth
10.1021/ac070215s.s001
https://acs.figshare.com/articles/journal_contribution/Immobilization_of_Malarial_i_Plasmodium_falciparum_i_Dihydrofolate_Reductase_for_the_Selection_of_Tight_Binding_Inhibitors_from_Combinatorial_Library/2999251
A simple procedure for selection of tight-binding inhibitors
of mutant dihydrofolate reductases from <i>Plasmodium
falciparum</i> (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.
2007-07-01 00:00:00
DHFR
PfDHFR
guanidine hydrochloride treatment
binding
enzyme
Plasmodium falciparum
affinity
gel
ligand
inhibitor