Fast, isotope-free methods for the assay of thiamine-binding proteins and for the determination of their affinities to thiamine-related compounds

A fast, isotope-free method for the determination of parameters for the int eractions of proteins with thiamine and related compounds was developed. Th e free and bound forms of a ligand (thiamine or a fluorogenic analogue) wer e separated by ultrafiltration using commercially available centrifugal pro tein microconcentrators (Nanosep(TM), Pall Filtron). The free thiamine conc entration in the filtrate was analysed by (i) a pre-column derivatisation o f thiamine to thiochrome with the use of alkaline potassium hexacyanoferrat e(III) followed by reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate, pH 7.8. 5% tetrahydrofuran) with fluorometric dete ction (excitation at 365 nm, emission at 430 nm), or (ii) an ion-pair rever se-phase HPLC (isocratic, ODS column, 0.08% trifluoroacetic acid-0.08% sodi um octanesulfonate-25% tetrahydrofuran) with post-column derivatisation and fluorometric detection. The 'saturation-binding' version (single ligand ad ded in increasing doses to the protein samples) of this method allowed the determination of low micromolar concentrations of thiamine-binding proteins and of the dissociation constants of their complexes with thiamine or fluo rogenic thiamine analogues in the range of 0.3-10 mu M. Using the other, 'c ompetitive displacement' version (constant amount of thiamine plus increasi ng doses of a competing ligand), dissociation constants at least one order of magnitude higher could successfully be determined. (C) 2000 Elsevier Sci ence B.V. All rights reserved.