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 interactions of proteins with thiamine and related compounds was developed. The free and bound forms of a ligand (thiamine or a fluorogenic analogue) were separated by ultrafiltration using commercially available centrifugal protein microconcentrators (Nanosep, Pall Filtron). The free thiamine concentration in the filtrate was analysed by (i) a pre-column derivatisation of thiamine to thiochrome with the use of alkaline potassium hexacyanoferrate(III) followed by reverse-phase HPLC (isocratic, analytical ODS column, 10 mM potassium phosphate, pH 7.8, 5% tetrahydrofuran) with fluorometric detection (excitation at 365 nm, emission at 430 nm), or (ii) an ion-pair reverse-phase HPLC (isocratic, ODS column, 0.08% trifluoroacetic acid-0.08% sodium octanesulfonate-25% tetrahydrofuran) with post-column derivatisation and fluorometric detection. The 'saturation-binding' version (single ligand added 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 fluorogenic thiamine analogues in the range of 0.3-10 microM. Using the other, 'competitive displacement' version (constant amount of thiamine plus increasing doses of a competing ligand), dissociation constants at least one order of magnitude higher could successfully be determined.