Figure 6: Detection of TPP and OxPP bound to PfPDH and PfOxoDH.

HPLC analysis showing the major metabolite detected from strep-tagged affinity-purified PfPDH from 3D7-PDH⁺ parasites (a–c) and PfOxoDH from 3D7-OxoDH⁺ parasites (d–f) incubated in normal RPMI-1640 (a,d), normal RPMI-1640 in the presence of 5.2 mM oxythiamine (b,e) and thiamine-free RPMI-1640 in the presence of 11 μM oxythiamine (c,f). In b and c, the dashed lines in the background show the separation of the standards (OxPP, TPP, oxythiamine and thiamine) to enable the identification of the metabolite. The dashed lines in b, c, e and f represent an identical sample as that shown by the solid line, but which had been ‘spiked’ with 1 μM TPP (peak indicated by the asterisk) to illustrate that the cofactor bound to the target protein when the parasites had been treated with IC₅₀ concentrations of oxythiamine is distinct from TPP. The traces are representative of those obtained in two independent affinity-purification experiments.