A, activity of unmodified bifunctional DNA glycosylases and DNA glycosylases conjugated to pyridoxal phosphate. 1 and 2, control (AP substrate, no enzyme); 3 and 4, DenV; 5 and 6, Nth; 7 and 8, Nei; 9 and 10, Fpg; 11 and 12, NEIL2; 13 and 14, NEIL1. AP substrate (S, 20 nM) is cleaved by the enzymes (500 nM) treated (even lanes) or not treated (odd lanes) with PLP for 30 min in the presence of NaBH4 to produce cleavage products of β-elimination (Pβ) or β,δ-elimination (Pδ). B, inhibition of NEIL2 by PLP in the absence of NaBH4 reduction. 1, fully active enzyme; 2, enzyme pre-treated with PLP but not reduced with NaBH4; 3, enzyme pre-treated with PLP and reduced with NaBH4; 4, no enzyme. The reaction conditions were the same as in Panel A except the absence of NaBH4 where indicated. C, Binding of unmodified and PLP-conjugated NEIL2 to the uncleavable THF:C ligand. Arrows indicate the mobility of the free THF:C ligand (L) and the enzyme–ligand complex (EL). 1, no enzyme, 2–6, NEIL2-PLP (2–1000 nM), 7–11, unmodified NEIL2 (2–1000 nM). See Materials and Methods for the reaction conditions.