FIG. 2.

HPLC chromatograms of the products of flavokinase/FAD-synthetase assays. Assay mixtures containing 50 μM riboflavin, 3 mM ATP, 15 mM MgCl₂, and 10 mM sodium sulfite (Na₂SO₃) were preincubated at 37°C for 5 min. Pure wild-type RibC (A and B) or mutant RibC820 (C and D) (3 μg of each) was added, and the mixtures were incubated for another 5 (A and C) or 30 (B and D) min. An aliquot was removed and separated on an HPLC column (Nucleosil 10 C₁₈; 4.6 by 250 mm; Macherey & Nagel). The following solvent system was used at a flow rate of 2.5 ml/min: 25% (vol/vol) methanol–100 mM formic acid–100 mM ammonium formate (pH 3.7). The reaction was monitored with a fluorescence detector (excitation, 470 nm; emission, 530 nm; Waters Associates). The chromatograms show three clearly resolved peaks of riboflavin (8 min), FMN (5 min), and FAD (4 min). Numbers in parentheses indicate decrease of riboflavin and increase of flavin nucleotides during the enzyme assay.