Functional and structural features of recombinant wild-type and Cys338Tyr aldolase A. (A) FPLC profiles of the wild-type (black) and Cys338Tyr (blue) aldolase A enzymes. Both enzymes have a molecular mass of about 170 kDa, as expected for the histidine-tagged homo-tetrameric form of aldolase A. The dotted line and arrows indicate the molecular masses of the protein standards used in calibration, that is, β-amylase (200 kDa), chicken ovalbumin (44 kDa), and carbonic anhydrase (29 kDa) on a 50-cm by 1.6-cm Sephadex G-200 column (Amersham Biosciences, Milan, Italy) in 20 mM Tris (tris(hydroxymethyl)aminomethane)–HCl (pH 7.4), 20% (vol/vol) glycerol, with a flow rate of 0.2 mL/min. (B) Melting curves of wild-type (black) and Cys338Tyr (blue) aldolase A obtained by monitoring molar ellipticity at 222 nm. Inset: the CD spectra of wild-type (black) and Cys338Tyr (blue) aldolase A, recorded at 20°C with 0.4 mg/mL protein. (C) Specific activity represents μmol of hexose substrate cleaved × minute (μmol × minute^-1) × mg of enzyme. The hexose fructose 1,6-biphosphate cleavage rate (μmol × minute^-1) was determined spectrophotometrically by measuring nicotinamide adenine dinucleotide (NADH) oxidation at 340 nm in a coupled assay with α-glycerolphosphate dehydrogenase/triosophosphate isomerase.