Protein content in biological samples was determined

using the Coomassie Blue dye-binding procedure of Bradford (1976). Proteins were separated in 7.5% SDS-PAGE (Gallagher et al., 1992), and the resolved proteins were stained with Coomassie Blue R250. Recombinant wild type and mutant apoforms of LH were activated on the addition of 4 mM CaCl2 and 200 μM PQQ followed by subsequent incubation at room temperature for 1 h. The LH activity was measured spectrophotometrically using horse heart cytochrome c as the electron acceptor (Hopper et al., 1991). A unit of LH enzyme activity is the amount capable of reducing 2 μmol of cytochrome c min−1 at 25 °C. Purified his4-tagged recombinant wild-type LH (2 μM) was reduced VEGFR inhibitor with 50 mM freshly prepared

DTT for 1 h, treated with 200 mM iodomethane under a nitrogen-flushed atmosphere and left in the dark for a further 1 h. The unreduced enzyme was alkylated similarly as the control. The samples were passed through a Ni-agarose column (0.5 mL bed volume) to remove DTT from the treated sample. The control sample was eluted with 100 mM imidazole (pH 8) and 100 mM EDTA, but the Forskolin in vivo reduced/alkylated sample was eluted using 8 M urea and rapidly diluted with H2O to 0.8 M as it could not be eluted from the column under standard conditions. The activated LH was reduced with varying amounts of DTT (0–5 mM), and CdCl2 ranging from 0 to 25 mM was added to the preparations and incubated for 1 h. Excess DTT and CdCl2 were removed by dialysis of the protein solutions on 0.2-μm Millipore sterile filters in 20 mL 10 mM Tris–HCl (pH 8) for 1 h in a sterile Petri dish. Free thiol content estimation of lupanine hydroxylase in either native (wild type) or DTT-reduced state was published earlier in Stampolidis et al. (2009). Reaction of LH with

Ellman’s reagent occurred following the reduction of the thiol groups, but not in the unreduced state of the molecule, implying the potential presence of a disulphide bond. This initial observation formed the basis for the investigation presented in this paper. To determine whether the two Cys residues present in LH are disulphide bonded, a purified preparation of the recombinant wild-type enzyme was treated with iodomethane. Measurement Amylase of the specific activities of LH preparations of the reduced and unreduced alkylated enzyme had specific activities of 182 and 169 (± 5%) A555 min−1 mg−1 protein with 83% and 77% relative to control sample, respectively. However, the reduced and alkylated enzyme had a specific activity of 19 (± 5%) A555 min−1 mg−1 protein and only 9% activity relative to control sample (Table 1). The loss in activity of reduced/alkylated form indicated that Cys residues of LH must form a disulphide bond that could play a role in the activity and/or the stability of the enzyme.