CHAPTER 2 Materials and methods
2.6 Molecular biology methods
2.7.4 Copper binding studies and the bicinchoninic acid assay
Copper binding and the oxidation state of copper bound to the recombinant proteins was assessed using the bicinchoninic acid (BCA) release assay. For this assay protein-copper complexes are disrupted by acid denaturation and the released copper detected in solution (Brenner and Harris, 1995). BCA is highly sensitive and specific for reduced copper, Cu(I), forming an intense violet complex detectable at 354 nm (extinction coefficient, ε = 4.58 ± 0.1 x 104 M-1 cm-1 at 354.5 nm). If oxidized copper, Cu(II), is present in solution then ascorbic acid must be added to the sample, following acid denaturation, to yield the Cu(I)-BCA complex. The formation of the Cu(I)-BCA complex in the absence of ascorbic acid, suggests the protein- bound copper ions were already in the reduced state. Recombinant proteins were also tested for their ability to reduce copper-catalysed ascorbic acid oxidation.
2.7.4.1 Materials
Ascorbic acid solution [100 mM Ascorbic acid]. Ascorbic acid (88 mg) was dissolved in 5 ml distilled water and stored at RT.
Copper stock solution 1 [10 mM CuCl2]. CuCl2.2H2O (8.5 mg) was dissolved in 5 ml distilled water and stored at RT. This solution was used for in vitro copper binding studies.
Copper stock solution 2 [1 M CuCl2]. CuCl2.2H2O (0.852 g) was dissolved in 5 ml distilled water and sterilised by filtration through a 0.22 µm syringe filter. This solution was used for in vivo copper binding studies.
Na-P buffer [0.1 M NaH2PO4, 0.01% NaN3, pH 7.5]. NaH2PO4 (11.998 g) was dissolved in 950 ml distilled water, titrated to pH 7.5 with NaOH and NaN3 added (1 ml of a 10% solution). The buffer was made to a final volume of 1 ℓ.
Reagent A [30% (w/v) Trichloroacetic acid]. Trichloroacetic acid (15 g) was dissolved in distilled water and made to a final volume of 50 ml. This reagent was stable at RT for up to 6 months.
Reagent B [2 mM Ascorbic acid]. Ascorbic acid (3.5 mg) was dissolved in 10 ml distilled water.
This reagent was prepared fresh before use.
Reagent C [0.15mM Bicinchoninic acid, 0.9 M NaOH, 0.2 M HEPES]. Purified bicinchoninic acid disodium salt (6 mg), NaOH (3.6 g) and HEPES (15.6 g) were dissolved in distilled water and made to a final volume of 100 ml. This reagent was stable at RT for up to 6 months.
H2Asc solution [240 µ M ascorbic acid, pH 4.5] . Ascorbic acid (2.2 mg) was dissolved in 45 ml distilled water, titrated to pH 4.5 with NaOH and made to a final volume of 50 ml
2.7.4.2 Method BCA release assay
The BCA release assay makes use of a HEPES-buffered BCA solution (Reagent C) with NaOH included to neutralise the TCA and bring the pH of the solution close to 7 (Brenner and Harris, 1995). For the assay, the desired protein solution (750 µl) was pipetted into a 2.0 ml microfuge tube and Reagent A added (250 µl). This solution was vortexed, to ensure mixing, and centrifuged (12 000 g, 2 min) to pellet the protein precipitate. The resulting supernatant was aliquotted (4 x 250 µl) into fresh microfuge tubes. For this particular study Reagent B (50 µl) was added to two of the tubes, whilst the remaining two tubes received an equivalent volume of distilled water. This solution was mixed and Reagent C (200 µl) then added to each tube, mixed and allowed to incubate at RT (2 min) before reading absorbance at 354 nm. Standards and blanks were prepared in the same final volume (500 µl).
In vitro copper binding
Each purified recombinant protein was used at 10 µM for in vitro copper binding studies. For copper binding, duplicate samples of each protein were prepared and each was mixed with a volume of copper stock solution 1, equivalent to a 20-fold molar excess. To one of the samples ascorbic acid solution was added to a final concentration of 10 mM, whilst the other sample received an equivalent volume of distilled water. Samples were mixed by brief vortex and incubated at RT (15 min) with occasional agitation. Unbound copper was removed by dialysis against three changes of Na-P buffer (> 100-fold volume excess) at 4°C, two by 2 h and one overnight. Following dialysis, protein samples were collected and bound copper measured using the BCA release assay, described above. Solutions of CuCl2 (Na-P buffer), with or without the addition of ascorbic acid, were used as standards and affinity-purified MBP served as a negative control. The statistical significance of copper binding was determined by Student's t-test.
In vivo copper binding
To test the ability of the respective proteins to bind copper in vivo, cells harbouring the relevant recombinant plasmid were induced in the presence of 0.5 mM CuCl2. This concentration of a copper salt has previously been shown to have no significant effect on the growth rate of E. coli cells (Lutsenko et al., 1997). Copper stock solution 2 was specifically added to recombinant E.
coli cultures 1 h post-IPTG-induced expression, to a final concentration of 0.5 mM. Standard procedures for optimal recombinant protein expression were followed (Section 2.7.1) and proteins were purified under identical conditions to the proteins expressed in the absence of copper (Section 2.7.2). Following purification, pooled His6-PyCox17 and GST-PfCox17 proteins were dialysed against three changes of Na-P buffer (>100-fold volume excess) at 4°C, two by 2 h and one overnight. MBP-fusion proteins were analysed for copper binding directly proceeding purification. All proteins were adjusted to a concentration of 10 µM, for consistency, and then analysed for bound copper using the BCA release assay described above. The statistical significance of copper binding and the effect of copper on recombinant protein yield were determined by Student's t-test.
Ascorbate oxidation assay
The ability of each recombinant protein to reduce the copper-catalysed oxidation of ascorbic acid was tested in vitro. In a typical experiment a mixture was prepared containing H2Asc solution to a final concentration of 120 µM (500 µl), copper stock solution to a final concentration of 8 µM (0.8 µl) and 5 µM recombinant protein (variable) made to a final volume of 1 ml with distilled water. The absorbance of this solution was monitored at 255 nm with readings taken every 5 s for 300 s. Reactions were carried out at RT.