Electrophoresis study
Purity of the two tyrosinase forms was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Whereas no bands corresponding to a non-target protein were visible, both forms showed a single band at around 39 kDa (see Fig. 2A). The in-gel activity was measured following non reducing SDS–PAGE as the reduction is detrimental to the activity of the enzyme. In this non reduced form the enzyme displays a higher electrophoretic mobility, which is probably due to its more compact conformation. Under non-reducing conditions the intramolecular disulfide bridges are intact and can therefore stabilize the enzyme’s conformation (see Fig. 2B). Moreover, the IEF (both samples applied) resulted in two spots at a pI of 5.1 and 5.2, respectively (see Fig. 2C). This matches with the theoretically calculated pI of 5.25 (ProtParam, ExPASy.org) corresponding to the identified amino acid sequence jrPPO1(Asp101–Pro444) and pI of 5.35 for jrPPO1(Asp101–Arg445) (UniProt.: COLU17) as described below.