L tyrosine

Dr-Nb(III) was cloned, expressed, and purified as already reported [52 (link)]. The Dr-Nb(III) concentration was determined spectrophotometrically by measuring the absorbance at 407 nm (ε = 1.57 × 10⁵ M⁻¹ cm⁻¹) [52 (link)]. Apo-Dr-Nb was prepared, as already reported [53 ]. Peroxynitrite was obtained from Cayman Chemical (Ann Arbor, MI, USA). The concentration of Peroxynitrite was determined spectrophotometrically at 302 nm (ε = 1.705 × 10³ M⁻¹ cm⁻¹) [12 (link)]. L-tyrosine (obtained from Merck KGaA, Darmstadt, Germany) was dissolved in 5.0 × 10⁻² M Bis-Tris propane buffer, at pH 7.0 and 22.0 °C; the final L-tyrosine concentration was 1.0 × 10⁻⁴ M [25 (link),30 (link),36 (link),39 (link)]. All the other chemicals were purchased from Merck KGaA (Darmstadt, Germany). All chemicals were of analytical grade and were used without further purification.

A mixture consisting of 9 ml assay buffer (50 mM Tris HCl pH 8.5, 50 mM NaCl, 10% glycerol (v/v)), 1 ml of cell lysate, and 0.3 g zein from maize (Sigma-Aldrich, USA) was prepared in 15 ml polyethylene (PE) round-bottom tubes. The tubes were vortexed in order to resuspend the zein powder and incubated at 20 and 50 °C under constant mixing on a wheel for 48 h. Three replicates per samples were run in parallel. Hydrolysis and solubilization of zein was monitored by detection of TCA-soluble peptides. Standard curve was made with 0–5.5 mM L-tyrosine (Sigma-Aldrich, USA). Soluble L-tyrosine and L-tryptophan containing zein peptides specifically react with Folin-Ciocalteu reagent (Sigma-Aldrich, USA) to produce a color change with a maximum absorbance at 660 nm. The tubes were centrifuged at 12,000×g for 10 min in order to pellet insoluble proteins. Samples were taken avoiding the top carotenoid-rich layer and further diluted in order to fit within the standard curve range. Measurements were done in triplicate in a Hidex Sense microplate reader (Hidex, Finland).

Both the acellular fraction and the haemocyte lysate were subjected to a similar analysis of PO. Three substrates specific for PO enzymes were tested: (i) L-tyrosine (monophenol; Sigma-Aldrich) metabolized by tyrosinase only; (ii) L-DOPA (o-diphenol; Sigma-Aldrich) metabolized by the enzymes catecholase, tyrosinase and laccase; and (iii) p-phenylenediamine (PPD; p-diamine; Sigma-Aldrich) metabolized by laccase only [15 (link)].
We used 10 mM of L-DOPA (3-4-dihydroxy-L-phenyl-alkaline) (D9628, Sigma-Aldrich), L-tyrosine (CAS number 60-18-4, Sigma-Aldrich), and PPD (CAS number 106-50-3, Sigma-Aldrich) diluted in PBS immediately before the experiment to avoid oxidation.
One-hundred microlitres of cell-free haemolymph, 50 μl of substrate solution, and 50 μl of PBS (total volume of 200 μl) were added to each well into a 96-well microtiter plate. As controls, we used 50 μl of substrate solution with 150 μl PBS. For each treatment, we tested samples, controls, and blanks (200 μl of PBS) in triplicate.
The reaction kinetics were measured for 2 h. Readings were performed every 20 s at 28 °C in a microplate reader (VersaMax Tunable Microplate reader, Molecular Devices, Sunnyvale, CA), using λ = 490 nm for L-tyrosine and L-DOPA, and λ = 465 nm for PPD. The results were plotted using the maximum reaction speed (i.e. the increase in the absorbance/min), according to Alves et al. [29 (link)].