Table 2.
Summarized chemical and biological properties of pyomelanin and commercial melanin.
| Production yield | PyoENZ | PyoCHEM | PyoBACT | MelSYNTH | MelSEPIA |
|---|---|---|---|---|---|
| 1.25 ± 0.11 g/g 2,5-DMPA |
0.31 ± 0.03 g/g 2,5-DMPA |
0.55 ± 0.09 g/L 0.24 ± 0.04 g/g 4-HPA |
– | – | |
| Polymer size | |||||
| MW (g/mol) | 5400 | 2300 | 5700 | 2000 | NDf |
| Mn (g/mol) | 360 | 350 | 470 | 250 | – |
| Mp (g/mol) | 3600 | 2500 | 4600 | 1600 | – |
| Ð (MW/Mn) | 15.3 | 6.64 | 11.9 | 7.95 | – |
| Monomer composition | HGA, BQA, GA, GALDa | HGA, BQAa | HGA, BQA, GA, GALDa | DHI, DHICAb | DHI, DHICAb |
| Elemental analysis | (%) | (%) | (%) | (%) | (%) |
| C | 49.15 ± 0.08 | 54.61 ± 0.11 | 40.58 ± 0.03 | 49.69 ± 0.10 | 34.10 ± 0.03 |
| H | 3.40 ± 0.02 | 2.59 ± 0.10 | 3.15 ± 0.03 | 2.83 ± 0.05 | 3.23 ± 0.02 |
| N | 2.75 ± 0.02 | 0 | 3.65 ± 0.05 | 6.34 ± 0.03 | 6.31 ± 0.08 |
| Od | 44.70 | 42.80 | 52.62 | 41.14 | 56.36 |
| C/O | 1.10 | 1.27 | 0.77 | 1.20 | 0.6 |
| C/N | 18 | – | 11 | 8 | 5.4 |
| Formulac | C221H140N11O151 | C105H59O61 | C193H179N15O187 | C83H57N9O51 | – |
| Calc. mass (g/mol) | 5362 | 2295 | 5697 | 1995 | – |
| Solubility | |||||
| NaOH 0.05 N | ≤ 10 mg/mL | ≤ 10 mg/mL | ≤ 10 mg/mL | ≤ 10 mg/mL | ≤ 10 mg/mL |
| DMSO | ≤ 0.5 mg/mL | ≤ 0.5 mg/mL | ≤ 0.5 mg/mL | ≤ 0.5 mg/mL | ≤ 0.5 mg/mL |
| Other solvents | Insoluble | Insoluble | Insoluble | Insoluble | Insoluble |
| DPPH-antioxidant activity (EC50 µg/mL)e | 27.5 | 20.23 | 133.7 | 25.9 | > 300 |
| ROS scavenging activity (IC50 µg/mL) | 82.2 ± 5.5 | ND | ND | 284.1 ± 12.3 | > 500 |
| UV spectrum range | 200–700 nm | 200–700 nm | 200–700 nm | 200–700 nm | 200–700 nm |
| Fe3+-reducing activity | |||||
| % Related to MelSYNTH | 96 | 95 | 54 | 100 | 34 |
| In ng Fe2+/h/µge | 5.30 | 5.24 | 2.98 | 5.52 | 1.94 |
| Stability to T°C | ≤ 80 °C, 72 h | ≤ 80 °C, 72 h | ≤ 80 °C, 72 h | ND | ND |
| Cytotoxicity (c ≤ 500 µg/mL) | No | No | No | No | No |
| Phototoxicity (PIF) | No (< 2) | No (< 2) | No (< 2) | No (< 2) | No (< 2) |
Since the A400 nm value of solubilized pyomelanin depends on the size of the pigment, a spectrophotometric quantification by surrogate melanin for calibration will not be correct. Weighing precisely the final purified pigment remains the only valuable technique for the quantification of pyomelanin as well as other melanin.
aIdentified by 13C solid-state NMR (Fig. 2 and Table 1).
bTo confirm the global structure of MelSYNTH and MelSEPIA, assays on their degradation by alkaline-H2O2 were conducted similarly to those on human melanin41. After centrifugation, 10 μL of the reaction volume was injected into an RP-HPLC(DAD)-QToF system in negative mode and confirmed the L-Dopa melanin structure by the presence of the two markers PTCA and PDCA.
cElemental analysis (C, H, N, S) of the pigments was performed by combustion on a Thermo Finnigan EA 1112 analyzer equipped with an autosampler, all managed by the Eager Xperience software (THERMO SCIENTIFIC, France). The oven was set at 970 °C and the flash combustion at 1800 °C. In the formula CxHyNz, each index was deduced by (x, y, z) = %atom (data from elemental analysis) x Mw/Matom.
dThe index w (for oxygen Ow) was deduced from 100% – C – H – N.
eStandard deviations were < 5%. Composition (C, H, N) of MelSYNTH was similar to those described63, that of MelSEPIA close to the reported values42 (see sample Com).
fMolecular weight of the S. officinalis melanin could not be determined on the MCX column eluted in the same conditions (see Fig. 6S). GA, gentisyl alcohol; GALD, gentisaldehyde; ND, not determined.