The influence of iron on selected properties of synthetic pheomelanin

Abstract

It is believed that while eumelanin plays photoprotective and antioxidant role in pigmented tissues, pheomelanin being more photoreactive could behave as a phototoxic agent. Although the metal ion-sequestering ability of melanin might be protective, transition metal ions present in natural melanins could affect their physicochemical properties. The aim of this research was to study iron binding by pheomelanin and analyze how such a binding affects selected properties of the melanin. Synthetic pheomelanin (CDM), prepared by enzymatic oxidation of DOPA in the presence of cysteine was analyzed by electron paramagnetic resonance (EPR) spectroscopy, spectrophotometry, chemical analysis, and time-resolved measurements of singlet oxygen phosphorescence. Iron broadened EPR signal of melanin and increased its optical absorption. Iron bound to melanin exhibited EPR signal at g = 4.3, typical for high-spin iron (III). Iron bound to melanin significantly altered the kinetics of melanin photodegradation, which in turn modified the accessibility and stability of the melanin-iron complexes as indicated by the release of iron from melanin induced by diethylenetriaminepentaacetic acid and KCN. Although bound to melanin iron little affects initial stages of photodegradation of CDM, the effect of iron becomes more pronounced at later stages of melanin photolysis.

Keywords: Iron; Pheomelanin; Photoprotection; Phototoxicity; Singlet oxygen; Transition metal ions.

Fig. 1

The effect of iron on room temperature EPR signal of 2 mg/ml CDM. a Melanin signal amplitude plotted against square root of microwave power and fitted with the following function: f(x) = A·x·[1 + (2^1/ε – 1)·x²/P_1/2]^–ε [23] as described in “Materials and methods” section. Filled circles—control melanin without iron, open diamonds—melanin with 1% (w/w) iron. b Half power in samples containing 2 mg/ml CDM without (C) and with (Fe) 1% (w/w) iron after different incubation time

Fig. 2

Photograph of 0.1 mg/ml water solutions of CDM without (C) and with (Fe) 1% (w/w) iron

Fig. 3

UV–vis spectra of CDM diluted to 0.05 mg/ml in 1 M NaOH (a) and EPR spectra of 0.5 mg/ml CDM in 1 M HCl (b). Continuous line—CDM without iron, dotted line—CDM with 1% (w/w) iron

Fig. 4

7 K EPR spectra of CDM without (a–c) or with (d–f) 1% (w/w) iron. These EPR spectra were registered after dilution of CDM to 1 mg/ml in PBS, pH 7.4 (a, d), 45 mM DTPA adjusted to pH 7.4 (b, e) or 45 mM DTPA with 5 mM KCN (resultant pH 7.8) (c, f) and their incubation at room temperature for 1 h (continuous line) or 24 h (dotted line)

Fig. 5

EPR spectra of iron bound to CDM. EPR spectra a–c were registered at 77 K after dilution of CDM to 1 mg/ml in PBS, pH 7.4 (a), 45 mM DTPA adjusted to pH 7.4 (b), or 45 mM DTPA with 5 mM KCN (resultant pH 7.8) (c) and their incubation at room temperature for 1 h (continuous line) or 24 h (dotted line). d Wide scan carried out at 20 K at CDM concentration 2 mg/ml

Fig. 6

The effect of iron on effectiveness of quenching of singlet oxygen and on the yield of photoinduced generation of this reactive oxygen species. a Inverse of singlet oxygen lifetime plotted against the concentration of CDM without (filled circles) and with (open diamonds) 1% (w/w) iron. b Constant of quenching of singlet oxygen. c Action spectra of photoinduced formation of singlet oxygen by CDM without iron (continuous line) or with 1% (w/w) iron (dotted line). d Yield of genergbation of singlet oxygen by CDM excited with 365 nm (UV) or 425 nm (vis) light

Fig. 7

Results of chemical analysis of melanin subunits. a Analysis by HI hydrolysis: bar a—4-AHP, bar b—3-AHP. b Analysis by alkaline H₂O₂ oxidation: bar c—PTCA, bar d—PDCA, bar e—TTCA. The presented values are averages from two analyses

Fig. 8

Time-dependent changes of 77 K integrated EPR signal of CDM (a, b) and 350–550 nm integrated optical absorption (c) irradiated with 400 nm (265 mW/cm²) light. EPR measurements were carried out in PBS, pH 7.4 (a) or in 45 mM DTPA with 5 mM KCN, pH 7.8 (b). Optical absorption was measured in 1 M NaOH. Filled circles—control melanin without iron, open diamonds—melanin with 1% (w/w) iron

Fig. 9

77 K EPR spectra of iron bound to CDM photodegraded for 40 h except dotted line in c. EPR measurements were carried out after the dilution of CDM samples to 0.5 mg/ml in PBS, pH 7.4 (a), 45 mM DTPA with 5 mM KCN, pH 7.8 (b), 45 mM DTPA, pH 7.4 (c), water, pH 7.4 (d). Dotted line in c—0.1791 iron complex with citrate after addition of 45 mM DTPA. EPR signal of iron in citrate—DTPA system is multiplied by 0.2