The "Unconventional" Effect of Cysteine on the In Vitro Synthesis of Melanin

Abstract

This report details some of our observations regarding the impact of cysteine on the air-mediated oxidation of catecholamines, particularly epinephrine. The intent was to synthesize light-colored, pheomelanin-like materials. Pheomelanin is commonly described as a material generated from a mixture of catecholamines and cysteine. However, we observed that (1) the presence of cysteine resulted in a concentration-dependent delay in the onset of color formation and (2) the presence of cysteine resulted in darker, more eumelanin-like materials. These effects were particularly impactful in the case of epinephrine. More elaborate studies involving other amino acids or scaled-up reactions were conducted with epinephrine as the precursor. These studies show that other amino acids, e.g., methionine or serine, could lead to darker materials, but none were as impactful as cysteine. Although our results are in contrast to typical descriptions regarding the impact of cysteine on the synthesis of melanin, they may reflect crucial differences between the in vitrovsin vivo synthesis of pheomelanin.

Figure 1

Relationship between decay constant k from eq 1 and the ratio of absorbance at 650 nm over 500 nm (A₆₅₀/A₅₀₀) to distinguish EuMN-like materials from PhMN-like materials according to eq 2.

Figure 2

(A): Relative AUC, compared to the reaction without C, as a function of C (mM) for the reactions involving epinephrine at various concentrations of C from Figure S1D. (B): Relationship between k and A₆₅₀/A₅₀₀ for the reaction mixtures involving epinephrine, with or without C, from Figure S1D.

Figure 3

Results of the kinetic experiments (n = 3) of the oxidation of epinephrine (0.25 mM) in the presence of C (between 0 and 0.9 mM). (A): Change in absorbance at 350 nm as a function of reaction time. (B): Correlation between the concentration of C and the average lag time ± standard deviation (n = 3) between the start of the reaction and the observed increase in absorbance at 350 nm.

Figure 4

Values of kvsA₆₅₀/A₅₀₀ for reactions involving epinephrine and C, M, or other AA (S, T, P, 4-OH P, G, W, Q, or L). Experimental details are discussed in the Section 2. The dotted line represents the theoretical relationship between k and A₆₅₀/A₅₀₀ according to eq 2; “reference” represents the average value ± standard deviation (n = 6) obtained for the reactions involving epinephrine without any AA present in this set of experiments.

Figure 5

(A): UV–vis spectra of F_disp at 0.2 mg/mL obtained from reaction mixtures containing epinephrine without any AA or containing S or C as outlined in the Section 2. (B): Concentration-dependent fluorescence profiles of solutions of F_disp obtained from reaction mixtures without AA or in the presence of S or C. (C): FT-IR spectra, normalized for their absorbance at 1600 cm^–1, of F_disp from the reactions involving epinephrine, epinephrine and S and epinephrine and C. (D): FT-IR spectrum, normalized for the absorbance at 1600 cm^–1, of the F_prec from the reaction involving epinephrine and C.