Comparison of the Structural and Physical Properties of Human Hair Eumelanin Following Enzymatic or Acid/Base Extraction

Abstract

Eumelanin was isolated from a sample of black, Indonesian human hair using three different published procedures: two different acid/base extractions and an enzymatic extraction. The morphology and spectroscopic properties of the isolated pigments differ significantly. The acid/base procedures both yield an amorphous material, while enzymatic extraction yields ellipsoidal melanosomes. Amino acid analysis shows that there is protein associated with the isolated pigments, accounting for 52, 40 and 14% of the total mass for the two acid/base extractions and the enzymatic extraction, respectively. The amino acid compositions do not correlate with those of keratin or tyrosinase. Metal elemental analysis shows that the acid/base extraction removes a majority of many metal ions bound to the pigment. Chemical degradation analysis by KMnO4/H+ and H2O2/OH- indicates significant differences between the pigments isolated by acid/base and enzymatic extraction. After correction for the protein mass in the two pigments, the lower yields of both pyrrole-2,3,5-tricarboxylic acid and pyrrole-2,3-dicarboxylic acid, eumelanin degradation products, indicate acid/base extraction modifies the chemical structure of the melanin, consistent with the result of Soluene solubilization assay. While the optical absorption spectra of the bulk pigments are similar, the spectra of the molecular weight less than 1000 mass fractions differ significantly. The data clearly indicate that pigment obtained from human hair by acid/base extraction contains significant protein, exhibits destruction of the melanosome, and possesses altered molecular structure. The acid/base extracted hair melanin is not representative of the natural material and is a poor model system for studying the physical and biological properties of melanins. The enzymatically extracted hair melanin, on the contrary, retains the morphology of intact melanosomes and is an excellent source of human melanin.

Characterization of human hair melanin and its degradation products by means of magnetic resonance techniques

“…This is reasonable, since the very low pH used to hydrolyze proteins makes iron ions soluble and decreases the affinity of melanin for metal ions (coordination sites become protonated). [19] These results are consistent with the ICP measurement of iron content in CH-type and CP-type MGs (150 and 1300 ppm respectively). In the case of CP-type MGs, a further weak signal with six lines centered for g = 2.04 is sometimes detected, due to a small amount of Mn(II).…”

“…These treatments give melanin extracts of different morphology, structure, and chemico-physical properties. [19] Solubilization of melanin can only occur after (at least) partial degradation, involving cleavage of covalent bonds and modification of its chemical structure to yield a soluble derivative called soluble melanin or melanin free acid (MFA). [20 -22] Typically, this can be achieved by treatment with hydrogen peroxide in alkali.…”

The Ultraviolet Absorption Coefficient of Melanosomes Decreases with Increasing Pheomelanin Content

“…19 This is common to this class of organelle and has been widely observed. [28][29][30] Third, the outermost pigment surface of these melanosomes is pure eumelanin, despite the significant difference in their overall pigment composition, consistent with a "casing" model of mixed melanogenesis (the pheomelanin present in the melanosome is encased by eumelanin). 5,6,[31][32][33][34] Therefore, with increased pheomelanin content, the outer eumelanin coating will be thinner.…”

“…[35][36][37] This particular extraction method was widely used to extract melanosomes from tissue, but this approach is now known to both alter the physical and biological properties of the melanosome and result in isolation of a larger protein content than that associated with intact melanosomes. 29 As a specific example, melanin comprises ∼86% of the material present in enzymatically extracted melanosomes from black human hair, whereas the acid-base extraction of melanosomes from the same hair sample alters the melanosome structure, and the isolated materials contain 40-52% protein, depending on the method used. 29 Thus, although previous isolation techniques produced samples with a large protein content, enzymatic isolation of intact melanosomes reveals that the dominant constituent in the organelle is the melanin pigment.…”

“…29 As a specific example, melanin comprises ∼86% of the material present in enzymatically extracted melanosomes from black human hair, whereas the acid-base extraction of melanosomes from the same hair sample alters the melanosome structure, and the isolated materials contain 40-52% protein, depending on the method used. 29 Thus, although previous isolation techniques produced samples with a large protein content, enzymatic isolation of intact melanosomes reveals that the dominant constituent in the organelle is the melanin pigment.…”