Comparison of Structural and Chemical Properties of Black and Red Human Hair Melanosomes¶

Abstract

Melanosomes in black and red human hair are isolated and characterized by various chemical and physical techniques. Different yields of 4-amino-hydroxyphenolanaline by HI hydrolysis (a marker for pheomelanin) and pyrrole-2,3,5-tricarboxylic acid by KMnO(4)/H(+) oxidation (a marker for eumelanin) indicate that the melanosomes in black hair are eumelanosomes, whereas those in red hair are mainly pheomelanosomes. Atomic force microscopy reveals that eumelanosomes and pheomelanosomes have ellipsoidal and spherical shapes, respectively. Eumelanosomes maintain structural integrity upon extraction from the keratin matrix, whereas pheomelanosomes tend to fall apart. The black-hair eumelanosomes have an average of 14.6 +/- 0.5% amino acids content, which is attributed to the internal proteins entrapped in the melanosomes granules. The red-hair melanosomes contain more than 44% of amino acid content even after extensive proteolytic digestion. This high content of amino acids and the poorly reserved integrity of red-hair melanosomes suggest that some proteins are possibly covalently bonded with the melanin constituents in addition to those that are entrapped inside the melanin species. Soluene solubilization assay indicates the absorbance of melanin per gram of sample, adjusted for the amino acid content, is a factor of 2.9 greater for the black-hair melanosomes than the red-hair melanosomes. Metal analysis reveals significant amounts of diverse heavy metal ions bound to the two types of melanosomes. The amount of Cu(II) and Zn(II) are similar but Fe(III) content is four times higher in the red-hair melanosomes. (13)C solid-state nuclear magnetic resonance spectra and infrared spectra are presented and are shown to be powerful techniques for discerning differences in the amino acid contents, the 5,6-dihydroxyindole-2-carboxylic acid:5,6-dihydroxyindole ratio, and the degree of cross-linking in the pigment. Excellent agreement is observed between these spectral results and the chemical degradation data.

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

“…In this region, the most abundant proteins are α-keratins, which make microfibrils aligned along the fiber axis, and matrix proteins, which make an amorphous matrix embedding the microfibrils. [36] In general, all these proteins give 13 C-CPMAS NMR signals [37] that can be grouped for our purposes into four characteristic spectral ranges:…”

“…Both types of MGs show complex spectra, as only a part of the signals can be attributed to the melanin. In fact, model melanins (obtained from dopa, dopamine, or DHI) give the most intense 13 C NMR signals in the aromatic region (90-160 ppm range), with a weaker signal due to carboxyl/quinone groups in the 160-180 ppm range (typically at about 170 ppm), and a weaker signal in the aliphatic region (about 10-90 ppm) due to the uncyclized side chains of precursor catecholamines. [24] This is well exemplified by the spectrum of a synthetic L-dopa melanin shown in Fig.…”

“…[4] In several melanins of biological source, including melanin from human hairs, the true melanin polymer is tightly bound to protein-like material. [13,14] Owing to the wealth of catechol/quinone/semiquinone and carboxylic moieties exposed on the edge of melanin layers, melanins show a remarkable binding affinity for metal ions, and indeed melanins usually contain a noticeable amount of metals. [6,13,15 -17] The color of human black hair is due to the presence of eumelanin granules in the cortex at a level of 1-3% w/w of the total fiber mass.…”

“…[18] Melanin in black hair is organized into structures called melanosomes, that are rice-shaped ellipsoidal particles with a long axis of about 1 µm in length and a short axis of 0.3-0.5 µm in width. [13] Melanosomes are surrounded and tightly embedded into a proteic matrix, called the melanoprotein. The characterization of melanin is usually performed in purified samples rather than in intact hairs.…”

“…Moreover, signals from paramagnetic metals bound to melanin can be directly observed by EPR, allowing the characterization of the binding mode. [31 -34] Results and Discussion Solid state 13 …”