ABSTRACT
Chemically unaltered melanosomes from black hair were isolated using a mild enzymatic procedure reported by Novellino et al. involving sequential treatment of a homogenized hair sample with different protease enzymes. Time-dependent fluorescence studies show, under identical conditions, that the rate of bleaching upon NH3/H2O2 treatment of hair melanosomes is twice that of Sepia melanosomes. The structure and morphology of hair melanosomes are compared to Sepia eumelanin using ESEM and TEM imaging studies. Black hair melanosomes are aggregates of rice-shaped ellipsoidal particles (0.8-1.0 microm in length and 0.2-0.6 microm in width) surrounded by an amorphous material suspected to be made of non-proteinacious materials. Sepia eumelanin aggregates are larger (2-5 microm) particles with a "doughnut" shape comprised of 100-150-nm spherical particles. Time-dependent TEM imaging studies of ammonia-treated (pH 10) hair melanosomes showed an initial breakdown of melanosomal aggregates followed by rupture of the melanosomal membrane, releasing melanin nanoparticles and leaving a ghost membrane behind. After prolonged treatment with aqueous NH3, a total loss of characteristic melanosome morphology was observed leading to an amorphous material. By contrast, Sepia melanosomes under identical conditions of ammonia treatment did not show such changes, probably due to different surface properties and aggregation behavior. Sodium hydroxide or sodium carbonate at identical pH did not show similar changes to ammonia, suggesting that the changes are not merely due to alkaline pH, but, rather, are specific to ammonia. Co-treatment with ammonia and peroxide induced a faster disintegration of the melanosomes, resulting in a complete dissolution and discoloration of melanin in 30 minutes. The data suggest that ammonia helps to release melanin nanoparticles out of melanosomes, making them more susceptible to oxidative attack by H2O2.
