Zeise L, Murr B L, Chedekel M R
Facility for Advanced Instrumentation, University of California-Davis.
Pigment Cell Res. 1992 Sep;5(3):132-42. doi: 10.1111/j.1600-0749.1992.tb00009.x.
Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.
从乌贼墨囊中分离出的黑色素（乌贼黑色素）已被提议作为天然真黑色素的标准。目前尚无黑色素分离、纯化和储存的标准方法。因此需要采用温和的方法来保留黑色素的天然组成和结构。本研究以乌贼黑色素为研究对象，旨在开发一种温和且通用的黑色素分离纯化方法。众所周知，黑色素聚合物含有大量游离羧酸残基。这些阴离子残基导致了黑色素所具有的阳离子交换特性。已证实，将黑色素与盐酸一起回流加热会导致大量脱羧反应。实际上，仅加热就会导致脱羧，因此必须注意避免这种情况。类比阳离子交换树脂，黑色素应根据相关抗衡离子进行分离和命名（例如，乌贼黑色素——钾离子形式）。本文报道的方法避免了极端的pH值和温度条件，旨在得到钾离子形式的黑色素。还发现使用湿磨步骤对颗粒状黑色素进行物理解聚有助于去除大量吸附的蛋白质。采用以下物理参数来监测纯化过程并表征所得黑色素：pH值、电导率、粒径和漫反射光谱。
