Apr 06, 2023
Synthesis and preparation of DOPA pheomelanin and eumelanin
- Kazumasa Wakamatsu1
- 1Institute for Melanin Chemistry, Fujita Health University, Toyoake, Japan
Protocol Citation: Kazumasa Wakamatsu 2023. Synthesis and preparation of DOPA pheomelanin and eumelanin. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27j4qg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 06, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 80143
Keywords: ASAPCRN
Funders Acknowledgement:
From cancer associations to altered immunity in the pathogenesis of Parkinson's disease
Grant ID: ASAP-000312
Abstract
This is the protocol for synthesizing and preparing DOPA pheomelanin and DOPA eumelanin using tyrosinase.
Attachments
As precursor(s) of melanin, 1) l-dopa, 2) DHI, 3) a mixture of DHI and DHICA, 4) DHICA, 5) a mixture of l-dopa and l-cysteine, and 6) 5SCD in the presence of l-dopa is used.
To a solution of the precursor(s) (1 mmol) in 0.1 M sodium phosphate buffer, pH 6.8 (98 ml), add a solution of mushroom tyrosinase in the same buffer (2 ml) at 25˚C.
The amount of mushroom tyrosinase (Sigma-Aldrich, 4270 U/mg) is either 50,000 U or 100,000 U.
Carry out oxidation with vigorous (maximal, but avoiding splashing) stirring in a 500 ml Erlenmeyer flask.
Stop oxidation (1-4) at 4 h by adding 6 M HCl (2 ml) to make pH 1 and keep the mixture at 4˚C overnight to ensure complete precipitation of the pigment.
Collect the precipitate by centrifugation at 3,000 rpm for 10 min and wash with 0.1 M HCl (40 ml x 3).
For the preparation of pheomelanins (5 and 6), acidify the oxidation mixture with acetic acid (3 ml) to make pH 3, and wash the precipitate with 1% acetic acid (40 ml x 3).
Dry the melanin powder by lyophilization and equilibrate with moisture in a desiccator with a saturated CaCl2 solution.
Prior to treatment, suspend the synthetic DOPA pheomelanin (DOPA:Cys = 1:1) and DOPA eumelanin (DOPA:Cys = 1:0) in sterile PBS and sonicate overnight.
Protocol references
d'Ischia, M., Wakamatsu, K., Napolitano, A., Briganti, S., Garcia‐Borron, J. C., Kovacs, D., ... & Ito, S. (2013). Melanins and melanogenesis: methods, standards, protocols.Pigment cell & melanoma research,26(5), 616-633.