Document Detail

A reexamination of the melanin formation assay of tyrosinase and an extension to estimate phaeomelanin formation.
MedLine Citation:
PMID:  2515217     Owner:  NLM     Status:  MEDLINE    
This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.
P Aroca; J R Jara; A Blazquez; J C García-Borron; F Solano
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of biochemical and biophysical methods     Volume:  19     ISSN:  0165-022X     ISO Abbreviation:  J. Biochem. Biophys. Methods     Publication Date:  1989 Oct 
Date Detail:
Created Date:  1990-03-01     Completed Date:  1990-03-01     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7907378     Medline TA:  J Biochem Biophys Methods     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  327-37     Citation Subset:  IM    
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Murcia, Spain.
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MeSH Terms
Carbon Radioisotopes
Catechol Oxidase / analysis*
Cations, Divalent
Cell Line
Melanins / analysis*,  biosynthesis
Melanoma, Experimental / enzymology*
Mice, Inbred Strains
Monophenol Monooxygenase / analysis*,  metabolism
Nickel / pharmacology
Radioisotope Dilution Technique
Sodium Hydroxide / pharmacology
Reg. No./Substance:
0/Carbon Radioisotopes; 0/Cations, Divalent; 0/Melanins; 10028-17-8/Tritium; 12627-61-1/phaeomelanin; 1310-73-2/Sodium Hydroxide; 7440-02-0/Nickel; EC Oxidase; EC Monooxygenase

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