Detection of dopachrome isomerase on gels and membranes.
Dopachrome isomerase, which mediates the conversion of dopachrome to dihydroxyindoles, is an enzyme affiliated with the melanogenic pathway found in most vertebrates and invertebrates. We have recently reported an activity-staining procedure for detecting this enzyme after electrophoresis on tyrosinease-embedded polyacrylamide gels using dopa [K. Nellaiappan, G. Nicklas, and M. Sugumaran, (1994) Anal. Biochem. 220, 122-128]. The usefulness of this method is limited to the availability of good quality of tyrosinase. To overcome the limitations of nonavailability as well as batch to batch variation in the specific activity of commercially available tyrosinase which will affect the staining, we have developed a new stain technique using chemically made dopachrome for the detection of dopachrome isomerase. The new method employs the use of dopachrome generated in situ by the oxidation of dopa with periodate. Dopachrome isomerase initially appears as a bluish-purple (melanochrome) band against an orange-red background. Eventual transformation of melanochrome to dark insoluble melanin polymer produces a black band corresponding to dopachrome isomerase activity. Continuous washing of the gel clears the background. Usefulness of this technique to stain dopachrome isomerase (a) in different organisms and (b) on polyacrylamide gels, agarose gels, and nitrocellulose membranes is demonstrated. In addition, conditions to stain the enzyme on nitrocellulose membranes using mushroom tyrosinase are described. Comparison of the effectiveness of tyrosinase versus periodate staining procedure reveals that the former method is superior to the latter in detecting dopachrome isomerase. The periodate method overcomes the problems associated with the quality of commercially available tyrosinase and its nonavailability in certain regions.
Nicklas
G
G
Department of Biology, University of Massachusetts, Boston 02125, USA.
Sugumaran
M
M
D003101:Collodion; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019746:Intramolecular Oxidoreductases; D007527:Isoenzymes / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D013194:Staining and Labeling
D007527:Isoenzymes; D003101:Collodion; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase