On Thu, 30 Nov 2000, Christine Lee wrote:

> I have methods specifying L-Dopa for studying melanin precursors and
> methods specifying DL-dopa. What is the difference?.

The histochemical method is for detection of the activity
of the enzyme DOPA oxidase. (It has several other names too:
monophenol monooxygenase, etc. EC No. 1.14.18.1, and is
closely similar to catechol oxidase, EC 1.10.3.1). This
enzyme, which occurs in melanocytes, catalyses the oxidation
of L-DOPA by oxygen to DOPA-quinone. This product is then
non-enzymatically oxidized and polymerized to make melanin.

Although the substrate of the enzyme is L-DOPA, it's OK to use
DL-DOPA, which is a mixture of L-DOPA and D-DOPA and is less
expensive. (The enzyme doesn't recognize the D-form of the
amino acid.) DOPA may appear in catalogues as its more
complete name, which is 3,4-dihydroxyphenylalanine.

Negative controls are important because DOPA oxidase occurs
in tissues that already contain melanin. In mammalian skin
melanin is synthesized in melanocytes and donated to epidermal
cells, which store the pigment but do not synthesize it. The
melanocytes, at the dermo-epidermal junction, with some in the
dermis, do not contain as much pigment as the epidermal cells,
but they blacken when incubated in the medium containing DOPA.
For negative controls, (a) omit the substrate, (b) add an 
inhibitor (10-3M cyanide or azide; not very specific but
DOPA oxidase is one of the inhibited enzymes). Skin from an
albino rat or mouse is negative. White or pale skin from a
non-albino should contain lots of melanocytes. (I've never
done this stain on a piece of skin with black and white in
the same section, as in a Long-Evans rat. Perhaps someone
else has, and will tell us if the appearances are what we
should expect.) 

You say you're working with equine skin. Interesting results 
might be obtained with specimens from a piebald pony, but make 
sure its measurements are correct: 3.142 cm diameter.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
   Phone: (519) 661-2111
   FAX (Department): (519) 661-3936
   E-mail: kiernan@uwo.ca