I have Drosophila melanogaster which I am doing an eye pigmentation assay on in the future. To do this I will dissolve the heads, 10 of them removed from frozen whole flies, in acidified ethanol for at least 48 hours (in the dark at 25 degrees) and then measure the colour of the liquid using a nanodrop. Specifically, this measures how well the liquid reflects red light, quantifying how much red pigment is in there.

I would like to know what causes the red colour in the eye and if leaving it dissolving for extended periods of time will (or is likely to) result in changes of what I measure? (ie is the material causing the redness in the eye susceptible to deterioration, which would change the colour of the liquid it is dissolving in over time)

Have any methods type papers tested this by comparing those dissolved for longer periods to those over shorter periods?

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    $\begingroup$ Just on a detail - a red pigment doesn't absorb red light, it absorbs light at "all other" wavelengths. $\endgroup$ – Alan Boyd Oct 24 '13 at 20:04

There's been a fair amount of work on this subject but a lot of it is old - see for example this 1945 paper and this 1948 paper. That latter one in particular goes into some good stuff: color in the wildtype is due to red and brown pigments, which the authors separated by using acidic ethanol (after grinding up the flies). Here's a 1961 paper from JSTOR that goes into a bit more detail on some of the red pigments (drosopterin, and isodrosopterin, and neodrosopterin) and looks like a good reference for some other, more basic work on Dmel eye pigmentation. More to the point, here's a 2013 minireview on the chemical synthesis of drosopterins, which is useful mainly for referencing a lot of other work, in particular this overly detailed chapter from 1970. That last one has tons of info about some of these pigment-causing proteins in insects.

The only particular reference to stability I could find was from that chapter, on page 144:

The simple pterines (group 2) are chemically stable under normal physiological conditions, while the substituted (groups I and 3) and conjugated pterines are unstable. Light catalysed oxydation of the polyhydroxy side chain at C-6 is a normal phenomenon. The hydrogenated pterines are extremely unstable, especially at pH 7. Tetrahydrobiopterin, for instance, is oxidized within seconds in the light, but more slowly in the dark. [sic]

  • $\begingroup$ Thanks @amory - I spoke to my supervisor and we'll do a mini test of it, looks like it could be something we need to consider if it is likely to be unstable. I'll post the result in a few months! $\endgroup$ – rg255 Oct 24 '13 at 19:28
  • $\begingroup$ @GriffinEvo Good luck! $\endgroup$ – Amory Oct 24 '13 at 20:46
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    $\begingroup$ you never know, it may lead to the first ever Biology SE publication! $\endgroup$ – rg255 Oct 24 '13 at 21:33

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