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David Walker David at alegba.demon.co.uk
Sun Dec 8 22:34:24 EST 1996

CHAigle at aol.com wrote:
> I am a 15-year-old high school student and am working on a science project on
> photosynthesis.  I am trying to use a variety of plants to see which one's
> chloroplasts will survive and produce oxygen outside of their hosts.
> I found "photosyn at net.bio.net" while browsing the Intermet and thought that
> one of your members might be be able give me valuable advice.
> I have, I believe, successfully separated chloroplasts from spinach leaves
> using a modified Whatley and Arnon procedure at home.  For example, my bell
> jar is a cheese board cover of about 1400 cm3 volume.  I produced
> chloroplasts from 25 g spinach leaves with a .25 mol saline solution made
> from halite.
> My basic problem is determining whether oxygen has been produced.  I have
> tried using a glowing match to see if it flared.  I couldn't be sure if I was
> successful.  Do you have any suggestions to help me determine, at home,
> whether I have successfully produced oxygen?  Is there, for example, an easy
> chemical test I could use?  Are there any special things I should do, or not
> do, to help ensure success?  Any advice or suggestions for sources on the
> Internet you might have would be greatly appreciated.  I have access only to
> a medical centrifuge and microscope (100x) where my mother works.  My school
> has a spectrometer I could use.
> I thank you in advance for any assistance you might be able to give me.
> Sincerely,
> Corinne Milliken
> (I am using my dad's e-mail account
I spent some years of my professional life trying to isolate
chloroplasts  which would cary out CO2 -dependent O2  evolution at
good rates so don't be too disappointed if you don't manage it first
In a nut-shell, if you wish to have your chloroplasts do 'full'
photosynthesis they need to be 'intact' i.e. still retain their
outer membranes  (envelopes) which keep all of the enzymes concerned
with CO2 asimilation in place. These are best isolated in sugar
solutions rather than saline and measurement of oxygen is most
easily followed on an O2 electrode. If you can get hold of a copy

WALKER DA 1987 The use of the oxygen electrode and fluorescence
probes in simple measurements of photosynthesis.  Oxygraphics
Limited, Sheffield. pp 1-145

you would find lots of stuff in it about isolation of chloroplasts
and measurement of O2 . If you are unable to get a copy from your
library ask:-

richard at hansinst.demon.co.uk

It is much easier (saline is fine) to isolate ruptured chloroplasts.
These have lost most of their souble enzymes but retain the internal
membranes (thylakoids) that do the photochemistry. These will evolve
O2  in the Hill reaction but they need to be provided with something
other than CO2  in order to work (i.e to accept the hydrogens that
CO2 would accept if the appropriate enzymes etc were still there).
The easiest hydrogen acceptor (Hill reagent) for you might be
2,6-dichlorphenol indophenol. This is a blue dye which becomes
colourless as it accepts hydrogens. If you can access a colorimeter
or spectrophotometer you can measure this change but you can watch
it happen with the naked eye and get quite a good feel for how fast
it is going with not much more than a test tube and a stop-watch. Of
course this is not quite the same as watching O2 appear but it is
nice anyway if you aren't able to get to an O2 electrode

Good luck

david walker.

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