SUBJECT: Turnip peroxidase (enzyme assay)
DATE: 5/95

We are doing the turnip peroxidase experiment in our summer seesion labs. We
use 1% H2O2, guaiacol as an indicator, and turnip blended in distilled water -
about 1 g/100 ml. However, our results last week were really bad, the
baselines barely hit 0.2 absorbance after 10 minutes, even after I doubled and
tripled the amount of turnips I used. Has anyone ever had this problem? My
guess is that these turnips just have really low peroxidase activity because
the H2O2 and guaiacol were made up fresh the day of the labs. Any ideas or
hint?

Thanks in advance for your help or advice.

Rosemary E. Boone
Dept. of Biological Sciences, University of Pittsburgh
email: rmboone@vms.cis.pitt.edu
phone: 412-624-9325


The usual problem is that people use a "rutabaga"-type turnip (orange
inside), and not the white turnip... It's the problem of
common names in different parts of the country refer to different
beasts. Other than that, I can't think where you may be having
problems, since your recipe looks correct.

Graham R. Kent (gkent@smith.smith.edu)
Dept. of Biological Sciences
Smith College Northampton, MA 01063


Rosemary,

We do this experiment every semester in our majors course and its
beautiful. Turnips really do vary quite a bit. I always standardize our
solution to hit abs=1 in 120 seconds. Sometimes it takes a fraction of 1g
per 100ml sometimes as much as 5-6g per 100ml. Maximum activity comes from
using the skin and not just the inside of the turnip. Be sure you are
grinding it in ph 7 buffer or you will have problems.

Chris
V. Christine Mahaffey Minor (vcmahaff@iastate.edu)

Laboratory Coordinator
154 Bessey Hall
Iowa State University
Ames, IA 50011
515-294-8596


Rosemary,
I'm not too familiar with this peroxidase experiment, but I
notice that you're extracting the enzyme in distilled water
and I do know form sad experience that this can lead to
highly variable results. Try using a buffer solution. If your
lab uses pH as a variable, extract it in 0.01 M phosphate
and use 0.1 M for the experiments- that way the enzyme
will be protected but can still be changed to the
experimental conditions.

If you're not sure whether the problem is the turnip or the
extraction process, try putting a fresh thin slice of turnip
in your reaction mixture. If that gives a good color, the
enzyme's probably there and denaturing upon extraction.
If not, you at least will know it's the turnip.

John Dickerman
Northern Illinois University
T80JWD1@WPO.CSO.NIU.EDU


We always had great success with the peroxidase experiment as described
until I tried to get smart and clarify the enzyme solution either by filtration
or centrifugation. Then we discovered that, at least with mild homogenization,
the peroxidase activity is in the particulate fraction. I tried a couple of
ways to solubilize it but was unsuccessful, went back to being happy with a
cloudy enzyme solution. Good luck.
Bob morris
robert.w.morris@cyber.widener.edu


Hello. Just read the turnip discussion. We do the turnip exercise
also. However, we sometimes use fresh horseradish root. If available,
it's really powerful stuff. Does anyone have comments on
horseradish or other plants that can be used for this experiment?
I believe Jean Dickey does something with potato and catalayse. Thanks to
everyone
for their input about buffers, color of turnip and 0.01 M posphate.
<-* Sharron Clark *->

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