SUBJECT: Phenol oxidase from potato and other sources
DATE: 2/96; 6/96
Help please! I seem to have 'lost" a lab protocol for this lab. The
setup is a standard one and I thought was well known but I can't find any
detailed reference to it. The lab setup involves:
blended potato as a source of phenol oxidase, catechol as the substrate,
a coloured product - I believe it is benzoquinone. My questions are:
1. What is the concentration of the catechol
2. How do I stop the potato extract from oxidising (brown colour) and yet
not affect the oxidase reaction?
Any help with this lab or even a readily available reference would be
very much appreciated.
Peter Gardiner @cln.etc.bc.ca
Biology Dept.
St. Michaels University School
Victoria
British Columbia Canada
The typical protocol for using the potato extract to oxidize catechol uses
1%
(one percent) catechol. Preparing the extract is the touchiest part since
oxidation begins immediately within the extract itself. Blend the potato
in
ice water, strain the solution, pour it into the smallest squeeze bottles
you
can to minimize air in the bottle. Immediately place bottles in ice water.
Prepare this extract immediately before needed since it's somewhat of a
race
against time.
Many people use turnips as an enzyme source for a similar exercise. Is this
as
time-sensitive as the potato extract is?
Joy Perry
Univ. of Wisconsin - Fox Valley
P.O. Box 8002
Menasha, WI 54952
joyperry@uwcmail.uwc.edu
The lab I have used is based on one from "The Biolab Book" by
Lundy Petz
and is published by the John Hopkins University Press. I have the second
edition and don't know if there is a newer one?
From: antibusr@bluffton.edu (Antibus, Robert)
Keep it cold. We use cooled distilled water then place it with a stopper
on
ice. Another poster notes eliminating as much air as possible.
As I look over the lab I notice we use pH 7 buffer to dillute the mixture,
since other pH's may alter the results.... Next year I may try
cooled pH 7 buffer instead of distilled water.
At any rate, we make fresh for each lab section (it only takes a few minutes
so the TA for the previous lab tries to make it, otherwise it gets made
in the 10 minute passing period or during the pre-lab quiz).
We use Kull, R.C. Preparator's guide for the Laboratory Manual to accompany
"The Nature of Life" (by Postlethwait and Hopson). 2nd edition,
1992 McGraw
Hill publishers.
-----------------------------------------------------------------
Jeff Lewin, Lab Associate http://www.bio.mtu.edu/perspage/jclewin/home.html
jclewin@mtu.edu Department of Biological Sciences
(906) 487-3435 Michigan Technological University
Fax (906) 487-3167 Houghton, MI 49931
-----------------------------------------------------------------
Joy Perry, in discussing use of potato as a source of "phenol-oxidase"
for a lab activity commented that "....it's somewhat of a race against
time," with reference to the fact that oxidized products form in the
potato extract, and that precautions must be taken to prevent them.
She then asked if using turnips as an enzyme source presents similar
problems.
No. The turnip extract does not contain the oxidizable substrates
that turn brown present in potato (at least not in as high a concentration).
Consequently, there is no brown material to interfere with the benzoquinone
("brown pigment") to be estimated by measuring absorbance.
So why use potato?
Dave McNeely, Biology, University of Texas at Brownsville, 80 Fort Brown,
Brownsville, TX 78520; mcneely@utb.edu
Thank you to all who replied to my query.
Homogenising the potato with a little cold water in a blender works
perfectly. We found that squeezing the homegenate through cheese cloth
gave sufficient separation.
We are now working on a spectrometric assessment of the reaction rate
rather than the rough estimate of colour light brown etc.
Turnip does not seem to work at all.
Thank you again for your help.
Peter Gardiner @cln.etc.bc.ca
Biology Dept.
St. Michaels University School
Victoria
British Columbia Canada
Several people have written concerning using turnip as the source of
peroxidase for an enzyme lab in general biology, and some have complainedd
that having tried turnip they found it wanting as a source for various
reasons.
We've had success using turnip as follows:
2 grams of fresh turnip, homogenized for 20 seconds in
300 ml cold 0.1 M sodium phosphate buffer at pH 7.0.
extract filtered and kept cold (on ice) until used.
The extract should be prepared immediately before the lab
during which it is used.
A final reaction mixture of 8 ml, with 1 ml being extract
has given useful results for us. For example, two tubes as
follows:
Tube One -- 2 ml pH 5.0 phosphate buffer
1 ml original homogenization buffer
1 ml turnip extract
Tube Two -- 1 ml pH 5.0 buffer
2 ml 10 mM hydrogen peroxide
1 ml 25 mM guaiacol
Spectrophotometer readings should be begun IMMEDIATELY upon mixing
the two tubes together.
We've had very good and very consistent results with all parts
of the exercise, which investigates effects of substrate concentration
, enzyme concentration, pH, temperature, and boiling by varying
appropriate components.
Perhaps in the preparations where inconsistent results were obtained
there was some failure to control temperature or concentrations?
Good luck with the activity.
Dave McNeely, Biology, University of Texas at Brownsville, 80 Fort Brown,
Brownsvile, Texas 78520; mcneely@utb.edu
We were watching the discussion about which root to use for catechol oxidase
with interest because that lab was coming up in Cell. We had been using
potato
with the usual problem of the color changing with time so the possibility
of
turnips sounded good. We tested both:
Potato: The extract darkened by itself but the test results were
readable and consistent with expected results.
Turnips: The extract was colorless however the results were not
consistent. For temp, [substrate], and pH testing: It might be that the
turnip
extract has to be diluted more than the potato to keep from getting a maximum
(4+) reaction under all conditions. The color standards using a range of
enzyme
concentrations were not as differentiated as the potato.
If anyone has any additional thoughts/insights, I'll appreciate hearing
them.
Anyway, for now, we vote for potatoes.
Christine Case
Skyline College
case@smcccd.cc.ca.us
6/96
Here's my 2 cents worth on the peroxidase enzyme lab discussion:
I'd just like to echo Dave McNeely's comments, especially "So why use
potato?".
We've had good success using turnip extract as the source of enzyme and
a
mixture of guaiacol and hydrogen peroxide as substrates. Our protocol was
adapted from 2 sources:
1. Laboratory Outlines in Biology - IV by P. Abramoff and R.G. Thomson
(published by W.F. Freeman and Company in 1986), pages 171 - 176.
2. General Biology Laboratory Manual by C. Eberhard
(published by Saunders College Publishing in 1990)
The keys to the protocol are (1) the dilution of the extract and (2) the
pH of the buffer used to incubate the enzyme with its substrate.
We use a sol'n of 1 mL of extracted turnip juice and 400 mL of distilled
water as our stock solution. To investigate the effects of enzyme conc'n
the students prepare tubes containing 0.5 mL, 1.0 mL or 2.0 mL of this
diluted turnip juice with enough pH 5.0 buffer to bring the volume to
5.0 mL. At t = 0 seconds the students add 1.0 mL of a solution containing
10 mM hydrogen peroxide and 20 mM guaiacol. The students follow the
development of the colored product (oxidized guaiacol) using a Spec 20
(set at 500 nm), recording readings every 10 seconds for 3 minutes.
Like Dave McNeely we've successfully used this system to demonstrate the
effects of substrate conc'n. We've even used this
system (in another course) to determine K m and V max values. The
results were satisfactory. Competitive inhibition can be demonstrated
using this system with hydroxylamine as the inhibitor. Abramoff and
Thomson suggest that NaF can be used as an inhibitor.
I hope this information is of help to someone.
But now I have a question?
Does someone know of a good enzyme-substrate-inhibitor system that
nicely follows Michaelis-Menton kinetics for competitive and
non-competitive inhibition and doesn't involve expensive reagents?
Thanks,
Ron van der Heiden (rvdheid@redeemer.on.ca)
Biology Department, Redeemer College, Ancaster, ON Canada
For a Biochem course I taught recently, we used wheat germ acid
phosphatase (Sigma cat# P3627 $19/1g) and Sigma 104 phosphatase
substrate (O-nitrophenyl phosphate)(cat#104-0 $21/1g). Product
formation is measured spectrophotometrically (I think at 495 nm).
NaH2PO4 is used as a competitive inhibitor, and NaF is used as a non
competitive inhibitor. The experiment worked fairly well in our hands.
One can also partially purify the enzyme from wheat germ. I'm not sure
where the lab originated, by I learned about it from Steve Munroe at
Marquette University as a graduate student TA. If you would like more
details, please let me know.
Jeff
Jeffrey D. Newman newman@lycoming.edu
Department of Biology http://www2.lycoming.edu/~newman/
Lycoming College Phone: 717-321-4386
Williamsport PA 17701 Fax: 717-321-4073
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