Download Group II: Wheat Germ, Isolation

Tentative Schedule
These are notes that should help you through the lab. They would also be good to use as your
daily pre-lab notes that you put on the board for the students. This lab is kind of an “Independent
Research” situation and the time markers are not meant to be exact. Some students may work
faster, some may miss a day etc. but for the most part they seem to bond in their groups (Potato
People and Wheat Germ People) and work together at pretty much the same pace as suggested
below.
Experiment
Period
Check-In / Introduction
1
I divide the students into two groups based on where their lockers are, i.e. the left side of the
room (by the hoods) are the Potato People. I put notes for them on the right side of the board,
they will do “G-1-P: Enzymatic formation from Starch” first. The other side by the centrifuge
(towards the O. Chem. Lab) are the Wheat Germ People. About half way through the semester
they physically switch lockers and experiments so that all students end up completing both labs.
Glucose-1-Phosphate / Wheat Germ Discussion
The Potato People will spend most of their time isolating and purifying the product, G-1-P. The
Wheat Germ People spend about equal amounts of time isolating/purifying and doing Kinetics.
Tell the Potato People to bring russet/baking potatoes for Monday. They can go in as a group and
have one person buy a bag (sharing the cost), this usually works well as there are enough for the
whole class for the semester.
The Wheat Germ People should spend some time preparing a flow chart where they make it very
clear when to keep or throw away the various supernatants vs. pellets. There is a flow chart at the
beginning of the manual but they should make their own as it is critical not to throw away the
wrong thing.
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Group I: Glucose-1-Phosphate, Isolation
2
Group II: Wheat Germ, Isolation
Potato people need to be reminded to keep everything cold and work efficiently. Gary will
prepare the starch so they do not need to. The phenyl mercuric nitrate is TOXIC so they should
be careful with the little wax paper packets. They can stop after “Incubation of the Enzyme with
Starch”, leaving the 1L Erlenmeyer flask (250 mL enzyme solution, 250 mL starch, 250 mL
0.8M phosphate buffer) with a cheese cloth cover in the hood. You will be able to tell if the
reaction has worked by watching the color change from red-brown to blue-black. They will
measure Total Volume and remove a small aliquot for later assay after each section they
complete. After today they do not need to worry about keeping things cold as they are no longer
working with enzyme.
The Wheat Germ People should stop after “Water extraction of the enzyme from wheat germ”.
You need to have Gary give them a tutorial on using the free-standing refrigerated ultra
centrifuge. Also today and every lab period after they must store the enzyme frozen in a plastic
bottle. The enzyme, wheat germ acid phosphatase, is very heat sensitive. They should elect
someone from their group who arrives on campus early M and W to remove their frozen samples
from the freezer placing them on the lab bench. Otherwise when they come in at 1:00PM things
will be frozen and the only way to speed thawing is using cold tap water. Also warn them not to
stir or agitate the enzyme too vigorously. They will remove a small aliquot, which should be
stored frozen, for later assay after each section they complete.
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Group I: Glucose-1-Phosphate, Isolation
3
Group II: Wheat Germ, Isolation
If the Potato People do not have blue-black solutions they should start the experiment over.
Otherwise they will heat to 950C and leave the solution to cool until the next period.
The Wheat Germ People do “Salt fractionation of the enzyme with manganese chloride”. That is
manganese, light pink solution, not magnesium.
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Group I: Glucose-1-Phosphate, Purification
4
Group II: Wheat Germ, Isolation
The Potato People must filter the solution with the very large Buchner funnels, not centrifuge,
they may want to use a filter aide, celite. Then they remove excess phosphate using magneiusm
acetate, again filtering using celite. Some of the filter aide will get through so they should finish
with a careful filtration using just filter paper, they may need to repeat this multiple times until
the solution is clear (it will be colored but should not be cloudy).
The Wheat Germ People should do by “Salt fractionation of the enzyme with ammonium sulfate
(35% cut)”. Note: a. should read “…..add 54 mL of cold saturated (NH4)2SO4 (pH 5.5) for every
100 (NOT .100) mL of SII.”
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Group I: Glucose-1-Phosphate, Purification
5
Group II: Wheat Germ, Isolation
Potato People may need to finish magnesium acetate treatment, then they do their first assay,
Fiske-Subbarow. The theory behind Fiske-Subbarow is that it measures the Pi in the solution so
Unhydrolyzed measures free Pi and Hydrolyzed measures the Pi released from the G-1-P during
hydrolysis. So to calculate the [ ] of G-1-P they must subtract off the free Pi (unhydrolyzed)
from the Pi of the hydrolyzed solution. For hydrolysis they should add 1 mL of 1N HCl, then
heat the test tubes in a boiling water bath for 7 minutes. Following that add 1 mL of 1N NaOH,
no need to measure pH, they can assume it has been neutralized. I sent you a nice simplified
procedure for the assay that you will want to hand-out. The first time through they will perform
the assay in duplicate on aliquots of 0.05, 0.1, 0.2 and 0.5 mL after this they will only need to do
the assay in duplicate on one aliquot, usually 0.1 mL. Also they will need to prepare a Pi
standard curve, one for the group, so they can work together.
The Wheat Germ People should do “Precipitation of the enzyme by ammonium sulfate (57% cut)
and heat treatment (600C)”. They should use a hot water bath to bring the enzyme to 600C, then
cool using ice-water-NaCl bath to about 100C
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Group I: Glucose-1-Phosphate, Purification
6
Group II: Wheat Germ, Isolation
Potato People more assays. If there solution contains too much, more than 15%, free Pi they will
need to re-treat with magnesium acetate.
The Wheat Germ People do “Water extraction of the enzyme from PIV”. They should also
prepare a BSA standard curve, one for the group, using the Biuret assay.
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Group I: Glucose-1-Phosphate, Purification
7
Group II: Wheat Germ, Isolation
Potato People should decolorize with a tiny, tiny bit of charcoal, then carefully filter. Now they
treat with cation exchange resin, Dowex-50, to remove cations. They must be very careful to use
the “regenerated” resin and to carefully filter and put the used resin back in the recovery bottle.
Dowex-50 is very expensive……More assays……
The Wheat Germ People do assays. They need to do both the Biuret and Phosphatase assay on all
of the samples they have saved/stored. The Biuret assay will tell them how much protein is in the
solution but not how much enzyme or if the enzyme is active. They will need to do the acid
phosphatase assay using the artificial substrate, PNPP. If they have enough active enzyme the
PNPP will be converted into PNP (yellow color that can be read using the specs) and Pi. Then
using Beer’s Law: ABS = [ ] x l (1 cm) x  (18.8 x 103 L mole-1cm-1) they can determine
concentrations of wheat germ acid phosphatase. The PNPP is expensive and should be stored
frozen, removed from the freezer in the AM along with the other samples. They should use 370C
heat blocks rather than water baths to do the acid phosphatase assays. Again there is a simplified
hand-out.
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Group I: Glucose-1-Phosphate, Purification
8
Group II: Wheat Germ, Isolation
Potato People finish assays and pack their columns with Amberlite IR-45, anion exchange resin.
At this point they have removed excess free Pi and cations now their G-1-P will stick to the
Amberlite allowing all other impurities (starch) to wash through, kind of the final purification.
They need to wash the IR-45 resin with a lot of water through the column checking the pH of the
effluent (water) until it is 9.0 or lower.
The Wheat Germ People should do “Methanol fractionation of the enzyme” followed by “Water
extraction of the enzyme from PVI”.
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Group I: Glucose-1-Phosphate, Purification
9
Group II: Wheat Germ, Isolation
Potato People need to run their G-1-P solution through the column twice, this will be slow. They
need to check the effluent to make sure most of the G-1-P has stuck to the column. They should
store the sealed columns in the cold box.
The Wheat Germ People should prepare for dialysis. The dialysis apparatus will be set-up in the
cold box.
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Group I: Glucose-1-Phosphate, Purification
10
Group II: Wheat Germ, Kinetics
Potato People should wash the column with water checking the water (effluent) for starch, I2/KI,
blue color. They elute the G-1-P with 5% KOH, 500 mL total, collected in 10 (labeled 1 through
10) 50 mL Erlenmeyer flasks, so ~50 mL per flask. Each flask must be assayed and the four
flasks, with the most G-1-P are “pooled”. In a 1 L erlenmeyer flask they put the “pooled” G-1-P
(~200 mL) and 600 mL of MeOH. Leave the flasks in the freezer.
The Wheat Germ People should carefully remove their enzyme from dialysis. They will do
assays to determine how much enzyme they need to use, i.e. the dilution factor needed, to give
an ABS of 0.15 to 0.50 in 5 minutes at 405 nm using the acid phosphatase assay. This will be
critical for performing the Kinetics which are fixed time assays that rely on using the correct
enzyme concentration. They should also finish any other assays they need to do.
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Group I: Glucose-1-Phosphate, Purification
11
Group II: Wheat Germ, Kinetics
Potato People should very carefully decant off most of the MeOH, then carefully filter, using a
small Buchner funnel, the fine crystals of G-1-P. Leave the crystals to dry.
The Wheat Germ People should do Kinetics I, time course, do the experiment one time through
in duplicate.
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Group I: Glucose-1-Phosphate, Characterization
12
Group II: Wheat Germ, Kinetics
Prepare a glucose standard curve, one for the group, using the Nelson’s assay (“G-1-P: Chemical
Characterization”). Potato People weigh the crystals using an analytical balance. Prepare a
standard G-1-P solution, 16-22 mg in 10 mL of dH2O. In duplicate do: 7-Min Hydrolysis, Total
Hydrolysis (1 mL 1N HCl, boiling water bath for 15 min), Unhydrolyzed all Fiske-Subbarow
assay. Do the Nelson’s assay which measures G-6-P contamination. Don’t do Chromatographic
Characterization.
The Wheat Germ People should do Kinetics II and III, time course and inhibition, do the
experiment one time through in duplicate.
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Group I: Utility Day (Finish G-1-P)
13
Group II: Utility Day (Finish Wheat Germ)
Report should include:
Glucose-1-Phosphate: Enzymatic Formation from Starch
“Report of Results” Answer Questions 1-4
“Exercises” SKIP
Glucose-1-Phosphate: Chemical Characterization
“Report of Results”
Rf Values SKIP
Answer the questions following the Table 16-1:
How many moles of inorganic phosphate contaminate your sample?
How many moles of glucose-6-phosphate contaminate your sample?
The Wheat Germ People
Report should include:
Proteins: The Extraction and Purification of Wheat Germ Acid Phosphatase
“Calculations and Questions” Answer Questions 1-3
Proteins: The Kinetic Properties of Wheat Germ Acid Phosphatase
Kinetics I
“Calculations and Question” Answer Questions 1-5
Kinetics II
“Calculations and Question” Answer Questions 1-6
Kinetics III
“Calculations and Question” Answer Questions 1-6
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Group I: Wheat Germ, Isolation (Lab Report Due)
14
Group II: Glucose-1-Phosphate, Isolation
The two groups physically switch lockers and you go through the same procedures/pre-lab notes
as above.
Group I: Wheat Germ, Isolation
Group II: Glucose-1-Phosphate, Isolation
This course has recently been submitted as “writing intensive” and this GWE activity is part of
that. It should be graded primarily for syntax.
Writing Assignment: A paper word processed, double spaced, 250 words or less, addressing
the following tasks is due on Wednesday, May 14th (50 pts).
Unexpected challenges in our lives sometimes bring out the best in us. For example, we act
heroically or courageously in emergencies, we are charitable when disasters affect our friends,
or we are unselfish and patient when misfortune strikes our friends or relatives. Pick an
instance in your own life when such a challenge stimulated in you a response that pleased
you. Describe the situation, explain how you responded, and tell what you learned about
yourself from the experience.
This paper will be judged on rhetoric, how effectively do you address the writing tasks and
syntax, grammar.