EXPERIMENT 10
TRANSAMINATION (ROUND PAPER
CHROMATOGRAPHY EVALUATION)
PRINCIPLE
Transamination reactions are catalyzed by transaminases (aminotransferases). In
this process the α -amino group of most amino acids is transferred to α
-Ketoglutarate to form glutamate and the corresponding new α-Keto acid. Every
transamination reactions are catalyzed by specified transaminase. Transaminases are
widespread in each organs of organism.
In this experiment, liver homogenate is under water bath with glutamate and
pyruvate,while alanine aminotransferase catalyzes the transfer of the amino group of
glutamate to pyruvate, thus alanine yields. Using Round paper chromatography to
evaluate the existing of alanine and to prove transamination in the tissue.
In order to prevent pyruvate would be oxidized or reduced by other enzymes in
the tissue, iodoacetic acid may be added to inhibite some enzymes of glycolysis
pathways.
PROCEDURE
1 The preparetion of liver homogenate:Obtain fresh animal liver 1g, add precooling
0.01mol/L(Ph7.4) 5ml phosphate buffer saline ,triturate it in a morta.
2 Transamination reactions:Get 2 tubes, perform according to the following table:
addition(drops)
determination tube
control tube
liver homogenate
10
10 (bath in boiling water for 10 minutes)
0.25% iodoacetic acid solution
5
5
1% glutamate solution
10
10
bath in 40℃ water for 10 minutes
5% trichloroacetic acid solution
2
2
bath in boiling water for 5 minutes
After cooling the tubes, filter with filter paper or 2000 rpm centrifuge for 3-5 minutes,
transfer the upper liquid to the new tubes marked with the same number.
3 Paper chromatography evaluation:
(1) Obtain a sheet of 12 cm diameter round filter paper.Draw two 2 cm vertical
lines
passing its center . Use the terminal point of the two lines as spot application and
mark “determination”、“control” 、“glutamate” 、“alanine” on the edge of the
paper corresponding to each point.
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(2) Use 4 capillary tubes, absorb one drop of determination solution、0.1%
glutamate
solution、control solution、0.1% alanine solution respectively. Dot the solution at the
corresponding points of the lines. Pay attention to the diameter of the spot less than
0.3 cm. While the spot is dried, dot the solution again, Each spot may dot for 2-3
times.
(3) Stab a hole (1mm diameter) through the center of the filter paper using a pin,
Get another filter paper strip (0.5×2.5cm). Roll it into a cylinder and twist it tightly
as a lampwick, insert it into the hole from the reverse side of the dotting spot.
(4) Add about 1 ml chromatography solvent to a 5 cm diameter watch-glass
placed in a 10 cm diameter Petri dish. Put filter paper flatly on the Petri dish in order
to soak the lampwick in the chromatography solvent. Cover the Petri dish with
another one of the same size . Solvent rise along the lampwick to the filter paper
and diffuse in a circle.( chromatography time is approximately
45-60
minutes).Allow the solvent diffuse to about 1 cm distance from the edge of the filer
paper.Remove it from the Petri dish. Draw the edge of the solvent with a pencil.Dry
it on a electric stove.
(5) Development: Put filter paper flatly on the Petri dish.Spray 0.1%
indene-triketon ethanol solvent.Dry it on the electric stove.. Purple arc patches then
appear on the filter paper.
(6) Draw the outline of the patches with a pencil. Record relevant data
according to the following table. Calculate the Rf values.
determination
the distance from the spotting point
to the center of the patches(cm)
glutamate
alanine
control
the distance from the spotting point
to the edge of the solvent(cm)
Rf value
Contrast the Rf values of the patches of “determination”and“control” with the Rf
values of the known amino acids, infer what amino acid they are. Explain
transamination reactions on these grounds.
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