CATHOLIC UNIVERSITY OF SANTA MARIA

FACULTY OF PHARMACEUTICAL, BIOCHEMICAL AND SCIENCES

BIOTECHNOLOGICAL

Professional School: Pharmacy and Biochemistry

Course: Biochemistry - Practices

Report N°3: amino acid degradation

Transamination

Students:
Bernedo Mamani, Gladys Soraida
Condo Charca, Fredy
Díaz Bernal, María Luisa
Jala Gutierrez, Enza Gianfranco
Mita Callahuanca, María Elena
Ocsa Laura, Fabiola Annie

Teacher: Yenny López Valencia

2021
 PRACTICE N o 3

DEGRADATION OF AMINO ACIDS: TRANSAMINATION

LIST OF EXPERIMENTS
1. Transamination using an enzymatic extract of pigeon liver

INTRODUCTION
Transamination is the process that consists of the reversible transfer of the
amino group from an amino acid to a keto acid, resulting in the conversion of the
first into a keto acid and the second into a new amino acid. This reaction is of
great importance in the metabolism of amino acids and It is catalyzed by
enzymes called TRANSAMINASES or Aminotransferases, present in most
animal tissues. The cofactor of these enzymes is pyridoxal phosphate and for
almost all transaminases alpha-ketoglutarate is the acceptor of the amino group,
therefore forming glutamic acid.

NH3 HI > EITH

Yo C=O ER
R-CH-COO- ( YO II
Enzyme

h NH3
<I I 'OOC-CH2- CH2-CH-
RC-COO- * HC-NH3 COO-
Enzyme

L-amino acid + alpha-ketoglutarate 4 alpha-keto acid + L-glutamic

Clinical interest is focused especially on two transaminases: L-alanine: alpha-
ketoglutarate aminotransferase (Glutamic Pyruvic Transaminase or TGP) and L-
aspartate: alpha-ketoglutarate aminotransferase (Glutamic Transaminase
Oxalacetic or TGO). These enzymes have an eminently intracellular action, so
serum activity under normal conditions is low or absent. An increase in activity
will be evidence of a deterioration of the tissues in which they are found, of which
the heart and liver are particularly important.

It has been observed that after a myocardial infarction, a marked increase in

TGO activity occurs in serum, due to the release of this enzyme, so abundant in
the myocardium, into the bloodstream. In this case there will be no increase in
serum TGP activity or it will be minimal.
In viral hepatitis or other forms of liver disease involving tissue necrosis, there
will also be a considerable increase in serum transaminase activity, even before
the appearance of clinical symptoms such as jaundice. In this case TGP will be
the predominant enzyme due to its high concentration in liver tissue. High
transaminase activity can also be detected in other conditions such as:
accidental or surgical trauma, acute pancreatitis, muscular dystrophies, etc.

TRANSAMINATION IN THE PRESENCE OF A PIGEON LIVER

PREPARATION

Aim:
Application of the thin layer chromatography technique to study transamination in
the presence of a pigeon liver preparation as an enzyme source, using alanine
and aspartate as substrates.

Procedures:

Preparation of the enzyme source (Acetone powders):

The livers recently extracted from 4 pigeons are homogenized in 10 volumes of
cold acetone for one minute. After filtering and washing several times, the
residue obtained is dried at room temperature, thus obtaining dry powders that
are easy to preserve.
On the day of the practice, 700 mg of acetone powder is ground in a mortar with
7 ml of distilled water and then the preparation is centrifuged at 2000 rpm. For 10
minutes. The supernatant is decanted and the volume is made up to 35 ml with
distilled water.
Acetone, due to its dehydrating action, facilitates the extraction of enzymes from
the tissues. In this form, acetone reduces the denaturation of proteins (enzymes)
and concomitantly produces the disintegration of cellular structures and the
disruption of lipoprotein bonds.

Preparation of the transamination experiment.

In four test tubes (13 x 100 mm), previously washed with concentrated HNO 3 ,
measure the following:
 SYSTEMS
Yo II III IV
REAGENTS
Alanine 0.05 M 0.2ml - - -
Glutamate 0.05 M - 0.2ml - -
Aspartate 0.05 M - - 0.2ml 0.2ml
alpha-ketoglutarate 0.1 M 0.2ml - 0.2ml 0.2ml
Pyruvate 0.1 M - 0.2ml - -
Enzyme extract 0.2ml 0.2ml 0.2ml -
boiled enzyme extract - - - 0.2ml

37ºC
Mix the contents of each system and then incubate in a water bath at for one
hour.

Identification of reaction products by thin layer chromatography:

Each group will have a glass plate with Silica Gel, 200 x 200 mm, on which the
points of origin have been marked 15 mm from the lower edge, with a distance
between them of 25 mm and in the following order: One for each of the four
experiment tubes and three for the three standards of: Alanine, Glutamate and
Aspartate. These standard systems must be prepared in the following way:
Measure 0.2 ml of the Amino Acid (Alanine, Glutamate or Aspartate) that is
previously at a 0.05 M concentration and add 0.8 ml of water.

POINTS OF ORIGIN
Sample or Standard Glutamic Standard
System No 1 2 3 4 To the girl Standard Aspartic
or
RF x 100
Apply using glass capillary tubes and at the corresponding point of origin, 10 ul of
each incubated system and 2 ul of the standard amino acid solutions in aliquots
of no more than 1 ul at a time, allowing each area to dry after each application. .
At the end, place the plate in the chromatographic chamber containing 75
volumes of phenol and 25 volumes of water as solvent. The chromatography
ends when the solvent reaches the line marked 10 cm from the points of origin.
Then take out the plate, dry it in the oven at 110 °C for 3 minutes, and develop
the chromatogram by spraying a solution of 0.1% ninhydrin in n-butanol
saturated with water and then letting it dry. The schematic representation of the
results obtained after developing the chromatogram is shown on the next page.

Expression of Results:

Calculate the Rf x 100 of each stain, complete the previous table and interpret
the chromatogram with the results obtained. Comment on each of the prepared
systems:

RF RF
Spot No. 1: 27 Spot No. 5: 12
Spot No. 2: 56 Spot No. 6: 27
Spot No. 3: 27 Spot No. 7: 12
Spot No. 4: 56
Identify each of the spots by indicating which amino acid it corresponds to based
on its Rf values. Note that the Rf values for the amino acids: Alanine, aq.
Glutamic and ac. Aspartic are 58, 26 and 11.2 respectively, for the solvent used
in the present chromatography.

Amino acid Amino acid

Spot No. 1: glutamate Stain No. 5: aspartate

Spot No. 2: alanine Spot No. 6: glutamate
Spot No. 3: glutamate Stain No. 7: aspartate
Spot No. 4: alanine
 BIOCHEMICAL QUESTIONS
1. What is RF
The relationship between the distances traveled by the solute and the eluent
from the origin of the plate is known as Rf, and has a constant value for each
compound under certain chromatographic conditions (adsorbent, solvent, cuvette
size, temperature, etc. .).
The Rf front ratio is calculated for each component by measuring the distances
from the seeding line to the center of the spot corresponding to each analyte and
from the seeding line to the solvent front (FM).

Results analysis
◄ Solvent front
Obtained.

System1:
There is a transamination because
glutamate and alanine are found.

System 2:
There is a transamination of alanine
and α acetoglutarate as pyruvate. . . .
is converted to alanine.

System 3:
Aspartate and glutamate stain found

System 4: there is denaturation

2. What are the reactions

catalyzed by TGO and TGP?
TGO and TGP are enzymes
that are normally measured
to evaluate liver health, which
is why they are called
 transaminases.

TGO or GOT, known as oxalacetic transaminase or AST (aspartate

aminotransferase), is produced in various tissues, such as heart, muscles and
liver, and is located inside liver cells. They are located in the process of
deamination and combined amination in the amino radical of an amino acid
that is reversibly transferred to an alpha keto acid, this being transferred to the
original amino acid.

TGP or GPT, known as pyruvic transaminase or ALT (alanine

aminotransferase), is produced exclusively in the liver; For this reason, when
there is any alteration in this organ, an increase in the circulating amount of
this enzyme in the blood can be observed. It catalyzes the transfer of an
amino group (NH2) from alanine to α-ketoglutaric acid, giving rise to pyruvate
and glutamate. The reaction is reversible.

3. What role does pyridoxal phosphate play in transamination reactions?

What is the functional group of the cofactor? Explain your mechanism
It acts as a transporter of the amino group between substrates, alternating its
structure between the aldehyde form (pyridoxal phosphate, PLP) and the
amino form (pyridoxamine-5-phosphate, PMP).
Transamination consists of transporting an α-amino group from a donor α-
amino acid to the keto carbon of a receptor α-keto acid. This process takes
place in two stages and is catalyzed by aminotransferases specific for each
substrate.

a) In the first stage, an α-amino acid that will act as a donor transfers the α-
amino group to the transaminase enzyme, producing the corresponding
αketoacid and the enzyme will be aminated.

b) In a second stage, the amino group is transferred to a different acceptor α-

keto acid from the first stage, forming a new amino acid and regenerating the
enzyme. In this process, the PLP is regenerated and a new amino acid is
produced due to the transfer of the amino group (which comes from the
 substrate amino acid) to the second substrate, the new incoming α-keto acid.

The aminotransferase reaction occurs through a ping-pong type mechanism.

4. What is the purpose of system 2

The objective of system 2 is the formation of alanine and also the reversible
reaction with the enzyme TGP (Alanine Aminotransferase ).

5. Explain the mechanism that allows the separation of substances by thin

layer chromatography
The mobile phase contains various solvents and moves in a certain direction,
and the stationary phase is the silica gel that is used to separate more polar
substances such as: alcohols, amines, acids and others.

6. Is free ammonia released into the medium during a transamination

reaction? Support your answer
Yes, because when glutamate deamination occurs, alpha-ketoglutarate is
formed, but the amine that is lost can enter the urea cycle.

7. Predict the effect of a vitamin B6 deficiency on degradation of the

amino acids. The degradation of the 20s
amino acids would be affected?
As for the effect, it would cause the degradation of amino acids, since the
enzymes Alanine Amino Transferase (TGP) and aspartate aminotransferase
(TGO) use vitamin B6 as a cofactor.

8. What will be the cellular distribution of TGO and TGP?

TGO is a bilocular enzyme, it is distributed in the cytoplasm and mitochondria
of cells, together with TGP it plays a diagnostic and monitoring role in
diseases with hepatocellular and muscle damage.
There is no evidence of increased transaminase synthesis in liver and muscle
diseases.
9. If in the transamination experiment that you perform you will place
pyruvic acid uniformly labeled with C14, what amino acids do you expect
to find preferentially labeled? Fundamentally.
Glutamate would be marked since it is the second compound that results from
Alanine + α-ketoglutarate and since it is reversible we will also find Alanine
marked.

10. If glucose with C14 is administered to an animal, the labeled carbons

will be incorporated into a protein. Point out some sequences of
reactions that explain this phenomenon.
Glucose comes from undirected pyruvic acid, therefore, excess insulin passes
into the blood.

11. Do transamination reactions participate in the biosynthesis of amino

acids?
Transaminases catalyze transamination reactions, important especially for the
synthesis of non-essential amino acids and for the degradation of most amino
acids, which lose their amino group by transamination, except for the amino
acids lysine and threonine, for which this reaction is not possible.

12. What would you think of an assay in which a biological sample such as
serum will be mixed with aspartate, alpha-ketoglutarate, NADH, and
excess malate dehydrogenase? What would you think of to determine
TGP activity by a similar method?

Aspartate aminotransferase (TGO) catalyzes the transfer of the amino group

from L-aspartate to 2-oxoglutarate, forming oxaloacetate and L-glutamate. The
catalytic concentration is established, using the coupled attitude of malate
dehydrogenase (MDH), from the rate of disappearance of NADH, measured at
340 nm. In the attitude medium there is also sufficient MDH to consume
endogenous ketoacids, thus avoiding their interference. So what will be
measured is the glutamic oxalacetic transaminase or GOT method.
Only malate would have to be changed for lactate to make it a glutamic
pyruvic transaminase method since the reaction catalyzed by TGP is shifted
towards the formation of pyruvate which reacts immediately with LDH (Lactate
 Dehydrogenase), so that the oxidation rate of NADH, measured at 340 nm is
proportional to the TGP activity in the sample. The excess LDH in the medium
is to consume keto acid of endogenous origin, thus avoiding its interference.

13. If leucine and a-keto glutarate were used to study transamination; What
products could be identified? What name would you propose for the
enzyme involved?

Leucine + alpha-keto glutarate ---------------> ketomethylbutarate +

glutamate
—
Amino acid transferase

The enzyme involved is amino acid transferase and the products formed are
ketomethylbutarate and glutamate.

14. Is the identification of keto acids by chromatography feasible? How?

No, because ninhydrin is a developer that reacts with amino acids

EXPERIMENT 2

QUANTITATIVE DETERMINATION OF ASPARTATE AMINOTRANSFERASE

GOT (AST)
Goals

• Study of transamination in normal and pathological sera

• Application of the Reitman and Frankel colorimetric method for the

determination of serum AST activity.

Procedure:

1. PREPARATION Working reagent (RT):

a) Mix: 0.5 ml. of (R2) Substrate + 2 ml. (R1) Buffer.
b) Pipette into a tube, mix, incubate 1 minute.

Reagents Systems
 1 2

RT (mL) 1,0 1,0

1 Sample (μL) 100 ---

2 Sample (μL) --- 100

2. Test conditions:
a) Wavelength: .......................... 340nm
Tray: ......................................1 cm light path
Constant temperature.............37ºC

Place the prepared solution in a cuvette and take it to the spectrophotometer.

Read the initial absorbance (A) of the sample, start the stopwatch and read the
absorbance every minute for 3 minutes.
 Absorbances
1 Sample 0.400 0.345 0.330

2 Sample 0.011 0.013 0.008

Expression of Results:
Calculate the average absorbance increase per minute (ΔA/min): ΔA/min x 1750
= U/L of AST
Units: The international unit (IU) is the amount of enzyme that converts 1 μmol of
substrate per minute, under standard conditions. The concentration is expressed
in units per liter (U/L).

∆Ab M1= 0.035 x 1750= 61.25 U/L

∆Ab M2= 0.005 x 1750= 8.75 U/L

Results
1 Sample U/L 61.25
2 Sample U/L 8.75

REFERENCE VALUES
25°C 30°C 37°C
Men Up to 19 U/L 26 U/L 38 U/L
Women Up to 16 U/L 22 U/L 31 U/L

Sample 1 is elevated, relating to heart damage

Sample 2 is below normal values
 BIOCHEMICAL QUESTIONS

1. Write the reactions catalyzed by TGP and TGO.

The TGO
Aspartate + α-ketoglutarate ⇔ oxaloacetate + glutamate

The GPT
Alanine + α-ketoglutarate ⇔ pyruvate + glutamate

2. What role does pyridoxal phosphate play in transamination reactions?

Explain its mechanism of action.

Transaminases need pyridoxal phosphate to carry out their function; It acts as a

transporter of the amino group between substrates, alternating its structure
between the aldehyde form (pyridoxal phosphate, PLP) and the amino form
(pyridoxamine-5-phosphate, PMP). Transaminases catalyze transamination
reactions, important especially for the synthesis of non-essential amino acids and
for the degradation of most amino acids, which lose their amino group by
transamination, except for the amino acids lysine and threonine, for which this
reaction is not possible.

3. Is free ammonia released into the medium during transamination

reactions? Support your answer.

No, in extrahepatic tissues it is GLU that performs this role by collecting

ammonia in the form of amide nitrogen from GLN, and this passes to the liver
mitochondria. In skeletal muscle, ALA is responsible for transporting the amino
group to the liver. Both GLU, GLN, and ALA are released from the amino group
in the liver so that there they become part of urea, the excretion product of amino
acid N.

4. How is the determination of TGP activity useful in the diagnosis of

pyridoxine deficiency? If serum TGP activity is decreased in a patient, is
this information sufficient to diagnose him or her as pyridoxine deficient?
Because?

The diagnosis of vitamin B6 deficiency is usually clinical. There is no single

accepted laboratory test to define vitamin B6 status; The most common measure
is serum pyridoxal phosphate levels.

Correct, if a patient has a decreased TGP it means that he or she has vitamin B6
deficiency.

5. It is mentioned that in the hepatocyte TGP is an enzyme, exclusively

cytosolic. Do you agree with this statement? What would be the
intracellular distribution of TGO? Support your answer.

Alanine Amino Transferase (TGP): It is a cytosolic enzyme, which is found in

high concentrations in the liver.

Aspartate aminotransferase (TGO): It is present in the cytosolic and

mitochondrial isoenzymes.

6. If in the transamination experiment that you have carried out, you placed
pyruvic acid uniformly labeled with C-14. Which amino acids would you
expect to find preferentially labeled? Support your answer.

Glutamate is marked since it is the second compound that results from alanine +
α-ketoglutarate and since it is a reversible reaction we could also find marked
alanine.

7. If C-14 glucose is administered to a laboratory animal, will the labeled

carbons be incorporated into a protein? Point out the sequence of
reactions that explains this process.

When pyruvic acid is not broken down, glucose is formed, therefore insulin is
directed to the blood.
8. Do transamination reactions participate in the biosynthesis of amino
acids? Cite some examples.

If they participate:
TGP
Alanine + alpha-ketoglutarate pyruvate +glutamate

TGO
Aspartate + alpha-ketoglutarate or xaloacetate +
glutamate

9. What other method, in addition to the one used in this practice, could
you use to detect the products of the transamination reaction?

• HPLC
• Thin layer chromatography
• Spectrophotometry

10. What would you think of an assay in which a serum sample is mixed
with aspartate, alpha keto glutarate, NADH, and excess malate
dehydrogenase? What would be its foundation? The activity of what
enzyme would be being measured?

TGO

• Aspartate + α-ketoglutarate ---------------• oxaloacetate + glutamate

DEHYDROGENASE

• NADH + α-ketoglutarate malate + NAD

BIBLIOGRAPHY
Lemos, M. (2020, October 13). TGO and TGP: what they are, what they are for
and normal values. Tua Saúde. https://www.tuasaude.com/es/tgo-tgp/

Wikipedia. (2021, February 3). Alanine aminotransferase. Wikipedia, the

free encyclopedia. https://es.wikipedia.org/wiki/Alanina_aminotransferase
BIOCHEMISTRY-2nd PHARMACY. (s. F.). University of Alcala. Retrieved
September 17, 2021, from http://www3.uah.es/bioquimica/Tejedor/BBM-
II_farmacia/tema13.htm
TGP. (s. F.). TGP. Retrieved September 17, 2021, from http://www.med-
informatica.net/lab-clinico/analisis/f_z/TGP.html