Studying Of Transaminase Activity In Heart Tissue Biology

Essay add: 16-06-2017, 16:19   /   Views: 8

The aim of this project is to investigate the reaction catalysed in some amino acids. In which the reversibility of the enzyme was to be determined. And to determine wether the reaction goes to equilibrium in the forward and reverse reaction.

Introduction

Key facts: Transaminases catalyse reversible reactions which inter-convert an α-amino acid and an α-keto acid into corresponding α-keto acid and α-amino acid respectively.

In transamination the α-amino acid group of an α-amino acid is transferred to an α-keto acid producing a new α-amino acid and a new α-keto acid.

Tissue transaminase can be investigated by incubating a homogenate with various amino acid/ keto acid pairs. Transamination is demonstrated if the corresponding new amino acid and keto acid are formed, as revealed by paper chromatography. Reversibility is demonstrated by using the complementary keto acid/amino acid pair as starting reactants. In transamination reactions the α-amino group from an α-amino acid is transferred to a the α-carbon of a corresponding keto acid. As the transimation reaction is basically a transfer of an amino group from an amino acid to a keto acid there is no net loss of amino groups.

All aminotransferases enzymes have the same reaction mechanism and the same prosthetic group, which is pyridoxal phosphate (PLP) the PLP acts as an intermediate carrier of the amino group in the transamination reactions. The transferase enzymes catalyse their reactions in a ping-pong fashion, which means that the first substrate reacts and its product leaves the active site before the second substrate can bind.

Image ch23fu4.jpg

Figure 2: Reaction mechanism of amino transferases [] 

The PLP found in all transaminases are derived from vitamin B6. PLP is bound to the transaminese through a schiff base linkage with a Lys residue on the enzyme. The aldehyde on the PLP is an important functional group which forms covalent Schiff-base intermediates with amino acid substrates; the aldehyde component also forms a Schiff base with a specific lysine residue on the enzyme [] .

In this project experiment a heart homogenate was used to investigate the forward and reverse reactions of amino acids. After the incubation, each reaction mixture is spotted directly onto a paper chromatogram. This is developed with a solvent which separates amino acids, and then treated with ninhydrin to locate the spots.

The remainder of each reaction mixture is treated with 2,4- dintrophenylhydrazine (DNP) to form the dinitrophenylhdrazone derivatives of all the keto acids present. These derivatives are extracted into a small volume of ethylacetate and this solution is applied to a second paper chromatogram that is developed with a different solvent (i.e., one suitable for separating the DNP derivatives of keto acids)

Method

Test tubes were collected and filled with reagents according to table 1. Thereafter another two tubes were prepared to act as markers for the forward reaction and they were set according to table 2. The 9 test tubes were thereafter incubated in a 37°C water bath for 30 minutes.

Two chromatogram papers were prepared for the forward and reverse reactions respectively. Followed by the preparation of another two test tubes to be used as markers for the reverse reaction, the reagents that were used in the marker solutions were distributed according to table 3. 2 ml of DNP was added to the markers for the reverse reaction then mixed and left to stand for 10 minutes. After the 10 minutes 1.5ml of ethyl acetate was added and the solution was then gently shaken for 30 seconds to allow the ethyl acetate to mix with the DNP hydrazones. Thereafter the tubes were left to stand for the two layers to separate. The markers made for the reverse reaction were covered with Parafilm as they were not being used immediately.

After the 9 test tubes had been incubated in the water bath for 30 minutes, they were taken out and applied to the chromatogram paper using fine capillary tubes. Each solution was applied three times on to the chromatogram paper and between every application a hair dryer was used to speed up the drying process. When all the initial seven solutions were plotted as well as the two markers for the forward reaction, the paper was stapled together and put aside.

The previous seven tubes were then used to make DNP derivatives in order to detect for carbonyl functional groups and hence detect the α-keto acids. To each of the seven solutions 2 ml of DNP was added, they were then mixed and thereafter left to stand for 10 minutes. After the 10 minutes 1.5 ml of ethyl acetate was added to all of the tubes, they were then shaken and thereafter left to stand until the two layers were separated. When the separation had occurred, the top layer solution was extracted using capillary tube and then transferred to small holders. The solutions in the small holders were applied to the second chromatography paper using a fine capillary tube. Each solution was applied five times to the chromatogram paper and once again a hair dryer was used between each application. The marker solutions for the reverse reaction were also applied to the chromatograph paper in the same manner mentioned above. The paper was stapled together in a top corner. The chromatograms were run over night by the technical staff.

Part one tables:

Table 1: The seven test tubes were set up as indicated in the table.

Forward Reaction

Enzyme blank

Reverse reaction

1

2

3

4

5

6

7

Glutamate (ml)

0.5

0.5

-----

Pyruvate (ml)

0.5

-

0.5

----

Alanine (ml)

----

0.5

0.5

-

α-ketoglutarate (ml)

----

0.5

-

0.5

Phosphate buffer (ml)

-

0.5

0.5

1.0

-

0.5

0.5

Enzyme (ml)

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Table 2: How the marker solutions for the forward reaction were set up.

Markers for the forward reaction

Sodium glutamate (ml)

Alanine (ml)

Phosphate buffer (ml)

Distilled water (ml)

Glutamate marker

0.50

-

2.0

2.50

Alanine marker

-

0.50

2.0

2.50

Table 3: How the marker solutions for the reverse reaction were set up.

Markers for the reverse reaction

Pyruvate (ml)

α-ketoglutarate (ml)

Phosphate buffer (ml)

Distilled water (ml)

DNP solution (ml)

Ethyl acetate (ml)

Pyruvate marker

0.50

-

0.5

1.0

2.0

1.50

α-ketoglutarate marker

-

0.50

0.5

1.0

2.0

1.50

Part two tables:

Table 4: The seven test tubes were set up as indicated in the table.

Forward Reaction

Enzyme blank

Reverse reaction

1

2

3

4

5

6

7

Glutamate (ml)

0.5

0.5

-----

Proline (ml)

0.5

-

0.5

----

Asparate (ml)

----

0.5

0.5

-

Oxaloacetate (ml)

----

0.5

-

0.5

Phosphate buffer (ml)

-

0.5

0.5

1.0

-

0.5

0.5

Enzyme (ml)

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Table 5: How the marker solutions for the forward reaction were set up.

Markers for the forward reaction

Sodium glutamate (ml)

Asparate (ml)

Phosphate buffer (ml)

Distilled water (ml)

Glutamate marker

0.50

-

2.0

2.50

Asparate marker

-

0.50

2.0

2.50

Table 6: How the marker solutions for the reverse reaction were set up.

Markers for the reverse reaction

Proline (ml)

Oxaloacetate (ml)

Phosphate buffer (ml)

Distilled water (ml)

DNP solution (ml)

Ethyl acetate (ml)

Proline marker

0.50

-

0.5

1.0

2.0

1.50

Oxaloacetate marker

-

0.50

0.5

1.0

2.0

1.50

Results

First attempt

Table8: Amino acids chromatogram results. See appendix (picture 5) for chromatogram paper.

Forward reaction

Enzyme blank

Reverse reaction

Markers

Test tubes

1

2

3

4

5

6

7

Alanine marker

Glutamate marker

Sample distance moved (cm)

4.5 & 5.5

5

3.8

3.7

5.7

5.5

3.6

5.4

4.9

Solvent distance moved (cm)

12.2

12.2

12.2

12.2

12.2

12.2

12.2

12.2

12.2

Rf value*

0.37 & 0.45

0.41

0.31

0.30

0.47

0.45

0.30

0.44

0.40

* Rf value was calculated by dividing the distance the sample has moved by the distance the solvent has moved.

Alanine marker Rf value is

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