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Transamination Using Glutamate Pyruvate Transaminase Biology Essay

Transamination is an enzyme coupled reaction that, if not in the presence of the specific transaminase will not occur. The transamination performed by Glutamate-pyruvate-transaminase (GTP; EC 2.6.1.2) functions on α-keto acids and amino acids to synthesis new amino acids and a new α-keto acid. GPT functions on pyruvate and glutamate to create alanine and α-ketoglutrate. The enzyme requires the cofactor pyridoxal-5-phosphate which acts a coenzyme by transferring the amino group between the substrates. TLC was performed to indicate whether or not transamination occurred. This was done by spotting the substrates individually, a sample in which the enzyme was present and a control in which no enzyme was present to act as a control. The ninhydrin reaction in which Ruheman purple development occurs when in the presence of amino acids was used as the amino acid detection method. Semicarbazide was used as the α-keto acid detection agent as in the presence of an α-keto acid it forms a semicarbozone which fluoresces under UV light. Successful transamination was noted as well as enzyme saturation due to presence of substrate in the reaction sample spotted.

Introduction

Transamination is an integral function of amino acid deamination to synthesise new amino acids from precursor α-keto acids. It creates an α-keto acid from the original amino acid and a newly synthesised amino acid from the precursor α-keto acid (Voet et al., 2008). It is a reaction which involves the transfer of an amino group from an amino compound to a keto or aldehyde compound without the intermediate formation of ammonia. It has been seen that although the most common of reactants are mono- and dicarboxylic α-amino acids and α-keto acid, several other amino acids and amines have been seen to undergo a transamination reaction (Sallach and Fahien 1969). The reaction is catalysed by a group of enzymes referred to as transaminases, which transfer the amino group between the compounds, and is one of the most common enzymatic activities that involve amino acids. Transaminases have been known to require a vitamin B6 derivative as cofactor, namely pyridoxal-5’-phosphate. This cofactor functions as a coenzyme in the presence of the enzyme and functions in the formation of a Schiff base with the amino acid, followed by electron rearrangement in which the amino group is transferred to the coenzyme to form pyridoxamine phosphate. The pyridoxamine phosphate then has the ability to react with the keto acid transferring the amino group, resulting in the regeneration of the pyridoxal phosphate upon reaction completion (Conn and Stumpf, 1966). Transaminases show extremely high specificity for natural substrates (Sallach and Fahien 1969).

Glutamate-pyruvate-transaminase (GTP; EC 2.6.1.2) also referred to as alanine transaminase (ALT) and functions in the alanine glucose-alanine cycle. It plays a unique role in the transfer of nitrogen form peripheral tissues to the liver. Alanine, one of the 20 common amino acids found in the body, is transferred to the circulation by muscle in which alanine is formed from pyruvate. Alanine build up occurs in the liver and the alanine reverses the transamination which occurred in the muscle, releasing nitrogen to increase urea production. Muscle alanine is produced in two distinct pathways: directly through protein degradation, and through the transamination of pyruvate by GTP. The enzyme uses the 2 substrates, an α-amino acid glutamate and an α-keto acid pyruvate to create and α-amino acid alanine and an α-keto acid α-ketoglutrate (King 2010).

Figure : Equation taken from King 2010

GTP

Ninhydrin also referred to as triketohydrindene hydrate is an oxidising agent. It results in the oxidative deamination of α-amino groups. It has uses in the quantitative analysis of amino acids. The reaction between α-amino acids and ninhydrin results in the production of Ruheman’s purple, a blue-violet dye, which has an absorbance maximum at 570 nm. Below is the reaction between ninhydrin and α-amino acid.

Figure : Diagram showing the formation of Ruheman's purple

Taken from the following URL: http://utenti.multimania.it/Pasquale_Petrilli/aastruc/aareac.htm

Semicarbazide, a reducing agent has applications in keto acid detection. Once in contact with a keto acids and pyruvate, they react immediately form insoluble semicarbazones (Umbargar and Magasanik, 1952). Illumination with UV light at 254nm results in both compound fluorescing as a blue-purple spot on a TLC plate(Dewhurst and Smallman, 1988).

Materials and Methods

The developing solution was made of the following reagents in an 80:6:14 part ration to a volume of 100ml; ethanol, 25% ammonia and dH2O and was placed into the TLC tanks and left to allow the solution to saturate the tank. Two silica gel G25 ½ plates were used to perform the TLC procedure. They were prepared with a starting line 2 cm from the bottom of the plate being draw. The plates were divided into 2 as the procedure was performed in duplicate on each plate. Six evenly placed dots were marked on the base line of each half to indicate the position for the spotting of the samples. Two test tubes were prepared with the following reagents being pipetted in the following quantities:

Table 1: Reagent composition of test tubes used to indicate the transaminase reaction catalysed by GPT as well as a control tube to indicate no reaction occurs without the enzyme

Reagent

Tube 1 – reaction tube (ml)

Tube 2 – control (ml)

0.05M TRIS/HCL pH 7.6

1.5

1.5

0.1M L-Glutamate

0.5

0.5

0.1M L-Pyruvate

0.5

0.5

GPT Enzyme

0.02 (20μl)

-

dH2O

0.5

0.5

The tubes were then placed in a water bath at 37˚C for an incubation period of 45 minutes. During this time the reagents were collected and the respective dilutions were performed. (See addendum 1 for calculated dilution factors). After the 45 minute incubation period the plate was spotted 10 times per lane according to the following list:

0.0167 M L-Glutamate ( form a 0.1M stock solution – See addendum 1)

0.0167 M L-Alanine

0.0167 M L-Pyruvate ( form a 0.1M stock solution – See addendum 1)

0.0167 M α-ketoglutrate

Tube 1 (reaction mixture)

Tube 2 (control mixture)

The plates were placed into the TLC tank and left for an hour and a half until the developing solution had migrated to within a distance of ± 0.5 cm from the top of the plate. The plates were removed and the migration front was immediately marked. Two detection solutions were made of which the first (detection solution 1) contained a 1:1 ratio of 0.2% Ninhydrin solution (Merck) in acetone (Rochelle chemicals) and 0.1% chlrorophenol red solution (Aldrich) in ethanol, and the second (detection solution 2) a semicarbazide solution made with 0.75g sodium acetate and 0.5g semicarbazide (Sigma) made to a final solution volume of 500ml with dH2O. The first plate was sprayed with the ninhydrin solution to detect the amino acids and was placed in a sealed oven at 110˚C for ± 2 minutes. The second plate was sprayed with the semicarbazide solution and placed in the oven for the same duration. Once the plates had cooled, circles where drawn around the ninhydrin rings visible under normal light while the second plate, which had been sprayed with semicarbazide require placement under UV light to cause the samples to fluoresce before the markings could be made. Distances from the base line to the migration front and to the centre of each ring were made to calculate the Rf values.

Results

Table 2: Table showing migration front, migration distance average and Rf values for plate sprayed with semicarbazide solution

Lane

Sample

Concentration (M)

Migration front (mm)

Migration distance 1 (mm)

Migration distance 2 (mm)

Migration distance Ave (mm)

Rf value

1

L-Glutamate

0.0167

68

-

-

-

-

2

L-Alanine

0.0167

68

-

-

-

-

3

L-Pyruvate

0.0167

68

57

59

58

0.85

4

α-ketoglutrate

0.0167

68

45

46

45.5

0.67

5

Reaction tube (1) *

0.0167

68

47

55

49

56

48

55.5

0.71

6

Control tube (2)

0.0167

68

55

57

56

0.82

*there were 2 bands present for this lane

Table 3: Table showing migration front, migration distance average and Rf values for plate sprayed with ninhydrin solution

Lane

Sample

Concentration (M)

Migration front (mm)

Migration distance 1 (mm)

Migration distance 1 (mm)

Migration distance Ave (mm)

Rf value

1

L-Glutamate

0.0167

70

35

30

32.5

0.46

2

L-Alanine

0.0167

70

30

30

30

0.43

3

L-Pyruvate

0.0167

70

-

-

-

-

4

α-ketoglutrate

0.0167

70

-

-

-

-

5

Reaction tube (1)

0.0167

70

35

38

36.5

0.52

6

Control tube (2)

0.0167

70

36

42

39

0.56

Discussion

As stated above, the semicarbazide reacts with the α-keto acids and pyruvate in creating a semicarbazone which fluoresces when under UV light (Umbargar and Magasanik, 1952). This allowed for the determination of the migration distance and hence a calculation of the Rf value. In table 2 it can be seen that there was no reaction with between the semicarbazide and amino acids, with the lack of spot development seen for the 2 amino acid samples. As seen in table 1, the reaction test tube contains the substrates glutamate and pyruvate which, due to the enzyme GTP, get converted into alanine and α-ketoglutrate. Glutamate and alanine, due to the lack of a reaction can be determined to be amino acids. Lane 5 which had the reaction solution should be expected to have the α-keto acid. However there were 2 distinct bands having Rf values of 0.71 and 0.82. These Rf values indicate 2 distinctly different molecules. In comparison to the rest of the table, there are similar Rf values for lane 3, 4 and 6. Lane 3 which was spotted with L-Pyruvate had an Rf value of 0.85 which has a similar value to the second band in lane 5 of 0.82. This indicates that in the reaction tube there was pyruvate. Due to due to the presence of pyruvate one could assume there was possible enzyme saturation as pyruvate is one of the substrates needed to be used in the transamination. In lane 6 a band was seen with an Rf value of 0.82. This was expected as the control tube was spotted in lane 6. The lack of enzyme in the control tube resulted in there being pyruvate in the solution and hence a band on the plate. The second band in lane 5 had an Rf value of o.71 which is similar to the band seen in lane 4, having an Rf value of 0.67, which was spotted with α-ketoglutrate. This is the product from the enzyme GPT hence one would expect there to be α-ketoglutrate in lane 5.

As above, ninhydrin reacts when in the presence of amino acids and develops the Raheman purple (http://utenti.multimania.it/Pasquale_Petrilli/aastruc/aareac.htm) colour, an indication of the presence of amino acids on plate 2. In table 3 one can see there are no recorded migration distances in lane 3 and 4 which is expected as ninhydrin does not react with α-keto acids such as pyruvate and α-ketoglutrate. There are bands seen in lane 1 and 2 which are expected as both alanine and glutamine are amino acids. Lane 6 would also be expected to have a band corresponding to glutamate as in the control test tube there was no enzyme hence no reaction took place. However the results do not correlate. In tube 1 the band has an Rf value of 0.46 which would be expected to have a corresponding band in lane 6, yet the Rf value was 0.56. This is an example of experimental error due to a poor spotting technique.

In lane 5 one expected to find a band for alanine and as a result of the data captured in the table 2 a band for glutamate as a result of enzyme saturation. However there was a single band seen with an Rf value of 0.52 which is far greater than the Rf value for alanine of 0.43 and the Rf value for glutamate of 0.46. It must be said though that there is a small difference between the Rf values of alanine and glutamate one could expect a large band with a equal to a band stretching from the bottom of the alanine band to the top of the glutamate. This however was not seen and due to the Rf values not having any similarity it is another case of experimental error due to poor spotting technique.

The Rf values attained were expected to follow a specific trend. The developing solution used in the experiment was a nonpolar solution made up of ethanol, ammonia and dH2O. It would hence be expected to readily dissolve nonpolar molecules while not having the same ability for polar molecules. The stationary phase was a silica G25 gel which is a polar stationary phase. From this one would expect the non polar molecules to remain in solution in the mobile phase and migrate the almost as far as the developing solution due to instantaneous polarity of the molecules resulting in a slight retardation of the molecule. The polar molecules would be expected to have migrated a short distance and no longer be in solution due to the “like sticks with like” principle in which non polar solutions do not dissolve polar molecules. Therefore the molecules would be on the plate not in the solution (Voet et al., 2008).

It must be noted that the substrate L-glutamate shows levorotatory stereochemistry which is the accepted stereochemistry by GPT. The D-glutamate which shows dextrorotatory would not be an acceptable substrate hence it can be determined that the GPT enzyme shows sterospecificty in the substrate choice. It must also be noted that if a substrate for an enzyme shows levorotatory stereochemistry, the product too will show levorotatory stereochemistry seen in the product of L-alanine (Voet et al., 2008)

Conclusion

Glutamate-pyruvate-transaminase (GTP; EC 2.6.1.2) a transamination enzyme successfully converted pyruvate and glutamate into alanine and α-ketoglutrate as seen from the results taken in tables 2 and 3. The enzyme also displayed saturation due to higher substrate concentration. As seen in the results from lane 6 in table 2 and 3 the lack of the enzyme in the tube resulted in no reaction as in vitro transamination can’t occur unless there is the presence of the specific transamination enzyme.

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