Heart Homogenate Using Paper Chromatography

Published Date: 02 Nov 2017

Transamination, alpha-keto acid

aminotransferase, alpha-keto acid, paper chromatography, Heart homogenate.

Aims: The aim of this experiment is to observe the reaction catalyzed by aminotransferase with different amino acids and to identify the reaction status whether it is catalyzed in a forward fashion or in a reverse way.

Methods and results: In this project experiment a heart homogenate was used to investigate the forward and reverse reactions of amino acids. Tissue transaminase can be investigated by incubating a homogenate with various amino acid/ keto acid pairs. For this project, substrate solution was well prepared (Sodium Glutamate, Alanine, Proline, Oxaloacetate, alpha-ketoglutaric acid, Sodium Pyruvate and Aspertate at 0.10 M in each solution taken here). Enzyme was used for this purpose extracted from heart tissues according to the protocols. All mixtures were mixed gently and incubated for 1 hour at 37⁰ C. After that, marker solutions of Sodium Glutamate and Alanine were used for amino acid Chromatography. For alpha-keto acid Chromatography, keto acid marker solution was taken like previous one. After overnight running, desired results were visible. Rf value for each sample was calculated from Chromatogram results. Finally, the primary purpose was to identify the occurrence of transamination and then to measure the reversibility of this process.

Conclusions: Transamination plays a crucial role in biochemical processes of living organisms. This depends on aminotransferase enzymes. Any type of abnormality in these enzymes may halt the biochemical processes.

INTRODUCTION

Transamination (or aminotransfer) is a chemical reaction between two molecules. One is an amino acid, which contains an amine (NH2) group. The other is a keto acid, which contains a keto (=O) group. In transamination, the α-amino 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 as follows:

R1.CHNH3 + .COO- R1.CO.COO-

+ +

R2.CO.COO- R2.CHNH3 + .COO-

A number of transamination reactions of the ribosome has been established catalyzed by the so-called ribozymes which are (RNA enzymes). Ribozyme plays important role in different RNA processing reactions such as RNA splicing, viral replication, transfer RNA biosynthesis. Some ribozymes also play important role as therapeutic agent as biosensor, gene discovery.

Transaminases into the blood is significant in the identifing the pathway of many kinds of diseases.

Two significant transaminase enzymes which are aspertate transaminase, AST in short (SGOT) and alanine transaminase, ALT in short (SGPT).

The test for AST determines the amount of the enzyme in blood. This enzyme is found in RBC, muscle , tissue,pancreas. When heart disorders occur additional AST release in the blood. The extent of the damage is directly related to the amount of the enzyme. The another enzyme alanine transferase also render crucial role for the determination of organ damage. ( perkov et al 2008)

These enzymes also play important role as an indicator of liver damage due to toxic substances. AST serve as a crucial indicator during the period of liver transplantation. Not only that the ratio of AST and ALT are found to be increased in alchoholic liver diseases. Serum AST is a good indicator of ethanol consumption. For these purposes these enzymes are of great importance in diagnostic field.( Cook et al 2002)

AST is of highest activity among other aminotransferases. It is preset in almost all mammalian tissues. It is the most commonly used aminotransferase for clinical purposes.

L-aspartate +Oxo-glutarate Oxaloacetate+

Glutamate.

In transamination reaction pyridoxal phosphate

(PLP) plays crucial role. That result of the transamination reactions are based on the accessibility of the alpha-keto acids. Their equivalent alpha-keto acids are also produced from side to side metabolism of the fuels. Threonine and serine are the two types of amino acids those do not forever undergo the transamination .

Most transaminations are freely reversible but cases of unidirectional transamination are known to be irreversible. .

One of the best characterized plant aminotransferase is glutamate-oxaloacetate transaminase (GOT) (or aspartate aminotransferase (AAT) [EC 2.6.1.1]) which catalyzes the reversible interconversions of glutamate and aspartate, and their 2-keto analogs: ( jones et al 1980)

Glutamate + oxaloacetate (OAA) <---> 2-oxoglutarate + aspartate

Aminotransferases (ATases) initiate catabolism of aromatic, branched-chain, and sulfur-containing amino acids to cheese flavor compounds . α-Keto acids are the intermediates and amino group acceptors in an ATase reaction. They are of interest in determining the role of these reactions in Fatty acid,FA production.) unwavering that each have 12 ATases. Though, the position of single ATases into the metabolism of the amino acid and keto acids is indistinct. α-keto acids. ( yonaha et al 1985)

Tissue transaminase activities 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. ( Boomer et al 2013)

Chromatography is an effective technique for separating amino acids. This separation technique is characterized by two phases as mobile phase and stationary phase. Different substances as paper, silica gel, are used in the stationary phase. The mobile phase is the medium where the substances move through the stationary phase. In the mobile phase both liquid and gas can be used as a medium. Usually the paper chromatography is used to identify the composition of stationary phase. ( watanabe et al 2008)

Different factors influence the separation techniques. The separation of each amino acid correlates with their affinity for stationary and mobile phase. As different amino acid have different molecular structures, they have different affinities for these two phases. The affinity of amino acids for each phase depends on several other factors such as shape of the molecule, water solubility, molecular weight. The water solubility of amino acids and pH of the solution also influence the separation technique.

In the present 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-dinitrophenylhydrazine (DNP) to form the dinitrophenylhydrazone derivatives of all the keto acids present. These derivatives are extracted into a small volume of ethyl acetate and this solution is applied to a second paper chromatogram that is developed with a different solvent.

References:

www.macalester.edu/kuwata.

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081430/

http://www.biocyc.org/META/NEW-IMAGE?type=ENZYME&object=MONOMER-2966

Materials and Methods

Reagents and Materials used

1. Phosphate buffer, 0.l0 M, pH 7.4

The solution of Phosphate buffer contain:

Name

Amount (M)

Sodium dihydrogen phosphate (NaH2PO4.2H2O)

1.0M = 156.0 g/litre

0.1M = 156.0/10 g/litre

0.1M = 15.6/4 g/litre = 3.9 g/250ml/ water

Disodium hydrogen phosphate (Na2HPO4)

1.0M = 172.0 g/litre

0.1M = 172.0/10 g/litre

0.1M = 17.2/4 g/litre = 4.3 g/ 250ml/water

2. Substrate solutions, all prepared in the above buffer:

Name

Amount (M)

Sodium glutamate

0.10

Alanine

0.10

Proline

0.10

Oxaloacetate

0.10

Sodium pyruvate

0.10

Asparate

0.10

α -ketoglutaric acid

0.10

3. Enzyme: 10 g heart was homogenised in 90 ml of ice-cold water. The cell debris was removed by centrifugation and the supernatant fluid stored at 0°C overnight.

4. Saturated solution of 2,4-dinitrophenylhydrazine (DNP) in l.00 M HCl, in a burette.

5. Paper Chromatogram.

Method

The detailed methods are as follows:

1.Incubation

Seven test tubes were set up for incubation as follows to table 1 and 2. They were mixed well and incubated for a period of 1.00 hr. at 37°C

2. Chromatography of amino acids

(a) 0.01 M 'marker' solutions were prepared of sodium glutamate and alanine by diluting the 0.10 M solutions provided, as follows: 0.50 ml sodium glutamate was pipette into a test tube, 2.0 ml buffer and 2.50 ml water were also added. This procedure was repeated with alanine. The solution was prepared with following specifics

Sodium glutamate

1.0M 169.1 g/litre

0.1M = 169.1/100 g/litre

0.1M = 1.691 g/100 cm3

• α- ketoglutaric acid

1.0M 168.08 g/litre

0.1M = 168.08/100 g/litre

0.1M = 1.68 g/100 cm3

• Alanine

1.0M 89.1g/litre

0.1M = 89.1/100 g/litre

0.1M = 0.89g/100cm3

Sodium Pyruvate

1.0M =110g/litre

0.1M = 110/100g/litre

0.1 M = 1.1g/100cm3

(b) A 25 cm square of Whatman No. 1 paper was taken, handling it as little as possible and only at the edges. (beware-sweat from the fingers contains amino acids). The paper was labeled clearly in pencil with name and draw a line across it 2.5 cm from the edge. Total 9 origins were marked, 2.5 cm apart on the line, and labelled them as 1, 2, 3, 4, G, A, 5, 6 and 7.

Sodium glutamate

1.0M 169.1 g/litre

0.1M = 169.1/100 g/litre

0.1M = 1.691 g/100 cm3

Aspartate

1.0M = 133.1g/litre

0.1M = 133.1g/litre/ 100

0.1M = 1.33g/100cm3

•Proline

1.0M = 115.1g/litre

0.1M = 115.1g/litre/100

0.1M = 1.15g/100cm3

•Oxaloacetate

1.0M = 132.07g/litre

0.1M = 132.07g/litre/ 100

0.1M = 1.32g/100cm3

(c) A fine capillary tube was applied with one small spot (less than 5 mm diameter) of each incubation mixture to its own numbered origin. A single small spot was also applied of marker alanine solution to A, and glutamic acid to G. Between applications, the capillary tube was rinsed twice with distilled water and then with the next solution to be applied, allowing it to rise up the capillary tube and blotting

on tissue. The spots were allowed to dry and place the paper in a frame.

(d) The solvent consists of butanol/acetic acid/water, in the ratio 3:1:1 (v/v/v). Next morning the papers will be removed and allowed to dry.

(e) The paper was passed through a dip tray containing a solution of 0.20% (w/v) ninhydrin in acetone. It was allowed to dry in the fume cupboard and then heat in an oven for a few minutes to develop the spots.

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

3. Chromatography of α-keto acids

(a) A 25 cm square Whatman No. 1 chromatography paper was outlined above for the amino acid chromatography, labelling the origins 1, 2, 3, 4, KG, P, 5, 6 and 7.

(b) The 'marker' solutions of the DNP derivatives of the ketoacids. Pipette 0.50 ml of the 0.10 M pyruvate solution into one test tube and 0.50 ml of the 0.10 M α-ketoglutarate solution into another.

0.50 ml buffer and 1.00 ml water were added to each tube. Subsequently, add 2.00 ml of the DNP solution was mixed and left for 10 min. 1.50 ml ethyl acetate was shaken gently from side-to-side for 30 sec. to extract the DNP hydrazones into the ethyl acetate layer. The two layers were allowed to separate for several minutes. The upper ethyl acetate layer should now be bright yellow and the lower aqueous layer nearly colourless. If not to be used immediately thereafter, the tube was covered with Parafilm.

(c) After applying the spots to the amino acid chromatogram, DNP derivatives were prepared in the following manner: to each of the 7 incubation mixtures add 2.0 ml of DNP solution, and then proceed exactly as with the ‘marker’ solutions in 3(b) above (leave for 10 min., etc.).

d) The first tube was taken and carefully insert a fine, dry, capillary tube, running its tip down the wall of the tube until the tip just meets the meniscus of the ethyl acetate layer. The yellow solution will at once rise into the capillary, which should be withdrawn and examined carefully to check that it contains no aqueous layer. Then, from the capillary, small spots were applied of solution to the corresponding marked origin on the chromatogram.

It is needed to multiply spot several portions for each sample. Each spot is allowed to dry before applying the next, and kept their diameter below 5 mm. It is continued until a bright yellow coloured spot was appeared on the base-line. This is essential since no colour development is achieved with the α-keto acids: they are simply identified by means of the yellow colour. Any remaining solution from the capillary was expelled, wash it twice by drawing up ethyl acetate, and dried with paper tissue. It was repeated with each tube in turn, but with those containing 'marker' solutions of α-ketoglutarate (KG) and pyruvate (P) applied only 2 spots to the chromatogram. When all the spots have been applied, the paper was mounted in a frame.

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

(e) The solvent consists of butanol/ethanol/0.50 M ammonia, in the ratio 7:1:2 (v/v/v) by volume. After overnight incubation the papers will be removed and allowed to dry. The yellow spots will be visible without further treatment.

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

C:\Users\abadir\Documents\project pictures\New folder\IMAG0967.jpg

Figure: Picture 2: Chromatogram paper of the α-keto acids.

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 Part B: 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

DNP solution (ml)

Praline Marker

0.50

-

0.5

1.0

2.0

1.50

Oxaloacetate Marker

-

0.50

0.5

1.0

2.0

1.50

Results

Chromatograms show the presence of amino acids and keto acids produced by transamination reactions and give Rf values.

Many different methods have been devised for separating mixtures into their components. In the present experiment a method called paper chromatography was used. It is used quite widely for making small-scale separations and identifications. The method works because of the differences in the ways various components of a mixture are absorbed and re dissolved from a piece of paper. First, the mixture that is to be separated is dissolved (if it is not already in liquid form.) Then, a small drop of the solution is applied to a piece of special chromatography paper, which is a porous paper similar to filter paper. This drop makes a tiny spot on the paper. Many such spots (of different materials) may be placed side by side on the same piece of paper. Next, the spotted paper is made to stand in a small amount of some special solvent (liquid), in such a way that only the bottom edge of the paper is submerged in the liquid, not the spots. The solvent then slowly rises up the paper, reaches the spots, and begins to dissolve them and carry the substances up the paper with it.The key to the separation is that the different components of the mixture in each spot will rise at different rates, and so will be found (after a few minutes) to have reached different heights on the paper. This happens because some components are more strongly attracted to the paper (and so move more slowly), while others are more strongly attracted to the solvent. Any particular substance always moves at the same rate, no matter what else it may have been mixed with originally. For that reason, it can always be recognized and identified, because it will always rise to the same height, relative to the heights that the other components rise. The movement of any spot on the paper can be quantified by calculating its Rf (retention factor) value after the experiment and the paper has dried. ( deng et al 2011) The distances used in calculating Rf values are measured as shown in the following Figure 4.

Figure 4: Method to calculate Rf value.

To determine the distance traveled by the solute, measure from the point at which you originally applied the spot to the center or densest part of the spot. The distance traveled by the solvent front is measured from the original point of application of the spot to the limit of movement of the solvent front (which must be marked immediately after the paper is removed from the beaker, because it may be nearly invisible after the solvent evaporates).

Figure 5: Pyridoxol

Vitamin B6 (noted as vitamin B6) is an natural nutrient of vitamin B complex. The pyridine imitative, vitamin B6 can pass on to any of 3 chemically connected and water-soluble types: pyridoxine (PN) with pyridoxol (PL-figure:5), and the pyridoxamine (PM). Pyridoxine is one kind of alcohol and which is also known as adermin and pyridoxol, as pyridoxal is the aldehyde and the pyridoxamine is one type of amine. Enzymes needy on the PLP center a large variety of compound reactions mainly connecting amino acids. These reactions passed out by PLP-related enzymes that take action on the amino acids comprise transfer of amino group and the decarboxylation, racemization, along with beta or gamma removal or substitute. Such adaptability arises from the aptitude of PLP to covalently attach this substrate, and after that to act as the electrophilic catalyst, thus stabilizing dissimilar types of carbanionic chemical reaction intermediates. The pyridoxal phosphates are worried in about all metabolism of the amino acid, from that synthesis to breakdown. ( coles SJ et al 2011)

Transaminase enzymes are required to break down the amino acids are needy on the attendance of the pyridoxal phosphate. The correct action of these enzymes is vital for the procedure of moving the groups of amine from one to another amino acid.

Transsulfuration: the pyridoxal phosphate is the coenzyme required for the correct job of the enzymes named cystathionine synthase and the cystathionase. Those enzymes are working to change methionine into the cysteine.

Conversion of the tryptophan to the niacin: Vitamin B6 is too required for that exchange of the tryptophan to the niacin and the low vitamin B6 rank will damage this exchange. ( Yo X et al 2012)

PLP is too used to make physiologically energetic amines by the decarboxylation of the amino acids. Some outstanding examples of that comprise: histadine to the histamine and tryptophan to the serotonin, the glutamate to the GABA (gamma aminobutyric acid), also the dihydroxyphenylalanine to the dopamine. In this experiment, the results derived from chromatogram are shown. Tube 4 was blank and there was no solution accept enzyme. It ensured about the presence of any contaminant in a reaction tube. ( Mendoza et al 2011)

The forward and reverse reaction were analyzed for Alanine ( non-polar) and Glutamate ( polar) markers. The Rf values for these two markers were found nearly same. By observing the reaction color the distance for sample and solvent were analyzed. The test tubes were prepared for experiment where tubes 1,2,3 were observed for forward reaction. Tube 4 was used as a control. Tubes 5,6,7 were for reverse reaction.

Table7 Part A: Amino acids chromatogram results.

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 Glutamate marker Rf value is

Table 8 Part A : α-keto acids chromatogram results.

The forward and reverse reaction were analyzed for α-keto glutarate and Pyruvate markers. The Rf values for α-keto glutarate marker was found quite different from the previous amino acids and pyruvate. The Rf values for these two markers were found nearly same. By observing the reaction color the distance for sample and solvent were analyzed. The test tubes were prepared for experiment where tubes 1,2,3 were observed for forward reaction. Tube 4 was used as a control. Tubes 5,6,7 were for reverse reaction.

Forward reaction

Enzyme blank

Reverse reaction

Markers

Test tubes

1

2

3

4

5

6

7

α-ketoglutarate

marker

Pyruvate marker

Sample distance moved (cm)

4.7 & 0.9

10.8

4.6

10.8

0.7

10.8

0.9

1.1

4.8

Solvent distance moved (cm)

10.8

10.8

10.8

10.8

10.8

10.8

10.8

10.8

10.8

Rf value*

0.44 & 0.083

1

0.43

1

0.065

1

0.083

0.10

0.4

* Rf value was calculated by dividing the distance the sample has moved by the distance the solvent has moved (as seen above).

Table 9 Part B: Amino acids chromatogram results

For aspartate and glutamate markers the Rf values were slightly different as both of them are polar and have side chains of carboxylic acids groups. The Rf values for these two markers were found nearly same. By observing the reaction color the distance for sample and solvent were analyzed. The test tubes were prepared for experiment where tubes 1,2,3 were observed for forward reaction. Tube 4 was used as a control. Tubes 5,6,7 were for reverse reaction.

Forward reaction

Enzyme blank

Reverse reaction

Markers

Test tubes

1

2

3

4

5

6

7

Asparate marker

Glutamate marker

Sample distance moved (cm)

4.6 & 6.3

4.5

6.2

13.4

3.6

3.5

5.9

13.4

4.8

Solvent distance moved (cm)

13.4

13.4

13.4

13.4

13.4

13.4

13.4

13.4

13.4

Rf value*

0.34 & 0.47

0.34

0.46

1

0.27

0.26

0.44

1

0.36

.

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

Asparate marker Rf value is Glutamate marker Rf value is

Table 10: Part B: α-keto acids chromatogram results.

For oxaloacetate and Proline markers the Rf value differs for their different structural features. The Rf values for these two markers were found nearly same. By observing the reaction color the distance for sample and solvent were analyzed. The test tubes were prepared for experiment where tubes 1,2,3 were observed for forward reaction. Tube 4 was used as a control. Tubes 5,6,7 were for reverse reaction.

Forward reaction

Enzyme blank

Reverse reaction

Markers

Test tubes

1

2

3

4

5

6

7

Oxaloacetate

Marker

Proline marker

Sample distance moved (cm)

8.9

8.8

8.9

8.9

3.8

8.8

3.8

8.9 & 10.2

8.9 & 10.2

Solvent distance moved (cm)

10.5

10.5

10.5

10.5

10.5

10.5

10.5

10.5

10.5

Rf value*

0.85

0.84

0.85

0.85

0.36

0.84

0.36

0.85 & 0.97

0.85 & 0.97

* Rf value was calculated by dividing the distance the sample has moved by the distance the solvent has moved (as seen above).

Discussion

Every component has a specific Rf value due to the characteristic properties of the specific components which leads to it spending different amounts of times in the mobile phase relative to the stationary phase and hence moving up the paper chromatogram at different rates. Rf value was therefore calculated in order to identify the specific components. ( Bera AK et al 2012)

Usually chromatography is a powerful technology for analyzing the amino acids . ( tian X et al 2013)

Out of all the seven test tubes only the first and fifth contained all the components needed for the occurrence of a transamination reaction. The solution in the first test tube contained the component needed for the forward reaction whereas the fifth test tube was designed for the reverse reaction. Upon examination of the chromatogram papers there was evidence of the presence of the corresponding amino and keto acids which indicate that a transamination reaction occurred.

However the occurrence of a transamination reaction was only indicated for the forward reaction and not for the reverse reaction. When the chromatogram for the keto acids was further examined, no enzyme debris could be found for the fifth test tube. However, on the amino acids chromatogram paper there was evidence of enzyme in the solution for test tube five. The results attained from the chromatogram indicate that this process is not reversible. Chromatogram papers was therefore compared with others in the class and as those reverse reactions did occur as expected it is concluded that this reverse reaction did not occur due to an unidentified error which occurred during the practical.

The presence of all four acid components for the amino and keto acid chromatogram papers is the first indication of the possibility that the reaction attained equilibrium. The intensity of the developed spots could also be used to determine whether the reaction reached equilibrium. The reactions tested in this practical should have reached equilibrium during the 30 minutes incubation at 37°C. In the paper chromatogram for the amino acids the intensity in the spots for the alanine and glutamate differ, which indicate that the reaction had not reached equilibrium at that stage, however in the keto acid chromatogram paper the α-ketoglutarate and pyruvate have the same kind of intensity. The two chromatogram papers might differ in their intensity due to the difference in time the tubes were left to stand. This time difference might be the reason for the difference in the intensity of the spots in the chromatograms.

The addition of DNP and ethyl acetate to the solutions was part of the preparations. DNP was used qualitatively to detect for the presence of ketones, the DNP undergoes a condensation reaction with the carbonyl group of a ketone or an aldehyde with the loss of water .

The solutions in tubes two, three, six and seven only contained enzyme solution and half the component needed for the forward reaction and reverse reaction, respectively. And as expected none of the corresponding acids showed up on the chromatograms. These were included in the chromatogram in order to determine whether the enzyme catalyzed a reaction with only one component. And also because the measurements of the Rf values is not always reproducible, identification of the components was therefore made on the basis of comparison with reference compounds [1] . Because the marker solutions did not contain any enzyme, there was a possibility that the components could have had different Rf values on the chromatograms depending on the presence of enzyme. With the inclusion of test tubes two, three, six and seven that possibility was taken into account.

Test tube 4 which contained only enzyme solution was included on the chromatogram to determine if the enzyme contained detectible amount of amino or keto acids. Which if it did there would be a reference spot and others like that spot could be disregarded as enzyme debris. The enzyme solution did contain detectible amounts of amino and keto acids, and it showed up on the chromatogram papers. As mentioned previously the occurrence of enzyme debris was used to determine whether or not enzyme solution was added to solution 5 in order to determine the reason the reverse reaction did not occur.

When the experiment was done with ninhydrin along with lysine it didn’t render enough positive reaction . The same result was also found for cysteine. The expected result for proline was found . Proline renders yellowish reaction . In case of tyrosine it is necessary to make the solution alkaline. otherwise false positive result can be found. Leucine, alanine and lysine canbe properly separated by chromatography. While working with cysteine some difficulties can happen due to their oxidized form. The color reaction are different for one amino acid to another. The results can be the acids which do not react, form complex with the colored pigment. The requirements for color development is ammonia and ninhydrin which is partially reduced. In some cases certain amines can render false positive results in the ninhydrin reaction.

For α- amino acids the Rf value for aspartate was found 1 . which indicated that this amino acid was separated properly. But for glutamate the Rf value was slightly different. For both oxaloacetate and proline marker the Rf value was same. Different reasons may be responsible for that. Temperature might influence the reactions. As temperature has effect on Rf value.( Burma et al 1951). The another reason might be the amino acids react differently fo their molecular structure, solubility properties and so on. The amino acids also have different mobility rate which also effects their Rf values. As paper chromatography is a qualitative method , Rf values can vary from run to run. The absorption of solvent system also role play for the variation of Rf values.

For these reasons the co chromatography can be done with standards at every time. This will help the better understanding of the separation of amino acids.

For comparing with other chromatographic results (http://www.personal.psu.edu/jpb5207/blogs/john_bugays_e-portfolio/Paper%20Chromatography.pdf) it has been observed that the Rf value was found between 0.5~ 1.00 . but in this experiment the Rf value was near 0.5 . The possible reasons may be the distance of solvent and sample were not measured properly. The solvent was not so polar to appropriately separate the mixture. Different solvent as iso- propanal was used there. By comparing these two results it can be said the more polar solvent is effective to separate the components.

In another experiment ( Macevoy –Boey et al 1961)

Paper chromatography was done to analyze the excreted amino acid from human urine. The excretion of glycine, alanine, glutamine was analyzed for different ethnic groups. Where different amount of these amino acids were found for different ethnic groups. Some precautions were taken for this reaction as specific temperature was given. Some deliberate control of humidity was maintained .These precautions were not taken in our experiments. The colour development for amino acids were carefully analyzed for European, chineese ethnic groups. The differences were clear when differenece of colour development were found. For the α- keto acids the absorbtion of color spectra was found slightly different. Overall the whole chromatogram technique was quite different from our experiment.

Conclusion

The results from the practical obtained by examining the chromatograms indicate that the reaction catalyzed by LDH goes to equilibrium. The practical results also indicate that the enzyme reaction is not reversible but because the reversibility of this enzyme is known it was determined that the results obtained occurred due to an unidentified error.

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