Transaminase Activities in Animal Tissues - Research Paper Example

Practicum 2: Transaminase Activities in Animal Tissues Student Name: Student Number: Actual Word Count: 1440 Abstract In this experiment, we used the rat as an experimental animal for transaminase activities. The primary aim was to determine the amino acids present in the tissues of rat heart and verify the hypothesis that there are many amino acids participating in the transamination reaction in this particular animal tissue. The presence of amino acids and transaminase reactions were determined using paper chromatography. After running the chromatography for 6 amino acid standards, 2 keto acids, and 8 samples from the tubes, it was found that the 8 sample solutions prepared from the tissue contained 6 amino acids, and two keto acids. The results obtained were compared with class data, which showed insignificant variation. This means that transaminase reactions are responsible for the synthesis of many amino acids in the heart tissue of rats. A statistical evaluation of class data showed that there is no significance variation in the values of Rf obtained as observed from the values of standard deviation. Introduction A transaminase or an aminotransferase in biochemistry refer to an enzyme that catalyzes a reaction that involves an amino group (from an amino acid) to an α-keto acid. Transaminase enzymes are important in the synthesis of different amino acids to form proteins (Cammarata & Cohen, 1950). In the field of medicine, the concentration of different transaminases in the blood is used to indicate tissue damage, usually the liver, and diagnosing many other diseases. Elevated transaminases present in the blood is an indication of damage of the liver and cardiac tissue (Stryer, 1988). There are more than 30 transaminase enzymes known that are specific for different amino acids. These transaminases are widely found in the cytosol and the mitochondria of cells. The two important transaminase enzymes in the body are glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT). GPT is found in high concentrations in the liver and it also occurs in many tissues, while GOT occurs in particularly in the heat and liver at relatively high concentrations (Wretlind, Orstadius, & Lindberg, 1959). These enzymes are responsible for the interconversion of amino acids, facilitating the combination of nitrogen from one amino acid with relevant carbon skeleton in order to synthesize another amino acid. Also, GOT and GPT show increased levels of blood serum in various diseases that involve tissue necrosis in both man and animals (Henry, Cannon, & Winkelman, 1974). Tissue damage, myocardial infarction, muscular disorders, and acute pancreatitis causes the release of transaminases into the bloodstream which can be used as a diagnostic tool. In this experiment, we investigated tissue transaminase activity in the heart of a rat by incubating a homogenate with different pairs of amino/keto acid. Transamination was demonstrated using paper chromatography. The main purpose of carrying out this experiment was to determine pyruvate acid, α-ketoglutarate acid, total amino acids present and the transaminase activity of rat heart tissue. Hypothesis There are many amino acids participating in the transamination reaction in rat heart. Materials and Methods Materials The following list of materials were used in the experiment: Heart tissues was obtained from rat. Crushed ice box for chilling the tissues. 0.01M phosphate buffer (pH 7.6) Weighing scale Scissors (or scalpel) All glass homoginiser Incubator with water bath at 38oC. α-keto acid Chromatography sheets and solvent Gloves Hair drier Pencil, clips, and P20 micro pipette for spotting. Procedure Tissue preparation – The heart was removed from the rat and chilled in crushed ice. The heart was drained of superfluous blood and fluids, and 1g of tissue weighed and chopped off with scissors before homoginisation. 10 ml of ice-cold 0.01M phosphate buffer was then added to the chopped tissue. The heart was then homoginised in the all-glass homoginiser placed in an ace bath. Incubation – The tubes for incubation for transaminase reactions were set as indicated in table 1 below: Table 1: Incubation for transaminase reactions Tube No. Phosphate buffer (ml) Tissue Homogenate (ml) Amino Acid (0.5 ml) α-keto acid (0.5 ml) 1 1.5 0.5 L-lysine α-ketoglutarate 2 1.5 0.5 L-aspartate α-ketoglutarate 3 1.5 0.5 L-arginine α-ketoglutarate 4 1.5 0.5 L-phenylalanine α-ketoglutarate 5 1.5 0.5 L-alanine α-ketoglutarate 6 1.5 0.5 L-glutamate pyruvate 7 2.5 0.5 - - 8 2.0 0.5 - α-ketoglutarate The 0.5 ml α-keto acid was added after shaking and placing the tubes and the α-keto acid in a water bath at 38oC for about 5 minutes. The solution was then shaken and incubated for another 1 hr. before applying the samples on a chromatography sheet. Paper Chromatography – Using the gloves, the chromatography paper was placed on a dry bench covered with a clean sheet of bench cover paper. A pencil line was then drawn 2cm from the bottom across the sheet, and even spaces for spots marked. These spaces were labeled on with the solutions to be applied on top of the sheets. These solutions were 6 amino acid standards, 2 keto acids, and samples from tubes in table 1. The sheet was placed such that the baseline was not in contact with the bench, before applying 1 l of each solution at the sports using the P20 micro pipette. The standards were applied only once, while the solutions from tube 1-8 were over-spotted 5 times, after careful drying between each spotting. Running and developing the chromatograms and determination of amino acids – After the spots were dry, the sheets were stapled into a cylinder and hung on a tank to equilibrate for 1 hr. The paper was then lowered into the solvent below the line of application of the spots. The chromatogram was then run for about 2 hours, removed and the solvent frond marked. The following morning, the dry sheet was dipped in a jar with 0.1% ninhydrin in acetone, drained and dried using a hair drier to develop color. Results Individual Data: The following photographs show individual results of chromatograms obtained after carrying out this experiment: Figure 1: Individual chromatogram The calculated Rfs of the solutions in the chromatogram shown in figure 1 are as presented in table 2 below: Table 2: Rf of the solutions in the individual chromatogram Solutions Distance moved by the solute from the origin [A] cm Distance moved by the solvent from the origin [B] cm Rf = [A/B] Standards: L-lysine 3.5 11 0.32 L-aspartate 1.3 “ 0.12 L-arginine 3.0 “ 0.43 L-phenylalanine 7.5 “ 0.68 L-alanine 5.6 “ 0.51 L-glutamate 1.8 “ 0.16 Pyruvate acid - - - α-ketoglutarate acid - - - Samples: 1 3.6 11 0.33 2 1.0 “ 0.14 3 2.9 “ 0.41 4 7.5 “ 0.68 5 5.4 “ 0.49 6 1.4 “ 0.13 7 - - - 8 - - - From the chromatogram results in table 2, it can be observed that there is a close relationship between the Rfs of the standard solutions and those of the sample solutions in tubes 1-8. This can help in the identification of the amino acids present in the samples. Form the data in table 2, the amino acids present in the sample solution are as follows: Table 3: Identification of amino acids Sample Amino acids and keto acids present 1 L-lysine 2 L-aspartate 3 L-arginine 4 L-phenylalanine 5 L-alanine 6 L-glutamate 7 Pyruvic acid 8 α-ketoglutarate acid Class Data: The following photographs show the chromatograms of the experiment obtained from class data: Figure 2 (a-j): Class data showing chromatograms obtained for standards and sample solutions. In table 3 below, we have presented the calculated Rfs of the standards and sample solutions obtained from class data. Table 3: Rf Class data of standards and sample solution Figure (Chromatogram) Calculated Rf. Value Standards Samples L-lys L-asp L-arg L-phe L-al L-gl 1 2 3 4 5 6 7 8 2a. 0.36 0.18 0.28 0.74 0.48 0.18 0.36 0.12 0.29 0.72 0.52 0.15 - - 2b. 0.34 0.16 0.36 0.68 0.53 0.15 0.33 0.16 0.34 0.75 0.48 0.13 - - 2c. 0.38 0.18 0.44 0.76 0.47 0.17 0.30 0.13 0.42 0.69 0.51 0.13 - - 2d. 0.32 0.12 0.38 0.75 0.56 0.21 0.37 0.16 0.32 0.77 0.50 0.16 - - 2e. 0.33 0.17 0.32 0.72 0.49 0.16 0.34 0.11 0.36 0.76 0.52 0.14 - - 2f. 0.35 0.16 0.30 0.72 0.47 0.14 0.29 0.16 0.31 0.76 0.48 0.15 - - 2g. 0.36 0.16 0.32 0.78 0.46 0.18 0.32 0.12 0.38 0.75 0.49 0.15 - - 2h. 0.32 0.18 0.42 0.71 0.53 0.15 0.30 0.13 0.34 0.78 0.53 0.12 - - 2i. 0.32 0.15 0.44 0.70 0.50 0.13 0.36 0.12 0.31 0.74 0.55 0.16 - - 2j. 0.34 0.19 0.38 0.75 0.48 0.19 0.33 0.14 0.42 0.74 0.52 0.14 - - Std Dev 0.021 0.019 0.061 0.031 0.034 0.024 0.030 0.020 0.040 0.027 0.024 0.014 The Rf values and the standard deviations calculated in table 3 for class data show a close relationship with the values obtained from the individual data. Thus, we can use these results to verify the individual data obtained in table 2. Discussion This experiment is concerned with the transaminase activity in the soluble fraction of rat tissue. From the results presented in table 1 and table 3, figure 1 and figure 2, it is evident that there are many normal transaminase activity in the heart tissue of rat investigated. This is an indication that many more amino acids are involved in the transamination reaction in rat heart. This findings are consistent with the results reported on other animals, and nominates the heart as one of the most likely tissue of pathological elevation in the serum transaminases in rat and other animals. The standard deviations in the values of Rf obtained in class data fall in the range 0.019-0.034. This variation may be attributed to the analytical methods employed in this experiment, which are certainly not very accurate. The distance travelled by the amino acid on the chromatogram sheet also depends on the experimental conditions under which the procedure was performed, and is affected by factors such as temperature, nature of the filter paper, and the nature of organic solvent (Stryer, 1988). It is apparent that the filter paper and the organic solvent were the same in all the experiments, and therefore, slight variations in temperature might have played a significant role in the variation of the Rf values. The demonstration from this experiment that the transamination reaction in animals includes many amino acids, gives this kind of activity a unique role in the animal metabolic processes. This is in agreement with our hypothesis, that there are many amino acids participating in the transamination reaction in rat heart. In addition to the interconversion of the major metabolites, transamination is recognized as being important in the synthesis of -ketoglutaric, pyruvic acids, aspartic acid, alanine, glutamate, lysine, arginine, and phenylalanine. In conclusion, the presence of six amino acids, in addition to -ketoglutaric and pyruvic acid, was determined in the tissues of rat heart. The six amino acids participate in the transamination reactions in the animal. References Cited Read More