Enzymes are biological catalysts, usually made up of protein molecules that act on one or more specific substrates, converting them to products with different molecular structures. Malate Dehydrogenase (MDH) is one example of an enzyme that catalyzes oxaloacetic acid and NADH into malic acid and NAD+ as part of the citric cycle. In order to perform the MDH assay, a solution of pure MDH was collected from bovine heart. A control assay was performed, along with three experimental groups to test concentration, pH, and temperature. Units of activity were calculated and showed that all three variables have an effect on enzyme activity. MDH was shown to have an optimum pH, concentration, and temperature at which enzyme activity was at its highest …show more content…
A control sample was taken so that it could be compared to the different experimental groups (enzyme concentration, pH, and temperature). The conditions used for the control sample were a MDH concentration of 1X, a pH of 7.5, and a temperature of 25 oC. The control solution was prepared in a cuvette with 10 μL oxaloacetic acid, 1.0 mL of phosphate buffer, and 10 μL NADH. The control sample was placed in the spectrophotometer in order to confirm that the original absorbance was above 0.6. There were then 10 μL of enzyme added to the solution and the spectrophotometer ran for one minute while the change in absorbance was recorded, giving the slope of the reaction rate. After completing the assay for the control, a set of assays was performed to test the effects of varying enzyme concentration, pH, and temperature on the reaction rate. The assay to test concentration was done by taking the original 1X solution of MDH and diluting the sample down to 0.5X and 0.25X. After completing the dilutions, the same steps done in the control assay were followed. The assays to test pH were performed using buffers with a pH of 4 and a pH of 10 and then using a spectrophotometer to record the data. Lastly, the effects of temperature on the reaction rate were tested by measuring three samples at varying temperatures. The three samples were tested in: a -1 oC ice bath, a 37 oC bath, and the third was …show more content…
It was hypothesized that as the enzyme concentration increases, the reaction rate increases. Also that as the pH moves away, both increasing and decreasing, from the optimum pH (7.5) the reaction rate decreases. Lastly, as the temperature moves away, both increasing and decreasing, from the optimum temperature (37 oC) the reaction rate decreases. All of the results, as described below, were consistent with the hypothesis that was made.
As the results show in Figure 1, as the concentration of enzymes is increased, the units of activity also increased with the maximum being 0.0622 µmol/min at 1X concentration. This may be because when the enzyme is in a higher concentration there is more catalyst present, causing the reaction rate to increase. As Figure 2 shows, altering the pH, either higher or lower, had a negative effect on the reaction rate. This indicates that the optimum pH for this reaction is approximately 7.5, giving a rate of 0.0622 µmol/min. It is possible that altering the pH to extreme highs or lows causes the configuration of the enzyme to be altered and inhibits the biochemical system. The results also show that temperature has an affect on the reaction
If temperature of the water(enzyme environment) is increased to 35°C, then the enzyme activity will
The normalized SDH activity of two homogenates can be compared by looking at the class statistics for the Liver and Kidney homogenate samples in the data sheet attached. The kidney exhibited higher enzyme SDH activity than the liver. This was in agreement with the proposed hypothesis. Comparing the same two homogenates in which malonate was present, it can be seen that the kidney exhibited higher SDH activity than the liver. Thus, both homogenates did in fact have a decrease in enzyme activity, as malonate inhibits the activity of SDH. In successive experiments more malonate was used, and class statistics, not the activity itself, showed lower amounts of enzyme activity/mg protein as reaction number increased and a greater significance. Thus, malonate’s effect did increase proportionally to its concentration. There was a significant difference in SDH activity between the liver and kidney homogenates (p=0.0001)0.05). (Figure 1)
As previously stated, temperature is another variable that can affect the enzyme reaction rate. At low temperatures there will be little to no activity but as the temperature is increased so the rate of activity will increase. However, just like pH, once the optimal temperature is reached and surpassed, the enzyme will begin to break apart. The enzyme will be experimentally tested using temperatures around room temperature (27℃). Due to the fact that there is a human application the enzyme will continue to be tested until temperatures of human death (45℃). After the testing is completed it can be deduced what temperatures create an optimum environment for the normal functioning of the enzyme. One
Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect
However, the rate of reaction only increases for a certain period of time until there is lesser substrate molecules than the enzyme molecules. The increase of enzyme concentration does not have effect if there are lesser substrate molecules than enzyme molecules initially.
These results shown from this experiment led us to conclude that enzymes work best at certain pH rates. For this particular enzyme, pH 7 worked best. When compared to high levels of pH, the lower levels worked better. The wrong level of pH can denature enzymes; therefore finding the right level is essential. The independent variable was the amount of pH, and the dependent being the rate of oxygen. The results are reliable as they are reinforced by the fact that enzymes typically work best at neutral pH
My results visibly show that the optimum for this enzyme is pH 7, the pH at which the highest rate of oxygen production was obtained, at 0.80 cm3 sec-1, corresponding to the highest peak in the graph. The other rate values obtained from more acidic pHs, for example at pH 2 and pH 4 were much less, measuring 0.02 cm3 sec-1, and 0.07 cm3 sec-1, respectively. This shows that a decrease in pH from the optimum decreases the rate of the reaction. Subsequently, an increase in alkalinity had a similar effect, a decrease in rate, at pH 10 and pH 12, yielded rate values of 0.08 cm3 sec-1, and 0.07 cm3 sec-1, respectively. This shows that an increase in pH from the optimum also decreases the rate of the reaction.
Effect of pH on Enzyme Activity. 1. Dependent Variable. amount of product (glucose and fructose) produced 2. Independent Variable. pH 3. Controlled Variables. temperature; amount of substrate (sucrose) present; sucrase + sucrose incubation time
In the following experiments we will measure precise amounts of potato extract as well as Phenylthiourea, combined with or without deionized water and in some instances change the temperature and observe and record the reaction. We will also investigate the different levels of prepared pH on varying samples of the potato extract and the Phenylthiourea and record the results. We will answer question such as what is the best temperature for optimum temperature reaction as well as the best pH level for the same reaction.
In this lab or experiment, the aim was to determine the following factors of enzymes: (1) the effects of enzymes concentration the catalytic rate or the rate of the reaction, (2) the effects of pH on a particular enzyme, an enzyme known and referred throughout this experiment as ALP (alkaline phosphate enzyme) and lastly (3) the effects of various temperatures on the reaction or catalytic rate. Throughout the experiment 8 separate cuvettes and tubes are mixed with various solutions (labeled as tables 1,3 & 4 in the apparatus/materials sections of the lab) and tested for the effects of the factors mentioned above (concentration, pH and temperature). The tubes labeled 1-4 are tested for pH with pH paper and by spectrophotometer, cuvettes 1a-4a was tested for concentration and cuvettes labeled 1b-4b was tested for temperature in four different atmospheric conditions (4ºC, 23ºC, 32ºC and 60ºC) to see how the enzyme solution was affected by the various conditions. After carrying out the procedures the results showed that the experiment followed the theory for the most part, which is that all the factors work best at its optimum level. So, the optimum pH that the enzymes reacted at was a pH of 7 (neutral), the optimum temperature that the reactions occurs with the enzymes is a temperature of 4ºC or
The independent variable in this investigation is pH. Each individual enzyme has it’s own pH characteristic. This is because the hydrogen and ionic bonds between –NH2 and –COOH groups of the polypeptides that make up the enzyme, fix the exact arrangement of the active site of an enzyme. It is crucial to be aware of how even small changes in the
The purpose of this experiment was to record catalase enzyme activity with different temperatures and substrate concentrations. It was hypothesized that, until all active sites were bound, as the substrate concentration increased, the reaction rate would increase. The first experiment consisted of five different substrate concentrations, 0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2. The second experiment was completed using 0.8% substrate concentration and four different temperatures of enzymes ranging from cold to boiled. It was hypothesized that as the temperature increased, the reaction rate would increase. This would occur until the enzyme was denatured. The results from the two experiments show that the more substrate concentration,
The use of multiple test tubes and Parafilm was used for each experiment. Catechol, potato juice, pH 7 phosphate buffer, and stock potato extract 1:1 will be used to conduct the following experiments: temperature effect on enzyme activity, the effect of pH on enzyme action, the effect of enzyme concentration, and the effect of substrate concentration on enzyme activity. For the temperature effect on enzyme activity, three test tube were filled with three ml of pH 7 phosphate buffer and each test tube was labels 1.5 degrees Celsius, 20 °C, and 60 °C. The first test tube was placed in an ice-water bath, the second test tube was left at room temperature, and the third test tube was placed in approximately 60°C of warm water. After filling the test tubes with three ml of the
Enzymes are high molecular weight molecules and are proteins in nature. Enzymes work as catalysts in biochemical reactions in living organisms. Enzyme Catecholase is found on in plants, animals as well as fungi and is responsible for the darkening of different fruits. In most cases enzymatic activities are influenced by a number of factors, among them is temperature, PH, enzyme concentration as well as substrate concentration (Silverthorn, 2004). In this experiment enzyme catecholase was used to investigate the effects of PH and enzyme concentration on it rate of reaction. A pH buffer was used to control the PH, potato juice was used as the substrate and water was used as a solvent.
Enzyme catalysis is dependant upon factors such as concentration of enzyme and substrate, temperature and pH. These factors determine the rate of reaction, and an increase in temperature or pH above the optimum will