Succinate Dehydrogenase Lab Report

Determining and understanding the enzyme activity in different types of Oncorhynchus mykiss muscle (heart, white and red) by comparing rate of reaction (V) of the succinate to the substrate concentration [S]

Question: How does changing enzyme concentration or temperature affect the reaction time of enzyme activity?

The enzyme’s rate did change over time. This compares to a real enzyme because an enzyme’s job is to speed up the reactions and as time allotted. That did happen since the enzyme in our lab was able to make more chainobeads as time progressed.

In this experiment we tested the effects that enzymes and substrate have on chemical reaction rates, which is the rate at which chemical reactions occur.. This experiment tested how different concentrations of enzyme and substrate affected the light absorption measurements on a spectrophotometer. The experiment also tested how temperature affected the light absorption, and in a separate test, the effect of the enzyme inhibitor hydroxylamine was also tested. In the first test conducted, 3 different concentrations of enzyme, and three different concentrations of substrate were measured in a spectrophotometer. For the enzyme and the substrate, the measurements got higher as the concentrations were higher, but the over measurements of the substrate were smaller than those of the enzyme. In the second test conducted, the medium concentration enzyme was tested under the temperatures; 4°C, 23°C, 37°C, and 60°C. The measurements in this test got higher as the temperature got higher, but did the measurements under 4°C were overall significantly higher than the other temperature measurements. Lastly, the last test conducted showed that the measurements of the substance with 0 and 1 drop of hydroxylamine inhibitor went up, but the measurements of the enzyme with 5 drops of hydroxylamine inhibitor stayed rather low and did not change much. In conclusion, these experiments showed that chemical reaction rates are sped up with higher concentrations of enzyme, substrate,

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)

Enzyme Concentration: Cuvette 2 gradually increased every twenty seconds for two minutes and contained 1 milliliter of the enzyme (figure 1). Cuvette 3 slowly increased but was a bit faster than cuvette 2 every twenty seconds for two minutes and contained 2 milliliters of the enzyme (figure 1). Cuvette 4 was started lower then cuvette 2 and 3 then made a significant increase every twenty seconds for two minutes and contained 4 milliliters of enzyme (figure 1). The average rate of absorbance for cuvette 2, 3, and 4 were .0013 au per second, .0028 au per seconds, and .0041 au per second.

The spectrophotometer as zeroed once 0.1ml of 16.2M ethanol was added. 0.1ml of the enzyme stock solution was added and the absorbance at 340nm was measured for two minutes. OD/min is then calculated from the graphs of the spectrophotometer. Concentration of the substrate was then calculated (Appendix 1) and Enzyme velocity was calculated (Appendix 1). The same procedure was repeated for 1.2ml of the buffer, 1.5ml of NAD+, 0.1 ml of ethanol and 0.2ml of enzyme solution was added and the velocity of the enzymes was then measured again.

We found that Tube 1 had an absorbance of 0.085, Tube 2 had 0.034, Tube 3 had 0.027, Tube 4 had 0.032, Tube 5 had 0.025, Tube 6 had 0.028, Tube 7 had 0.022, Tube 8 had 0.024, Tube 9 had 0.030, and Tube 10 had an absorbance of 0.022. We then graphed absorbance vs fraction number so that we could demonstrate the absorbance of each tube and found that Tube 1 had our highest value of absorbance. Basically, the first few tubes have the most protein and then the rest drop off as is shown in the graph.

The kinetic profile of tube 1 is represented by a horizontal line in graph 1. Tube 2 contained 0.01ml of enzyme and graph 2 represents the kinetic profile of tube 2. It shows that the absorbance of tube 2 increased over time at an exponential rate. The amount of enzyme added to tube 3 was 0.1ml. The absorbance of this tube increased at a steady rate over time. Graph 3 represents tube 3 and its enzyme kinetic profile is indicated by the positive linear line. About 0.5ml of enzyme was added to tube 4 and graph 4 represents its kinetic profile. The absorbance increased over time, but at the end it began to plateau. Comparing the kinetic profiles of 4 tubes, the appropriate enzyme concentration needed for the assay was determined to be

In this lab we experimented with pH, spectrophotometry, and enzymes like catecholase. We used spectrophotometry to detect how enzyme activity would change under different pH, temperature, enzyme concentration, and substrate concentration. Enzymes are important for biochemical reactions because they speed up the process and allow the organism to continue living. The enzyme used in the experiment is catecholase and it catalyzes a reaction between catechol and water. We analyzed the samples in the spectrophotometry and it is measured by absorbance and transmittance and this will allow us to see which sample will have the largest concentration of molecules. The first experiment involved enzyme concentration and enzyme activity; in this experiment

When the relationship between enzyme concentration and absorbance is graphed, it shows a rapid increase that eventually levels out.

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.

However looking at figure 1 the gray line of the graph pertaining to the concentration of tubes 6 and 7, there was a drop in activity visually on the graph due to an outlier of class data. Yet, looking at figure 2 the reaction rate of looking at the reaction rate or rate of change in the absorbance over the time of two minutes in 20 second increments. The slopes of all three lines show that the peroxidase looks directly proportional in data. Thus, supporting our hypothesis that the concentrations of peroxidase will increase the absorbance of the enzyme and not denature it because factors such as temperature, or pH were not

lysis of Cellular Respiration Under Various Conditions Introduction: These experiments provided insight into the process of cellular respiration in aerobic and anaerobic conditions with the variable factors such as the presence of succinate, temperature, and variable carbohydrates. Cellular respiration is the set of metabolic reactions that transform glucose and other reactants into useable energy (ATP) and waste products (Saylor, 1). In the first part of the experiment, the impact of succinate toward aerobic respiration is observed. By utilizing the suspension of mitochondria, the conversion of succinate to fumarate can be examined in the citric acid cycle. Using spectrophotometer, the transmittance of the color change is facilitated by

Abstract: Enzymes are biological catalysts. They are also proteins and there properties are determined by their structure. The reactant is a substrate and the resulting factor is the product. . Enzyme activity is influenced by many different things including: substrates, products, presence of cofactors, and inhibitors. The effect of inhibitors is measured as a percentage called percent inhibition.