Optimal Dh And Temperature Conditions Lab Report

From Figure 5, Vmax = 0.0172 and Km = 0.3144. At ½ Vmax (Figure 4), the rate of reaction increases steadily with an increase in substrate concentration. At this point, the rate of product formation is partially limited by substrate availability. As the curve approaches Vmax, the enzyme catalyzed saturation increases. In effect, the enzyme activity increases the rate of product formation up to a point whereby the reaction rate remains constant as more substrate is added (saturation).

The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of techniques including homogenization, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the overall amount of LDH present in the samples.

PH can affect the way fermentation occurs due to the irregularity of the acidity or alkalinity within the glucose solution. This is an enzyme-based reaction that is susceptible to pH. The aim of this experiment was to determine how pH affects the yeast fermentation rate by performing the experiment numerous times with a different pH of glucose solution which included pH 3, 5, 7, 9, 11. The hypothesis was ‘If the pH is lower than the neutral point then the fermentation reaction will occur faster?’ The experiment conducted was to measure the amount of C02 produced by the yeast going into fermentation, however varying the pH of glucose solution by using different pHs . To test this every 5 minutes the volume of gas in the test tube was observed and recorded until a period of 30 minutes had been. The end results

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]

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

6) See attached for the graph. Correlation appears to be negative with this data. It appears like the high temperature caused the enzyme to become denatured.

• Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.

To prevent fluctuation in the pH, a solution known as a “buffer solution” was used in the experiment. Buffer solutions are mixtures of at least two chemicals which counteract the effect of acids and alkalis. Therefore, when a small quantity of alkali or acid solution is added the pH of the enzyme doesn’t change.

The three tissues being analyzed in this experiment, those of the kidney, heart and liver, were taken from the animal Bos taurus. The tissue homogenates used were made by adding 1 gram of tissue to 20 ml of sucrose phosphate buffer. The buffer was composed of 250 mM sucrose, 50 mM NaPO4, with a pH of 7.4. This mixture was homogenized with a high-speed blender for 3 pulses for a duration of 10 seconds each. The homogenates were then filtered through a cheesecloth and stored at -70° C.

While it does have a relatively high specific activity value it is easily overshadowed by the specific activity of C2. This raises the question of whether the data collected for the latent C2 samples is

We then tested the last set of test tubes containing milk and lactase, we did this to find which ones would present the most glucose concentration results, when placed in different temperatures, 4°C, boiling and room temperature. What we wanted to know was how far temperature could affect lactase to perform its enzymatic activity. We hypothesized that if the lactase is placed in a high or low temperature outside its active range, the temperatures would have a negative impact on the functions of the enzyme. If the temperature has an affect on lactose then we would see some temperatures in which lactase will be function able. We came to a conclusion that enzymes work at a temperature that is closest to body temperature (25°C); boiling water (100°C) denatures the enzyme, while the enzyme is not able to function properly if

Acids and bases were added to the solution in order to achieve the desired pH. While there were complications leading to a shortage of data, the results pointed to a peak of enzymatic activity between 6 and 8 pH. This suggested that the hypothesis

The purpose of this lab is to examine the specificity of the lactase enzyme to a specific substrate and how it can denature due to the rise in temperature.

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

This indicates that oxamic acid is likely a competitive inhibitor, and that competition for binding of the LDH enzyme active site is occurring. The Vmax and Km values for inhibition of LDH enzyme by ⍺-ketoglutarate were very different from Vobserved(100µL) and Kapparent(100µL) respectively. This indicates that oxamic acid is likely a mixed inhibitor, competing with pyruvate for active site binding, in addition to binding to the enzyme-pyruvate transition state, inactivating