Lab Report On Luciferase

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

One gram of liver was sliced into pieces, then 10ml of homogenization buffer was added to it. The mixture of the liver and homogenization buffer was then placed into a homogenizer to make the liquid slurry. Once the slurry was made it was placed into a 50ml falcon tube to then be placed in the centrifuge. The slurry was centrifuged for 2 minutes at 2000rpm. Once the spin in the centrifuge was complete, the slurry had separated, the most dense particles to the bottom of the tube forming sediment and the lighter (liver succinate dehydrogenase enzyme) also known as the supernatant. The supernatant was extracted from the falcon tube and placed into a test tube. The tube was then kept at a low temperature, (in an ice bath) until it was required for use.

40 mL of a concentrated solution of sucrose was prepared at 200mg/mL. Using appropriate dilutions of the stock, 11 solutions including a control solution were made in plastic tubes. The enzyme reaction with sucrose was run in 2 mL volume at room temperature in water. The enzyme constituted half the volume of the stock solution. The substrate was added to the enzyme in order to start the reaction. Each reaction ran for 5 min after which 2 mLs of DNS reagent was added. The solution was boiled for 10 min and the results were read using a spectrophotometer.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

Cytoplasmic streaming was measured 4 times after flushing the cells with pond water, at 5 minute intervals

When lysing the cells, add 20 - 50 μl chilled lysis buffer (RIPA buffer with protease inhibitors and PhosSTOP), and keep the samples on ice for 30 min. Centrifuge the samples at 20,000 × g for 20 min at 4℃. Transfer the supernatant to new tubes,

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,

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

There were a couple of problems that our group encountered while conducting this experiment. Since this was our first experiment dealing with enzyme activity, the probability of human error increased. We found it difficult to go through the procedures with undefined roles for our team. The other problem that our team encountered was the getting the cuvettes into the spectrophotometer quickly once the enzyme was introduced to the cuvette.

Using an O2 sensor to measure how much additional oxygen is added into a sealed Nalgene bottle, we could determine the enzymes effectiveness. Three variations of temperature were tested: cold, hot and room temperature. This was done by taking test tubes with the three different samples and placing them in a Nalgene Bottle. It was determined that the enzyme is most effective when at room temperature although still functions when it is chilled. The heated sample was drastically less effective.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

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.

This ensured that the subsequent enzymatic reactions would occur at the correct rate for best observation. Following the optimization of Tyrosinase, the normal enzymatic activity was observed with L & D-DOPA. The objective of this step was to obtain the Km and Vmax of each enzymatic reaction. By obtaining and plotting the spectrophotometric data, the Lineweaver-Burk plot was used to calculate both the Km and Vmax for each substrate-enzyme reaction.

The products produced by the first reaction were used as a substrate for the second. In this case the enzyme used NADH, which resulted in the decreased absorbance due to the NADH oxidation to NAD+. In addition, the spectrophotometer was used as a measuring device to follow the change in absorbance of the NADH molecules at 340nm.

Researchers have taken a closer look at the structure of the active site that facilitates the bioluminescent reaction; they found the Arg218 residue plays a heavy role in the secure binding of luciferin. The guanidinium side chain of Arg218 is particularly responsible for binding with the benzothiazole ring of the luciferin substrate. Moreover, Arg218 establishes electrostatic interactions with the ground state product of the phenol group ionization of luciferin, yet Arg218 also stabilizes the final excited state phenolate ion of oxyluciferin (Branchini, B.R., Magyar, R.A., Murtiashaw, M.H., and Portier, N.C, 2001, 2415-2416). The proper binding of luciferin determines the color of the light emission and is dependent on the amount of rotation of the excited oxyluciferin molecule. To further understand the role of Arg218, researchers created a mutant enzyme without the Arg218 residue to determine its impact on substrate production. When researchers converted the Arg residue to Lys, there was a 15-fold increase in Km for luciferin; the Km value suggests the substrate binding affinity of the enzyme. The increase in Km shows that luciferase has a lowered affinity to bind with substrates. This shows that a manipulation of the Arg218 residue can affect the catalytic activity of the enzyme. Usually, the enzyme loses its high binding affinity with luciferin.