This lab involved several experiments over the span of many weeks. The overall intent of the lab was to be the successful purification of a protein. Specifically, the purification of the enzyme acid phosphatase from wheat germ. Through three major steps we were able to perform this purification. The steps involved were disrupting the source cell, selectively purifying the enzyme from contamination, and finally preserving the original structure of the enzyme and preventing degradation. Aliquots, or small volumes, of the solution were collected from each step in the purification process for enzyme activity and protein content. These calculated values were then used to determine the specific activity, which can be used to analyze the specific …show more content…
Enzymes play a role to catalyze numerous reactions and help in the creation of DNA, RNA, and other molecules. This experiment focuses on the enzyme acid phosphatase. Acid phosphatase is a ubiquitous lysosomal enzyme that hydrolyses organic phosphates at an acid pH, which means that they remove phosphate groups under slightly acidic conditions. (Henneberry, 1979). Acid phosphatase has become to be used commonly because of the practical applications such as being used extensively as a serum marker for prostate cancer (Bull, 2005). In this lab a primary focus is to successfully perform an isolation of acid phosphatase and prevent it from degrading while keeping it in its native structure. First, disrupted the cell used as a source via hypotonic shock. Then, through several methods including centrifuging and dialysis, we were able to purify it and protect it from breaking down further. Next, the substrate PNPP was used to follow the extent of which it was purified and characterization. A spectrophotometer was needed to measure the absorbance of the protein solution and compared to a standard curve. Using gel electrophoresis we determined the success that our purifications steps achieved. Absorbance was also measured at 405nm from the hydrolyzed PNPP. The use of these techniques of purification and analysis to determine overall …show more content…
This assay was used to measure the concentration of protein in a solution. Use it to determine the concentration of our saved supernatants 1-6. The total amount of protein is determined and so is the phosphatase activity. By combining the Bradford reagent with our diluted supernatants in a test tube and letting sit for 5 minutes to be read at 595nm we were able to determine the mg/ml of protein. The absorbance was read on a spectrophotometer which measures the light intensity. Using a standard curve we were able to get our readings. The procedure was taking the six supernatants and aligning them to six test tubes. A solution was created with 50x dilution for sup1, 10x for sup 2-3, and no dilution for sup 6. A Bradford reagent was then added to each. Afterwards analyzed using the A595 wavelength. A formula was then used to determine the amount of active protein. Sup 1 yielded 104.31 mg/ml, sup 2 yielded 22.33 mg/ml, sup 3 yielded 1.21 mg/ml, and sup 6 yielded 0.66
The purpose of this study is to analyze the activity of the enzyme, catalase, through our understanding
Catalase is a common enzyme that is produced in all living organisms. All living organisms are made up of cells and within the cells, enzymes function to increase the rate of chemical reactions. Enzymes function to create the same reactions using a lower amount of energy. The reactions of catalase play an important role to life, for example, it breaks down hydrogen peroxide into oxygen and water. Our group developed an experiment to test the rate of reaction of catalase in whole carrots and pinto beans with various concentrations of hydrogen peroxide. Almost all enzymes are proteins and proteins are made up of amino acids. The areas within an enzyme speed up the chemical reactions which are known as the active sites, and are also where the
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
One of the most primitive actions known is the consumption of lactose, (milk), from the mother after birth. Mammals have an innate predisposition towards this consumption, as it is their main source of energy. Most mammals lose the ability to digest lactose shortly after their birth. The ability to digest lactose is determined by the presence of an enzyme called lactase, which is found in the lining of the small intestine. An enzyme is a small molecule or group of molecules that act as a catalyst (catalyst being defined as a molecule that binds to the original reactant and lowers the amount of energy needed to break apart the original molecule to obtain energy) in breaking apart the lactose molecule. In mammals, the lactase enzyme is present
This is an experimental lab that tested if drinking water passes the United States maximum phosphate standard. The results of this lab can help the American who drink the water know if there are too much phosphate in the water. Each group made a Potassium phosphate dilution from a stock solution. The concentration of the solution that needed to made affected the amount of Potassium phosphate that was diluted. To create a calibration curve, each group used the different concentrated Potassium phosphate solutions in their test. The lab utilized a spectrophotometer to figure out the absorbance of the five different Potassium phosphate solution and the absorbance of an unknown concentration solution. The absorbance of the unknown solution was used
This pH homeostasis lab is used to show how acids and bases react when submerged into different solutions - water, a homogenate, and a buffer. A homogenate is blended up water and the cell tissue. They are used to show how much pH the cells have when adding an acid or base to it. A buffer is a solution that doesn’t change it’s pH level, even when acid or base is added to it. They are important to help all living things maintain homeostasis.
Enzymes are proteins that increase the rate of chemical reaction by lowering their activation energy. The enzyme glucose oxidase is one of the most widely used enzyme as an analytical reagent due to its ability to identify the presence of glucose, its low cost and good stability. This report discusses the role of enzymes concentration in biological reactions and the catalytic activity of glucose oxidase on D-Glucose. The activity was studied by spectrophotometry and the results were first tabulated and then plotted. The results of this experiment indicate that the enzyme concentration has no major affect on the rate of
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
PH can affect the way fermentation occurs due to the chemical differences between acid and alkaline elements, particularly within a solution. In this experiment an enzyme-based reaction was examined that in order to observe this pH trend. The aim of the experiment was to determine how pH affects the yeast fermentation rate by performing the experiment numerous times with a different pH (pH's 3, 5, 7, 9, 11) in different glucose solutions. 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 carbon dioxide (C02) produced by the yeast during fermentation whilst modifying the pH of the glucose solution. To test this every 5 minutes
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
From looking at the results I can conclude that when the pH was 3 and 5. No oxygen was produced, therefore no reactions were taking place. This was because the pH had a high hydrogen ion content, which caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place of the syringe. The enzyme lost its functional shape.
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
Introduction: Purifying proteins is an important part of biology because it can help identify the function of that protein. Once a protein’s function has been identified, it can be manipulated to see how the function would change if the protein was changed. A common way to purify a protein is through Ion Exchange Chromatography, which is where charged proteins will bind to the beads in the column to purify it from the solution (Berg JM, 2002). The purpose of this experiment is to use Ion Exchange Chromatography to purify cellulase.