Abstract: Ammonia is toxic to the human body and the complex process of removing it from the body is called the urea cycle. This experiment focuses on the intermediates and enzymes within the urea cycle This was done through spectrophotometry and assay techniques which helps show to us the varying intermediates and key steps involved in the urea cycle and allowed us to determine the activities of the proteins and enzymes and identify the proteins that do cause the production of urea in the cycle. Introduction: Ammonia and Amonium (NH3- and NH4+) are produced within the body and are highly toxic, especially to the brain and very reactive and so our bodies need a way of quickly processing this into a form for excretion, it does this by converting …show more content…
These five steps can be summarized as follows: 1. Formation of Carbamoyl Phosphate: Ammonia undergoes a condensation reaction with bicarbonate ions resulting in the formation of carbamoyl phosphate this reaction is then catalysed by the enzyme carbamoyl phosphate synthetase I, this step also requires Mg2+ and N-acetyl glutamate. 2. Synthesis of Citrulline Carbmoyal phosphate combines with ornithine by the transfer of a carbamoyl group to ornithine to form citrulline this is in the presence of enzyme citrulline synthase or ornithine transcarbamoylase and then diffuses through the mitochondrial membrane and into the cytosol. Phosphate is produced as a byproduct. 3. Synthesis of Argino-succinate: Once in the cytosol of the hepatocyte citrulline will comine with the ammino acid aspartate forming aginino-succinate this is catalysed by the enzyme arginine-succinate synthase. This process requires energy in the form of ATP which will be hydrolysed resulting in AMP and the use of the cofactor Mg2+ 4. Cleavage of …show more content…
Cleavage of Arginine: Arginine is hydrolysed to form urea and ornithine, under the enzyme arginase. Through this step Ornithine is regenerated and the urea cycle is completed with the formation of Urea. The overall reaction of the urea cycle is as follows: 2NH3 + CO2 + Aspartate + 3ATP → Urea + Fumarate + 2ADP + AMP + PPi + 2Pi The main methods used were that of spectrophotometry, using light and standard curves. A urea enzyme assay was performed via separating the proteins so as to observe which of these proteins were specifically causing an effect on the absorbance and produce urea. Another technique used in the experiment was the generation of a protein standard so as to determine the concentration of protein in the liver extract mixture The objective of this experiment is that the urea cycle substrates are amino acids integrated in cycle and that specific proteins and enzymes must be present to continue on and produce urea. This lab was based on the assumption that all enzymes were present and functional and so ultimately we were looking at the intermediates in the cycle and how well the enzymes moved them through the cycle and produced urea. Materials and
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.
The enzyme urease breaks urea down into NH3 and CO2. An orange broth containing urea is used for this test and needs to be inoculated with the gram negative bacteria. A pink color in the medium indicates a urease-positive organism, an orange or yellow is negative.
For the Urease test, I incoluated my Urea test tube with my unkown bacteria from a TSA plate using and inoculating loop. The Urea tube was then incubated at 37 degrees Celsius for 8 days to observe for a color change. The Urea tests for the ability of a bacteria grown in urea broth produces urease. This medium contains the pH indicator phenol red. If urease is produced the pH of the media will raise thus causing the phenol red to change from yellow to a pink color.
In contrast, there are four metabolic stages happened in cellular respiration, which are the glycolysis, the citric acid cycle, and the oxidative phosphorylation. Glycolysis occurs in the cytoplasm, in which catabolism is begun by breaking down glucose into two molecules of pyruvate. Two molecules of ATP are produced too. Some of they either enter the citric acid cycle (Krebs cycle) or the electron transport chain, or go into lactic acid cycle if there is not enough oxygen, which produces lactic acid. The citric acid cycle occurs in the mitochondrial matrix, which completes the breakdown of glucose by oxidizing a derivative of pyruvate into carbon dioxide. The citric acid cycle produced some more ATPs and other molecules called NADPH and FADPH. After this, electrons are passed to the electron transport chain through
Reaction 3- 1. Obtained a clean and dry test tube and placed a small amount ( about the size of a jelly bean) of ammonium carbonate into the test tube.
Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.
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.
Also, the enzyme was treated with different urea concentrations before it was added to the assay mixture. The amount of the second and third enzymes was essential. Likewise, this would have had an effect on the rate of the measured aldolase activity. Hence, the concentrations for both enzymes was measured and calculated prior to the experiment whilst preparing the assay mixture. Furthermore, in order to measure thiol group reactivity the base line was adjusted to zero for each cuvette. This was done to make sure that the results were
Police brutality, or the general brutality towards black people, is not a new issue in America. Over 700 unarmed African-Americans were murdered in 2015 alone. Michelle Alexander argues in “The New Jim Crow” that the criminal-justice system in America has purposely been used as a means for oppressing black people after the Civil Rights Act of 1964 was passed. In 1903, Hon. Frank Moss, a former police commissioner of New York City, published this paragraph:
Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).
Amylase experiment # 2 was done to see how the pH affected the efficacy of the enzyme. First we collected all of the materials that were necessary to make this experiment. We needed five clean test tubes, the following standard solutions, 1% Starch Solution pH 3,1% Starch Solution pH 5,1% Starch Solution pH 7,1% Starch Solution pH 9,1% Starch Solution pH 11
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
Aminotransferases are enzymes involved in catalyzing reactions between amino acid and α-keto acid. Amino acid such as L-glutamate contains an amine group whereas α-keto acid such as pyruvate contains a keto group. The amine group of L-glutamate is exchanged with the keto group of α-keto acid resulting in the α-keto acid becoming an amino acid and the amino acid becoming a keto acid. Aminotransferase catalyzes the reaction of transferring α-amino group of L-glutamate to pyruvate in exchange for the α-keto group of pyruvate where L-alanine and α-ketoglutarate are formed. Aminotransferases are specific for the amino acid formed and reactions catalyzed by aminotransferases are reversible. Aminotransferase used in the formation of alanine is called glutamate-pyruvate aminotransferase (GPT) where in the formation of aspartate, glutamate-oxaloacetate aminotransferase (GOT) is used. (Vroon, D., & Israili, Z, 1990)
In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube
The urea cycle is the process by which ammonia is converted to urea. During the urea cycle, the amino acid arginine is converted to ornithine another amino acid upon addition of water. During this reaction urea is produced (Nelson and Cox 2008).In this experiment, the enzyme arginase is present in the liver extract. The reaction was investigated by adding the liver extract containing arginase to solutions of arginine and water. The amount of urea produced was determined using spectrophotometric analysis with α-isonitrosopropiophenone.