Introduction 1.1 Aim: To determine the kinetic parameters, Vmax and Km, of the alkaline phosphatase enzyme through the determination of the optimum pH and temperature. 1.2 Theory and Principles (General Background): Enzymes are highly specific protein catalysts that are utilised in chemical reactions in biological systems.1 Enzymes, being catalysts, decrease the activation energy required to convert substrates to products. They do this by attaching to the substrate to form an intermediate; the substrate binds to the active site of the enzyme. Then, another or the same enzyme reacts with the intermediate to form the final product.2 The rate of enzyme-catalysed reactions is influenced by different environmental conditions, such as: concentration
Enzymes are biological catalysts made of proteins that accelerate chemical reactions by lowering their activation energy therefore increasing the activity rate of the enzyme and more substrates turned into products. The ‘Catalase’ enzyme that was used during this experiment was obtained from peroxisome found in celery which are organelles found in bacteria, plant, and animal cells. It is involved in the breaking down of certain substances and the diminish of reactive oxygen species and that includes hydrogen peroxide (H2O2) which can be a byproduct of the metabolism of oxygen. Hydrogen peroxide is toxic to the cell and so the catalase enzyme is utilised to break down H2O2 to form oxygen molecules and water free of free radicals. As with all
To ensure the constant rate between HCl and propanone, solutions of propanone and HCl were prepared by following next steps: 100 cm3 of 2M propanone was poured in 250 cm3 measuring cylinder (± 1.5 cm3) 50 cm3 of 2M HCl was poured on the top of propanone (±1.5 cm3) The mixture was poured between two 250 ml flasks Flasks were closed with corks The process was repeated for 5 times but every time the propanone was diluted by 10%. The amounts of propanone, distilled water and HCl were following: 1st solution 2nd solution 3rd solution 4th solution 5th solution CH3COCH3 (cm3) (±1.5 cm3) 100 90 80 70 60 H2O (cm3) (±1.5 cm3) 0 10 20 30 40 HCl (cm3) (±1.5 cm3) 50 50 50 50 50 Therefore, the concentrations of propanone were 2M, 1.8M, 1.6M, 1.4M and 1.2M. Half of the solutions were put in fridge to cool down the solutions and others were left to stay in room temperature over night. Afterwards, for temperatures, 29°C, 37°C and 45°C, solutions were put into water bath to keep temperature constant. Measuring the
The function of an enzyme is determined by its structure, thus the order in which the amino acids are in make up the enzymes specific shape. The precise way that the amino acids are twisted and folded creates a distinctive shape of the enzymes active site. This active site is now open for substrates which are reactant molecules. Once the substrates go into the enzymes active site they bond together and then leave the enzyme, making the enzyme ready for another set of substrates. The function of enzymes is to speed up reactions by lowering the amount of activation energy needed to get the reaction started.
They are proteins that are complexly folded to allow smaller molecules to fit into them; this active site is where substrate molecules bind. Enzymes must collide with one another at a precise position with enough activation energy. The active site must bind to the reacting molecule, or the substrate (1). Enzyme-catalyzed reactions require lower activation energy. The activity of an enzyme is affected by its environmental factors, and any change results in an alteration in the rate of the reaction caused by the enzyme (2).
Respiration is a series of biochemical pathways that take place in order to create the ATP needed for an organism to survive. ATP is created by either oxidation or reduction reactions depending on what type of respiration process is taking place. An oxidation reaction is when the biochemical pathway has to lose electrons, while in reduction reactions gain electrons to create ATP (Notes, 9/30/15). Aerobic respiration is a biochemical pathways that creates ATP through a series of oxidation reactions. In this type of process, the electron acceptor that would be used is NAD+ and the final electron acceptor has to be oxygen.
Throughout the urea cycle, the amino acid, arginine, is changes into ornithine- this is another amino acid when hydrated, that is when water was added. During this reaction, urea is the product formed (Nelson and Cox 2008). Figure 1 shows the urea cycle, occurs specifically in the mitochondria and cytosol in the liver. (Nelson and M.Cox 2008). Urea is made in the liver by means of enzymes in the urea cycle.
Then, putting of choloform in amount of 10 mL and solution of Hanus iodine as amount of 10 mL into conical flask is realized. Addition of these two substance into otheraflask also occurs for blank. Next, waiting for these two samples for exactly 30 minutes is realized. Afterthat, solution of potassium iodine in amount of 15 mL and 40 mL water being distilled are added. Titration of last mixture is performed in company with 0.1 M Na2S2O3 until the obtaining of color in yellow.
Additionally, there exists three domains of the enzyme namely C- terminal catalytic domain, an N- terminal regulatory domain and a tetramerization domain. Tetrahydrobiopterin (BH4) acts as a cofactor for the enzyme activity. Hence, the regulatory action by PAH enzyme involves activation by the presence of the amino acid phenylalanine, inhibition by the cofactor Tetrahydrobiopterin (BH4) and activation of the enzyme by phosphorylation. Cyclic adenosine monophosphate (cAMP) – dependent protein kinase helps in the phosphorylation of the amino acid serine that is present on the 16 position of the regulatory domain of the enzyme. This in turn helps in maintaining the activity of the enzyme by reducing the concentration of the phenylalanine
Once the gel hardened, .5X TBE (44.5 mM Tris base, 44.5 mM boric acid, and 1.0 mM EDTA) was added just until the gel was covered with the TBE buffer. Each sample was loaded into the gel as well as 10 μL of DNA size markers (1kb ladder, New England Biolabs) into a separate lane. The gel was allowed to harden at room temperature and then electrophoresed at 100 volts for 75 minutes. Using a UV imager, a photo was taken of the resulting traveled DNA fragments in the gel. Results Table 1.
Many organisms use energy to perform their cellular functions. That energy comes from the energy that is stored in food then converted to adenosine triphosphate or ATP. ATP can be obtained with or without oxygen, aerobic respiration and anaerobic respiration. Aerobic respiration produces carbon dioxide (CO2) as a by-product while anaerobic respiration produces Ethanol (C2H6O) or Lactic acid (C3H6O3). In aerobic respiration the “CO2 produced during cellular respiration can combine with water to produce carbonic acid.” While CO2 is produced, the amount of CO2 produced is different depending on the organisms, in this case crayfish.
32 100 μL of afore-prepared sample solution and the mixed reference standard were diluted 100 times with ethyl acetate. 50 μL of these dilution solutions were separated on the TLC plate coated with SNISG. The plate was developed with petroleum ether: ethyl acetate (4:1) and the movement of solvent was usually controlled at 1 cm from the upper edge. After completion, the plate was dried until no solvent smell remained. It was sprayed with an ethanol solution containing 10% sulfuric acid, and heated at an infra-red drier until obvious color came up, as shown in Fig.2 (B.ab).