Dehydrogenase Lab Report

Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect

J. Moldovan & B. Nilson, (2010), Lab 4 – Enzyme Kinetics, UBCO BIOL/BIOC 393, UBC Vista accessed Monday, November 8th, 2010.

From the stock substrate solution of 2.5 mM, each group serially diluted at least one different substrate concentration for a total of four different substrate concentrations to be investigated: 1.25 mM, 1.0 mM, 0.75 mM, 0.25 mM. The enzyme concentration was kept constant at 2.0 mM while experimenting on the affect of varying enzyme concentration on the rate and product formation of ONP. Enough 2.0 mM enzyme solution was prepared in the previous part of the project to supply this assay. Using similar procedure to collect absorbance data as the first part, 0.5 mL of 2.0 mM enzyme concentration was placed into the cuvette and used to calibrate the spectrometer at 420 nm. Data was then started, with the immediate addition of 0.5 mL of varying substrate concentrations. Each varying substrate concentration was split between the team and run for a total of 10 minutes, with the exception of the 1.25 mM run. Upon completion, data from each varying substrate concentration was copied to a single Excel sheet and used to produce an absorbance vs. time graph, product formation vs. time graph, Michaelis Menten plot, and Lineweaver-Birk plot. This analysis was used to calculate the V0,Vmax, and Km for β-Galactosidase

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.

This experiment is to study and measure the enzyme activity of β-galactosidase in the different concentrations of o-Nitrophenylgalactoside (ONPG) using a spectrophotometer. The spectrophotometer was also set at 420nm, a wavelength which is best for recording the absorbance values for the experiment. From the results, 0.9mM ONPG solution has the highest absorbance and 0.1mM ONPG solution has the least. Also, 0.5mM ONPG solution has the highest rate of enzyme activity and it is the most efficient as the enzyme activity of the ONPG solution continues even though the other concentrations of ONPG solution has already stopped the enzymatic reactions as the substrate is already used up.

The Measure of Enzyme Lab #1 Matthew Red Ashley Kaylan Dr. DaCosta 9/13/2015 II Introduction: An Enzyme is a substance produced by a living organism that acts as a catalyst to bring about a specific biochemical reaction. In simpler terms this is a certain substance that is procured from another living organism that is used by others to create a reaction out of another substance. Tyrosinase or TYR is what is called a oxidase or enzyme that catalyzes an oxidation-reduction with special interest with reactions containing oxygen on a molecular level all of this is for the controlling the making and production of melanin.

Used as a catalyst, Enzymes are a means of lowering the activation energy to speed up biochemical reactions, which make the organism more efficient, do to the use of less energy to get work done in the cell. With different enzymes such as lactase, catalase and amylase which each have different usages. The one used in this experiment is Amylase, which catabolizes starch molecules into sugar molecules. If this enzyme, or any enzyme, is not placed in their appropriate optimal temperature, then it will not work as efficiently or not at all. The iodine was important in the experiment conducted because the use of iodine was to determine the possible optimal temperature of the two amylases, the bacterial and fungal amylase.

Abstract What results would acquire when given certain changes to temperature; substrate concentration, enzyme concentration, and inhibitor existence are made considering the kinetics of a specific enzyme? While examining the results these variables significant to the enzyme kinetics will provide a understanding of the general enzyme activity. Observing the end products of each chemical reaction that included an enzyme produced a velocity of that certain enzyme involved, which is measured. These results in this experiment will show temperature change, substrate concentration change, enzyme concentration change, and existence of an inhibitor produce a positive or negative effect on the enzymes activity. Abbreviations [S]: Substrate concentration

The experiment was carried out to investigate the effects of the increase in the enzyme concentration on the rate of reaction. By using self investigative and experimental skills, the experiment was done in order to determine how the rate of reaction will be altered, whether it will increase, decrease or remain constant when the different concentration of enzymes added.

To begin the lab, part A was performed to determine the amount of enzyme that would produce a reaction rate that did not proceed too slow or too rapidly. As seen on page 13 of the Lab Handout, varying amounts of tyrosinase and phosphate buffer were added to a cuvette while the amount of the L-DOPA was constant. After all reagents were added to the cuvette, the cuvette was inserted into spectrophotometer and absorbance of product formation at 475 nm was recorded for two minutes at fifteen seconds interval. After absorbance of product formation was measured and recorded for each cuvette, as shown on page 14 of the Lab Handout, graphs of rate of production formation versus time, were made with the data of each cuvettes and the

Factors include temperature, pH, inhibitors and activators-all of which will be tested and observed for in this lab. The rate of enzyme-catalyzed is affected by concentrations of both substrate and enzyme. Increasing the temperature on a reaction increases its molecular movement. The rate of an enzyme-catalyzed reaction increases with temperatures but only up until the point of optimum temperature-the highest point before the eventual decline. Below the optimum temperature, the hydrogen bonds and hydrophobic interactions that make the enzyme its given shape can no longer be supported by its flexibility.

INTRODUCTION: Lactate dehydrogenase (LDH) is a tetrameric molecule categorised into several types, which are known as isozymes or isoenzymes. LDH activity is commonplace in all tissue and can be seen in higher quantities in the blood when tissue damage is present due to enzyme leakage, typically seen in myocardial infraction or liver disease (1, 2). LDH consists of either a heart (H) or a muscle (M) type subunit and can be made up collectively where five different proteins may be produced H4, M4, M2H2, M3H and MH3. Migration rates differ with H4 having the highest and M4 the lowest rate toward the anode (1).

Test tube four contained distilled water with the enzyme and catechol solution. After ten minutes the spectrophotometer read .313 nanometers and after twenty minutes the machine read .598 nanometers. The color was an orange-yellow after ten minutes and changed to a dark orange after twenty

Rat liver cells can be separated and analysed by Subcellular fractionation using homogenisation and then centrifugation to separate the cells into the subcellular fractions. When the fractions are separated succinate dehydrogenase is used to identify mitochondria. Succinate dehydrogenase is an enzyme that catalyses the oxidation of succinate to fumarate in the Krebs cycle and is usually on the mitochondria inner membrane and not found in other organelles in the cell(Rustin, Munnich, and Rötig, 2002). Care needs to be taken when separating the subcellular fractions as organelles can be damaged and can cause lysis giving incorrect results. Regulation of conditions like the temperature and the pH are important as incorrect conditions could cause the enzymes to denature. Also adding an isotonic buffer not water will help to avoid damage to cell organelles due to osmotic an imbalance (Greenawalt and Vasington, 1968). When it comes to measuring the SDH it is measured indirectly because the succinate dehydrogenase and the fumerate involved in the reaction are both colourless so it is impossible to measure any changes using a spectrophotometer. Instead the absorbance of formazan is measured at 490nm. The formazan is produced by reacting the FADH2 from the SDH reaction with INT which a tetrazolium salt. The formazan produced is red in colour and when dissolved into the ethyl acetate can be extracted and then read on the spectrophotometer (Smith and McFeters, 1997). As

We could go even depeer in the explanation of metabolism reaction but this is just an