Enzyme assay analysis of succinate dehydrogenase to resolve Km and Vmax values and to determine the affects of different variables on the oxidation of succinate to fumerate
Introduction
Enzyme kinetics studies chemical reactions where enzymes are involved; rate of reaction and other factors that affect it such as increasing substrate concentration, enzyme concentration, presence of an inhibitor, deviation of temperature and pH. This will highly aid the information about mechanism of an enzyme and its effect on the substrate, subsequently, acquiring a deeper understanding of the system in which enzyme acts upon on; e.g. how metabolism is controlled.
The mitochondria contain enzymes that are involved in cellular respiration where amino acids,
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This was done quantitatively by monitoring the reduction of an artificial electron acceptor, DCIP. As DCIP is initially coloured blue, the reduction gradually turns this to transparent colourless as the reaction continues. Absorption values were recorded at set intervals and used to construct a substrate concentration- velocity graph. This is used to find the Vmax which is the maximum velocity in which the enzymes present can work. The Michaelis constant, Km is the substrate concentration at 1/2Vmax is also calculated from this. Using these value along with the Michaelis Menten model, will aid the understanding of the mechanism by providing a measure of affinity of succinate dehydrogenase (SDH) with its substrate, succinate and subsequently, the efficiency of the reaction.
Results
δAbs at 5 min δAbs at 10 min δAbs at 15 min δAbs at 20 min δAbs at 25 min δAbs at 30 min
Tube A 0.042 0.323 0.592 0.717 0.830 0.892
Tube B 0.114 0.917 0.949 0.955 0.956 0.957
Tube C 0.093 0.543 0.625 0.919 0.947 0.952
Tube D 0.169 0.234 0.410 0.416 0.438 0.445
Tube E 0.044 0.057 0.106 0.115 0.201 0.227
Tube F 0.039 0.044 0.129 0.133 0.134 0.133
Tube G 0.045 0.046 0.064 0.105 0.134 0.134
Table 1 shows that the difference in absorption increases as time progresses.
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Tube B has the highest increase in absorbance due to the great amount of mitochondrion present whereas tube A and C has less. This would mean that there are more succinate-SDH complexes; in return, more succinate being oxidized to fumerate, thus the reduction of E-FAD to E-FADH2 (DCIP) is greater in tube 2. Malonate is a compound which acts as a competitive inhibitor, it’s chemical structure is similar to succinate, allowing it to bind to the active site of SDH, its presence is seen in tube 4; this restricts the formation of enzyme-substrate complexes being made and consequently, less reaction of succinate to fumerate resulting a significantly less increase of the absorbance recorded over time compared to tubes A,B and
However, at 3% substrate concentration, the hydrogen peroxide decomposition showed an immediate peak of up to 3.8 mm in height. As the substrate concentration slowly increased, enzyme
Overall, as the concentration of the substrate increases, the enzyme activity increases up to a 70% of solution, where the enzyme activity starts to level off. The curve is polynomial because of the fact that the enzyme activity exponentially increases as the concentration of substrate increase; additional evidence for this is the fact that the gradient graph is constantly changing. The polynomial curve is shown because until 70% (the saturation point); this is because there are more casein substrate molecules that can successfully collide with the renin enzyme molecule, therefore increasing the rate of reaction.
Catecholase is an enzyme formed by catechol and oxygen used to interlock oxygen at relative settings, and it is present in plants and crustaceans (Sanyal et. al, 2014). For example, in most fruits and vegetables, the bruised or exposed area of the pant becomes brown due to the reaction of catechol becoming oxidized and oxygen becoming reduced by gaining hydrogen to form water, which then creates a chain that is is the structural backbone of dark melanoid pigments (Helms et al., 1998). However, not all fruits and plants darken at the same rate. This leads to question the enzymatic strength of catecholase and how nearby surroundings affect its activity. The catecholase enzyme has an optimal temperature of approximately 40°C (Helms et al., 1998). Anything above that level would denature the tertiary or primary structure of the protein and cause it to be inoperable. At low temperatures, enzymes have a slower catalyzing rate. Enzymes also function under optimal pH level or else they will also denature, so an average quantity of ions, not too high or low, present within a solution could determine the efficiency of an enzyme (Helms et al., 1998). Also, if more enzymes were added to the concentration, the solution would have a more active sites available for substrates and allow the reaction rate to increase if excess substrate is present (Helms et al., 1998). However, if more
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: = == ==
Investigating the Effect of Substrate Concentration on Catalase Reaction. Planning -Aim : The aim of the experiment is to examine how the concentration of the substrate (Hydrogen Peroxide, H2O2) affects the rate of reaction. the enzyme (catalase).
Enzymes have the ability to act on a small group of chemically similar substances. Enzymes are very specific, in the sense that each enzyme is limited to interact with only one set of reactants; the reactants are referred to as substrates. Substrates of an enzyme are the chemicals altered by enzyme-catalysed reactions. The extreme specific nature of enzymes are because of the complicated three-dimensional shape, which is due to the particular way the amino acid chain of proteins folds.
The enzymes have active sites on their surfaces to allow the binding of a substrate through the help of coenzymes to form enzyme-substrate complex. The chemical reaction thus converts the substrate to a new product then released and the catalytic cycle proceeds.
Chemical kinetics is a branch of chemistry that involves reaction rates and the steps that follow in. It tells you how fast a reaction can happen and the steps it takes to make complete the reaction (2). An application of chemical kinetics in everyday life is the mechanics of popcorn. The rate it pops depends on how much water is in a kernel. The more water it has the quicker the steam heats up and causes a reaction- the popping of the kernel (3). Catalysts, temperature, and concentration can cause variations in kinetics (4).
In this lab, it was determined how the rate of an enzyme-catalyzed reaction is affected by physical factors such as enzyme concentration, temperature, and substrate concentration affect. The question of what factors influence enzyme activity can be answered by the results of peroxidase activity and its relation to temperature and whether or not hydroxylamine causes a reaction change with enzyme activity. An enzyme is a protein produced by a living organism that serves as a biological catalyst. A catalyst is a substance that speeds up the rate of a chemical reaction and does so by lowering the activation energy of a reaction. With that energy reactants are brought together so that products can be formed.
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.
Purpose: This lab gives the idea about the enzyme. We will do two different experiments. Enzyme is a protein that made of strings of amino acids and it is helping to produce chemical reactions in the quickest way. In the first experiment, we are testing water, sucrose solution, salt solution, and hydrogen peroxide to see which can increase the bubbles. So we can understand that enzyme producing chemical reactions in the speed. In the second experiment, we are using temperature of room, boiling water, refrigerator, and freezer to see what will effect the enzyme.
Enzymes are types of proteins that work as a substance to help speed up a chemical reaction (Madar & Windelspecht, 104). There are three factors that help enzyme activity increase in speed. The three factors that speed up the activity of enzymes are concentration, an increase in temperature, and a preferred pH environment. Whether or not the reaction continues to move forward is not up to the enzyme, instead the reaction is dependent on a reaction’s free energy. These enzymatic reactions have reactants referred to as substrates. Enzymes do much more than create substrates; enzymes actually work with the substrate in a reaction (Madar &Windelspecht, 106). For reactions in a cell it is important that a specific enzyme is present during the process. For example, lactase must be able to collaborate with lactose in order to break it down (Madar & Windelspecht, 105).
Chemical kinetics is the study and examination of chemical reactions regarding re-arrangement of atoms, reaction rates, effect of various variables, and more. Chemical reaction rates, are the rates of change in amounts or concentrations of either products or reactants. Concentration of solutions, surface area, catalysts, temperature and the nature of reactants are all factors that can influence a rate of reaction. Increasing the concentration of a solution allows the rate of reaction to increase because highly concentrated solutions have more molecules and as a result the molecules collide faster. Surface area also affects a
One vital process in the human body observed in chemistry is the idea of chemical kinetics. Chemical kinetics is the study of the rate of reactions, or how fast reactions occur.1 Three factors that affect chemical kinetics are concentration, temperature, and catalysis. As the concentration of a substance increases, the rate of the reaction also increases.1 This relationship is valid because when more of a substance is added in a reaction, it increases the likelihood that the
In this experiment, researchers used different measurements of catechol and 1cm of potato extract. Researchers hypothesized that the increase in substrate would level out the enzyme activity by