Lactate Dehydrogenase Synthesis Lab Report

Succinate dehydrogenase is an enzyme found in the mitochondrial inner membrane. The enzyme catalyzes the reaction of oxidizing its substrate, succinate, into fumarate via the removal of hydrogen ions from succinate. This oxidation is vital in the Krebs cycle.

The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by

Understanding the activity of enzymes in different muscle types can aid greatly in obtaining more information about other processes such as metabolism of the tissues (Anderson et al., 2012). There are many different methods in order to achieve this information based in two different major categories but the most convenient method is one called continuous assay. This process includes the use of a spectrophotometer to continuously monitor the assay. This method allows for an easy way to calculate the initial rate of reaction as one can establish time points easily. In the lab performed the continuous method was used in order to determine the measurement of the activity enzyme succinate dehydrogenase (SDH). SDH is most commonly found in mitochondria. It is present in many different muscle tissues including the heart, red, and white tissue. In the following lab the enzyme was tested and measured in these three muscle types in the Oncorhynchus mykiss in order to determine which type contained the highest and the lowest activity. This enzyme is involved in multiple processes such as involved in both the Krebs cycle and the electron transport chain (ETC) (Smith, 2014). Its role in

Lactic acid fermentation: Plant and fungal cells produce alcohol as a result of fermentation and animal cells produce lactic acid

One of the most significant reactions in Glycolysis is reaction one which involves the phosphorylation of glucose to form glucose-6-phosphate. Through the transfer of the hydrolysis of ATP, this supplies energy for the reaction and makes it essentially irreversible, having a negative free energy change, which allows for a spontaneous reaction in cells. Although the preparatory phase is energy consuming and uses up 2 ATP, the pay off phase synthesizes 4 molecules of ATP, with the transfer of 4e- via 2 hydride ions to 2 molecules of NAD+. Therefore, a net gain of 2 ATP is achieved through the glycolytic pathway alone. Following the glycolytic pathway, due to the absence of oxygen, as oxygen cannot be supplied fast enough to undergo aerobic respiration, the athlete will instead, undergo lactic acid fermentation. Lactic acid fermentation involves pyruvate that is formed from the glycolytic pathway to be reduced to lactate, with the aid of the enzyme, lactate dehydrogenase, while the coenzyme Nicotinamide Adenine Dinucleotide (NADH) is oxidised to NAD+. The product NAD+ then re-enters the glycolytic pathway in order to produce 2 ATP. This process of lactic acid fermentation produces 2 ATP for each cycle, and thus, rapidly supplies the body with a small amount of energy. However, with the buildup of lactic acid in the body, the athlete will eventually encounter the feeling of discomfort as this accumulation of lactate causes the body to

The independent variable in this investigation is pH. Each individual enzyme has it’s own pH characteristic. This is because the hydrogen and ionic bonds between –NH2 and –COOH groups of the polypeptides that make up the enzyme, fix the exact arrangement of the active site of an enzyme. It is crucial to be aware of how even small changes in the

enable the substrate to bind to the enzyme and form the enzyme substrate complex and

Enzymes are proteins that act as a catalyst in bringing about specific biochemical reactions when met with particular substrates. Substrates will merge into a suitable area of the enzyme called an active site – this becomes the enzyme/substrate complex. Once the substrate is attached to the active site, the substrate will undergo a procedure where the substrate is modified and released as a product. There are different types of this that can occur, where either a chemical bond is broken in a substrate to produce two separate products; as in the ‘Induced-Fit’ model illustrated in figure 1.a. Chemical bonds can also be built between two substrates to produce a single product.

Introduction Enzymes serve important roles in the biological processes that are undertaken in human bodies. Enzymes are most important as their role as proteins which speed up chemical reactions in the body, which make them catalytic proteins (Agarwal, 2006). The unique structure of enzymes allows them to fit into a specific type of chemical, called a substrate (Robinson, 2006). The functionality of an enzyme is determined by its active site. The active site of an enzyme is the location on the enzyme where the substrate binds to produce a substance (Robinson, 2006).

The spectrophotometer as zeroed once 0.1ml of 16.2M ethanol was added. 0.1ml of the enzyme stock solution was added and the absorbance at 340nm was measured for two minutes. OD/min is then calculated from the graphs of the spectrophotometer. Concentration of the substrate was then calculated (Appendix 1) and Enzyme velocity was calculated (Appendix 1). The same procedure was repeated for 1.2ml of the buffer, 1.5ml of NAD+, 0.1 ml of ethanol and 0.2ml of enzyme solution was added and the velocity of the enzymes was then measured again.

“Enzymes are proteins that have catalytic functions” [1], “that speed up or slow down reactions”[2], “indispensable to maintenance and activity of life”[1]. They are each very specific, and will only work when a particular substrate fits in their active site. An active site is “a region on the surface of an enzyme where the substrate binds, and where the reaction occurs”[2].

Without the presence of fermentation, anaerobic pathways can no longer continue to process. NAD+ is used as an electron acceptor and indirectly caused the hydrogens to cross over from molecules to molecules. Without fermentation, ATP and pyruvate molecules cannot be created by anaerobic systems. 6. Why do mammalian muscle cells perform lactic acid fermentation (instead of, say, ethanol fermentation)?

Optimum pH determination Enzymatic activity is effected by different environmental conditions; these environmental factors have a direct impact on whether the enzyme will catalyse the reaction tested or if the environment does not permit enzyme activity. One environmental factor that effects enzymatic activity is pH. All enzymes have an optimal environment where the enzyme is most efficient, this environment can even contribute to the function of the enzyme. For LDH the optimal pH would be a pH that results in a rapid conversion of pyruvate and NADH to lactate and NAD+. Based on the experiment the optimal pH of LDH shown in figure 2 is 10.5. This suggests that LDH for conversion of pyruvate to lactate works best in basic/alkaline conditions.

The purpose of this lab is to examine the specificity of the lactase enzyme to a specific substrate and how it can denature due to the rise in temperature.

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