Extraction and Characterization of Proteins

Temperature is a measure of kinetic energy. As this movement increases, collision rate and intensity, and therefore reaction rates, increase. This experiment was conducted to determine if there is a minimum temperature that increase kinetic energy and denature enzymes to slow enzymatic reactions or fail to catalyze them. The experimental results indicate an increase in temperature will increase reaction rates until proteins denature.

Thiol groups are important to protein folding and forming disulphide bonds that are essential to protein structure. Determining the number of thiol groups in a protein is important in determining the tertiary structure of the protein. The ovalbumin is the experiment was purified from egg white using centrifugation and ammonium sulphate precipitation and then the thiol groups identified using DTNB and spectroscopy. The ovalbumin was found to have one thiol group; from this we were also to infer that DNTB alkylates thiolgroups; whereas SDS keeps proteins denatured.

As stated in the introduction, three conditions that may affect enzyme activity are salinity, temperature, and pH. In experiment two, we explored how temperature can affect enzymatic activity. Since most enzymes function best at their optimum temperature or room temperature, it was expected that the best reaction is in this environment. The higher the temperature that faster the reaction unless the enzyme is denatured because it is too hot. Similarly, pH and salinity can affect enzyme activity.

is loss of its structure. This occurs when the ionics and hydrogens bonds of the protein

pH along with temperature and concentration is a limiting factor of enzyme activity. Enzymes, which are proteins, have a distinct pH range in which they work most effectively. Beyond or below this specific pH, enzyme activity begins to diminish and eventually the enzyme is rendered useless. The optimum pH of an enzyme is the pH at which it is most effective and causes the fastest rate of reaction(source). This decrease in enzyme activity beyond the optimum pH is attributed to the change in shape of the active site(explained earlier) of the enzyme. This change in shape can be brought upon when the surrounding pH moves away from the optimum pH, and it can prevent the enzyme from binding with the substrate.

Protein standards were previously loaded into the first rows of the well plate of which the concentrations were as followed; 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, and 0 μg/mL (PBS only). After making note of the rows used the plates were loaded onto the selected protein row, followed by the addition of 200 μL of Bradford reagent to each well using a multichannel pipette. The absorbance of the plate was read at 595nm and recorded. The NT-2 and LPS-2 protein sample tubes were labeled for easy identification in the following lab and the protein samples were stored in the freezer for 1 week.

Load the remaining protein mix into the column, and this will begin to bind within the column. Once the solution has settled, fill the column with NaCl a total of four times; as the wash is flowing through, collect and label the protein solution in fractions, and save for the

A protein’s tertiary structure, the compact, biologically active and most stable form of the protein, results from further folding of the amino acid chain. The environment in which a protein is synthesized and allowed to folded is a significant determinant of its final shape. If the tertiary structure of a protein is disrupted, the protein is said to be denatured, and it loses its activity. Based on their tertiary structure, proteins can be classified as globular, fibrous and membrane proteins. Globular proteins participate in sophisticated processes such as enzyme-mediated catalysis, transport of molecules, signal transduction, defense and regulation.

Separation of the proteins involved in both species blood and serum revealed that each was composed of a major protein. A prominent protein in the rabbit blood was found accumulating at approximately 13kDa, as shown in Figure 1 and Figure 2. It was concluded that this protein was most likely hemoglobin, given the weight and large amount present in all blood due to its necessary function of transporting oxygen and carbon dioxide throughout the body. Hemoglobin is a “tetrameric protein of about 64.5 kDa, consisting of two -chains and two -chains” which breaks down into each subunit having an approximate weight of 15kDa (Yu et al, 1997). This was in correlation with the data present in Figure 1, given that the major protein present in rabbit blood was approximately 13kDa, which would be the subunits that make up hemoglobin.

** The Low vs. High column shows how each protein band was influenced by the salt. If band was present in both conditions, it was noted what condition had and increase or if they were equal.

, n.d) If the salt concentration of a molecule (Metal salt) is close to zero, the charged side chains of amino acids of the enzyme (catalase) will attract each other and interact with each other. The enzyme will eventually denature and form a precipitate that is currently inactive. If the salt concentration is too high, the interaction of charged groups will be stopped and interfered with. New interactions will eventually occur, and again the enzyme will produce a precipitate (Deakin University,

To find out how modifying the variables of concentration, temperature, and pH effects enzyme activity.

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

The transport activity is expressed as nmoles of substrate transported during the incubation time per milligram of the reconstituted protein and it is calculated with the following formula:

The topics of these experiments are both important processes known as identification and quantification. They were also used to review and introduce new lab methods such as titration and the Bradford assay. The identification was used to determine the identity of unknown amino acid #11 and the quantification was used to determine the concentration of unknown protein #11. The identity of amino acid #11 was found to be lysine, and the concentration of protein #11 was calculated to be 0.51 ± 0.019 ug/uL with a 3.67% error, reflecting accuracy in the results.