expt3 - BC 367 Experiment 3 Purification and...

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BC 367 Experiment 3 Purification and Characterization of the Enzyme Lysozyme Introduction Enzymes are truly remarkable catalysts. For example, catalase can carry out the decomposition of up to 5x10 6 moles of H 2 O 2 per minute per mole of enzyme (the turnover number). Even though the turnover numbers of most other enzymes are substantially lower than this, most of the reactions they catalyze occur at rates at least a million times faster under physiological conditions in the presence of the enzyme than in its absence. In this experiment you will purify an enzyme, using its known activity to monitor the process. The isolation and purification of a specific protein or enzyme is generally a difficult task. First, the enzyme must be liberated from its source tissue in an active form. Fortunately, a wide variety of tissue disruption techniques have been developed. However, the enzyme to be purified is usually only a small percentage of the total protein in the crude extract of the tissue. The object of protein purification is to remove nonprotein contaminants as well as to isolate the protein in question from other proteins. The first objective is relatively easy to obtain, whereas the latter is more difficult. For example, it is not unusual for an enzyme to be 0.1% of the total protein in a crude tissue extract. To purify this enzyme to homogeneity, 99.9% of the protein must be removed, preferably with as little loss as possible of the desired enzymatic activity. This can be a difficult task for two reasons: 1. Enzymes are relatively labile molecules. Most enzymes are denatured by foaming, by heating, by organic solvents (particularly at room temperature), by drying at room temperature, and by concentrated acids or bases. Furthermore, proteins in aqueous solutions are excellent nutrient systems for microorganisms, and therefore, cleanliness of equipment and avoidance of unnecessary contamination are vital for successful purification attempts. 2. Differences between some of the various protein molecules in the tissue extract may be subtle. A single purification step is seldom adequate to purify an enzyme completely. Typically, several different procedures that exploit different properties of proteins must be used. Generally, either salt precipitation, organic solvent precipitation, or isoelectric precipitation is used at an early point in the procedure. Chromatographic procedures, such as ion-exchange, gel-filtration, or adsorption chromatography, are employed after the enzyme has been partially purified by one of the precipitation techniques. Unfortunately, the establishment of an enzyme purification procedure must be done largely by trial and error. Determination of the progress of a purification process is important. Specific activity and total activity are the critical parameters in enzyme purification. The activity of an enzyme is defined in some unit, usually micromoles of product formed per minute. The specific activity, then, is defined as activity units per milligram of total protein. As the enzyme is purified, the
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This note was uploaded on 02/09/2010 for the course CH CH242 taught by Professor Katz during the Spring '10 term at Colby.

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expt3 - BC 367 Experiment 3 Purification and...

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