BioS32 Lab 3

BioS32 Lab 3 - Analysis of Experimental Factors affecting...

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Analysis of Experimental Factors affecting Enzyme Kinetics and Production of β–galactosidase BioS 32 February 6 th , 2004 INTRODUCTION:
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Enzymes are regarded as the powerhouse of the cell. They are responsible for the perpetuation of almost all functions of cellular life. They act as catalysts to speed up reactions that may not occur under normal circumstances, sometimes “by several orders of magnitude” (Source 1). They help cells function in cellular conditions (concentrations, temperature, p H, and pressure) that would normally prevent the reaction from occurring at all (3). Our experiment studied the effect that various conditions would have on the subsequent production of the β–galactosidase enzyme. In our experiment we were supplied with a culture of Escherichia coli which acted as the source of the β–galactosidase enzyme. The β–galactosidase breaks down and “cleaves the sugar lactose to form glucose and galactose” (3) (Figure 1). CLEAVAGE OF LACTOSE BY β–galactosidase (2) We added o-nitrophenyl-β-D-galactoside (ONPG) to our test tubes. The ONPG is a colorless substance, but when the β–galactosidase enzyme is present the ONPG breaks down into o-nitrophenol and galactose. This degradation of ONPG is the key to the success of our experiment. The o- nitrophenol assumes a yellow hue and the absorbance, and thus concentration,
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can be measured by the use of a spectrophotometer. The absorbance reading directly correlates to the concentration and amount of β–galactosidase being produced. To better understand the qualitative limitations of the efficacy of β– galactosidase, we put the enzyme through a series of test. After synthesizing a solution containing the enzyme, and other substances (detailed later) we performed the following tests: Experiment A. Effect of Enzyme Concentration Experiment B. Effect of Temperature and Induction Experiment C. Effect of an uninduced sample Experiment D. Effect of 2 concentrations of the competitor Experiment E. Effect of changing pH of the buffer For the tests we will be able to determine the concentration of β– galactosidase through the use of the spectrophotometer (all measurements taken at 420 nm). To stop the process of the reaction we added NaOH (pH of 11) to denature the protein. HYPOTHESIS:
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BioS32 Lab 3 - Analysis of Experimental Factors affecting...

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