Bio5LAManual12f

The time sec slope a t of this portion of the trace is

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Unformatted text preview: s (of product) / unit time. There are many ways to calculate v0. The approach we will use is Absorbance vs. Time for Rxn. X based on the following: Begin by plotting absorbance vs. time for each trial in a manner similar to that shown at right. (Note: when analyzing your data, you should plot all of the traces for a given experiment on the same graph.) @ 410 nm 2 1.5 Z 1 Once this is done, you should see that each trace 0.5 Y exhibits a relatively long segment that falls in a straight line (or very close to straight). For the graph above, this 0 0 50 100 150 200 250 300 linear segment falls between points “Y” and “Z”. The time (sec.) slope ( A/ t) of this portion of the trace is related to (but not equal to) the v0 for Rxn. X. The reason that this slope does not represent the initial velocity of the reaction is that A/ t is a meaningless expression since absorbance is a unitless value. A step in the right direction is to use Beer’s law to convert the absorbances to concentrations of product. This conversion gives the change in concentration of product made vs. the change in time. However, this value does not tell us much more than A/ t. So, it is necessary to change the concentration values to moles by multiplying the concentrations by the reaction volume used in the experiments: moles/liter (concentration) x .005 liter (reaction volume) = moles of product. Once this is done, you end up with moles/ t which is an expression of initial velocity. QUESTION: If the product of Rxn. X is p - nitrophenol (E410 = 1.83 x 104 liters per mole x centimeter), what is the v0 of Rxn. X as plotted above? (ANSWER: 2.9 x 10-9 moles / sec.) Biology 05LA – Fall Quarter 2012 Lab 5 – page 4 Learning Goals/Intended Outcomes 1. Be able to define the following terms: enzyme, substrate, product, enzyme-substrate complex, and enzyme-product complex. 2. Be able to use the above terms to describe the catalytic cycle of an enzyme. 3. Be able to define the term chromogenic substrate. 4. Be able to explain the experimental strategy for our investigations of the activity of bovine alkaline phosphatase. Your response should include: a. an explanation of why a chromogenic substrate and a spectrophotometer were used. b. a definition of the term initial velocity (v0). c. an annotated outline of the procedure used for converting the measured rates of color change in the reaction tubes to the v0’s for these reactions. 5. Be able to explain the necessity of the “0.0 M substrate” trial in the [substrate] experiment. 6. Be able to present and explain the supporting facts that you used to support your hypotheses for the two studies that you performed. 7. Be able to interpret the data presented in the following graphs by filling in the blanks in the table below with the number of the plot that is the best match for the listed description / explanation of the data. You must be able to explain your choice. 2 5. 1 1 Time Time 2 6. Abs. Abs. Abs. 2 3. 1 Time Time 4. 2 Abs. 1 1 2 2. Abs. 1. Abs. 2 1 Time Description / Explanation The plot that depicts the slowest reaction rate The plot that depicts data for which a v0 cannot be validly calculated. The plot that shows the result of an experiment where the enzyme was denatured. The plot that depicts the fastest reaction rate. The plot that shows the result of an experiment where the substrate was contaminated with enzyme prior to the start of the experiment. The plot that shows the result of an experiment where the substrate was completely converted to product during the experiment. Time Plot # Biology 05LA – Fall Qtr. 2012 Lab 6 – page 1 LAB #6: FERMENTATION AND RESPIRATION All living organisms must continuously expend energy to maintain themselves and drive energy-requiring life processes. This energy is ultimately derived from the sun via photosynthesis. Because not all organisms are photosynthetic and the sun does not always shine, organisms need alternative sources of energy. Plants provide this energy in the form of organic molecules that contain large amounts of chemical potential energy. These molecules (e.g. sugars) are relatively stable and can be stored or transported. This energy is extracted by a stepwise, enzymatically-mediated oxidation of these molecules that results in the production of another energy-storing molecule, adenosine triphosphate (ATP). When the terminal phosphate of ATP is split off, the ATP is converted to ADP (adenosine diphosphate) and considerable energy is made available for “cellular work”. The ADP is then available for the regeneration of more ATP. Shown below are balanced summary equations for two classes of metabolism that provide most of the ATP that cells use. C6H12O6 (glucose) + 2 ADP + 2 Pi 2 CH3CH2OH (ethanol) + 2 CO2+ 2 ATP + heat Alcoholic Fermentation C6H12O6 + 6 O2 + 32 ADP + 32 Pi 6 CO2+ 6 H2O + 32 ATP + heat Cellular Respiration The experiments that you will perform in this exercise are made possible by two features related to the above equations. The first is that gaseous reactants and/or products are characteristic of both metabolic processes. The second is that it is relatively easy to determine number of moles of a g...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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