Instructions for Lab Report #2

This will be a less formal lab report than #1. Please follow the directions carefully for maximum credit. The data are linked HERE and also reproduced at the end of these instructions. See below those data for an explanation.

Please use a cover sheet with the report name and your name. The report must be corner stapled. Do not use report folders, plastic covers, spiral bindings, or anything other than a simple piece of paper stapled to the rest of the report.

Introduction:  You need to write an explanation of the exercise. First, talk briefly about the enzyme we used, the substrate we used, the product of the reaction, and how we detected and quantified the product of the reaction. The morning class varied the concentration of the substrate to show what effect that has on the velocity of an enzymatic reaction.  The afternoon class did a standard curve which relates absorbance to product concentration and also showed the effect of different conditions on enzyme velocity. You will need to write something about all of this.

Questions and Graphs: The remainder of the report is graph intensive, and if you have problems using either version of Excel (2003 or 2007) I would strongly suggest you ask for help. I will try to spell out what types of activities I am expecting you to do with a spreadsheet.  Make sure you use the xy scatter choice on the graph menu for all graphs unless specifically told to do otherwise.

1. The enzyme converted substrate to product, and the product absorbed light. Thus, data were collected with a spectrophotometer. These data need to be converted to an actual amount of product.  You are provided with a table showing the absorbance readings and the number of nmoles of product. Graph these to create a standard curve. Since you used the 0 nmole sample to electronically set the absorbance to 0, the (0, 0) point is fixed, and you should check the box in the trendline menu that does that. Your graph needs a title and proper axis labels. It DOESN'T need a legend (e.g. "Series 1") because there is only one thing being graphed. Be sure you include all the information about the graph (including wavelength used, name of product) either in the axis labels or as a separate Figure legend. You will also want to generate the equation of the line (from the trendline menu) because you will use this to convert all the data you collected from absorbance to nmoles of product.

2. What is the effect of substrate concentration on the rate of the reaction? First, what is a rate? To visualize what the effect of substrate concentration is on how fast the enzyme works, you will make a graph that shows some of the data. First, in Excel, use the equation of the Standard curve to convert all the absorbance units into nmoles of product. Check your work. Copy and paste the sampling times, the nmoles of product for 1 mM substrate (undiluted) and the nmoles of product for 0.0625  mM substrate (1:16 dilution) into a separate table. Graph these data.  Include title, axis labels, and change "series _" to something appropriate. Have Excel calculate trendlines for both sets of data, and force the lines through the origin (0,0). The point 0,0 is a known, correct, data point. Explain why.  Which trendline fits its data set better? Even though you are not graphing the other substrate dilutions, what do you think the trend would be, that is, would higher or lower substrate concentrations give you better fitting trendlines, and why?

3. This graph is convenient to visualize what is taking place, but to do further calculations, we will need to calculate specific numbers for the rates of the enzyme reaction.   It is critically important that you understand the meaning of "rate", because we will be dealing with the same concept shortly when we discuss bacterial growth. "Rate" is the amount of change over time. The slopes of the lines you just plotted are rates, the change in product produced (Y) versus time (X). A rate always compares a change in amount to a change in time. There is a function in Excel which will calculate rates for you, not surprisingly called "slope". To get the slope of a set of points, type in "=slope(y value range, x value range)" . For example if your times are A1 through A5 and your nmole data are in B1..B5, the formula will look like =slope(b1..b5,a1..a5). To copy and paste this to all your data for different substrate concentrations, make the following change:  =slope(b1..b5, $a1..$a5).  The $ fixes your second term so that it even when you paste the formula elsewhere, it still uses the set of data in your "time" column.
Output: generate a table that lists the different substrate concentrations and the corresponding rates of  reaction of the enzyme. Be sure to indicate the proper units. If you are copying and pasting to somewhere else in Excel, remember that you have to "paste special" and paste as a "value" or you will paste a misplaced formula.

To properly calculate enzyme velocities, it is very important to only look at initial velocities, that is, how fast the enzyme works at the very beginning of the experiment. When we do the "slope" calculation listed above, we are NOT doing that; we are cheating. If you understand initial velocities, I need to be honest with you, and you can ask me for more details. If you don't know what I'm talking about, that's OK, it won't affect the lab report or your grade.

When you were given the substrate concentrations, you were told that the most concentrated one was 1.0 mM and that all the other concentrations were serial 1:2 dilutions. To get all the other concentrations, you just have to keep dividing by 2. However, be VERY CAREFUL that you don't lose significant figures. I recommend that you keep 3 significant figures. To review the rules on significant figures, go to http://www.usca.edu/chemistry/genchem/sigfig.htm.

4. Now that you have a table of substrate concentrations and enzyme rates, graph them to show the effect of substrate concentration on enzymatic rate. Remember that the independent variable goes on the X axis. Properly label the graph and the axes. You may leave it as a scatter plot, or use the menu choice that smoothly connects the points or you may see if a logarithmic trendline fits the data (I didn't try it). The shape of this graph should be very characteristic of an enzyme. Comment in words on what this graph is showing and why. Remember, a graph with just one line on it doesn't need a legend.

5. It is difficult to get a slope from a graph of this shape. Lineweaver and Burk devised a way to graph such data so that valuable information could be obtained. Create such a graph by taking the reciprocal of the enzyme rates and of the substrate concentrations and make a graph of these values. When you create the trendline, note the menu option that says "forecast". This allows you to extend your trendline to the left to see where it crosses the X axis. Guesstimate the distance you need and type a positive number  in the box that says "backward". You may need to format the Y axis so that the minimum value is 0.  Review the enzyme kinetics slide in Lecture 03b or in other notes you have. Explain in your own words what the following terms mean: Vmax and Km. Calculate and report the values of Vmax and Km you determined for this enzyme at this temperature.  Be sure you use the proper units.

6. A number of things affect the functioning of the enzyme.  The afternoon class did experiments to explore two of these. Data are supplied for changes in amount of product over time at room temperature and on ice. Working as you did in #3 above, convert your absorbance data to nmoles, then use the "slope" function in Excel and calculate the rate of the enzyme at room temperature, on ice, and in the presence of the sodium fluoride. Use the data to produce a bar graph, (vertical please) showing the effects of the different "treatments" used in these experiments. The names of the treatments will be your X axis data and the enzyme velocities will be your Y data.

Conclusions: Write a paragraph summing up the experiment, discussing a) the effect of substrate concentration on enzymatic activity, b) the effect of temperature, and c) the effect of sodium fluoride. You are commenting on the data you graphed, so reflect that in what you write. This report needs to be only as big as it needs to be! Strive to make your graphs informative and visually appealing.

Due date: September 29.

 Time data are in minutes and product amounts are in units as indicated; all other data are in "absorbance units" collected at the wavelength described in your handout.