Student Information for the Enzyme lab report

You have two weeks to complete this report.  It is due the week of March 30th at your scheduled lab.  Don’t put off doing the report until the last minute or you will regret it.  The lab is worth 30 points. Review the lab manual for the basic report format.  This information is specific to the enzyme lab. Attach the cover sheet that is posted on Blackboard. The basic format here is similar to the lab manual, but modified for the enzyme lab.

1. Title:  This should include the important information about the experiment that you conducted, and should not be simply (“lab report”, or the title of the lab). What enzyme are you using?

2. Introduction: Be sure to include:  What is an enzyme? State your question to test.  What experiments were conducted? How can you cover this in a sentence or two? Leave the details for discussion. Separate the parts of the introduction, a, b & c as described in the lab manual.

3. Materials and Methods (protocol): see lab manual for directions.  You should include any deviations you made from the instructions in the manual. Include protocol from the graded worksheet you did during the previous lab if you have it or a copy, now with filled in tables. Since the exercise was done as a bench, you need a copy.  You do not need to replicate the protocol in the M & M section, just refer to it – use page numbers and include changes. For the Methods part of this, recap what you did, a short paragraph for each part. How these methods tested your hypothesis is in the discussion part b.

4. Results:  Remember to label all tables and graphs. If you use Excel, you can do it all on one sheet.  See the next part for details on the presentation of data. Note that all the data goes in a single table or tables organized so that the data can be easily prepared. Graphs follow the table(s)

Part B; STD Curve: include a table of the O.D. vs. concentration for the standards.  Plot data, and draw best fit line using Excel. You can then use Excel to help you plot all the data for the subsequent graphs. Note that while Excel can give you a trend line for the linear graphs, you may not want to use it. See lab manual for instructions on finding the initial velocity for enzyme activity. NO Least Squares used here.

Part C; Rate of Reaction:  Include a table of time and [Product – P]. Use the standard curve by equation or hand to convert O.D. in protocol to concentration of product [P]. Plot the [P] vs Time.  Draw the best-fit line using Excel and trend line equation.  Determine enzyme activity and initial velocity (see lab manual for details).  Include this in a table for rates of each part.

Part D; Enzyme Concentration:  Table of time, O.D. and [P] for all 5 enzyme concentrations, use the standard curve to convert O.D. to [P] as in B.  Plot [P] v. time.  Compare the line and equation Excel give you to initial velocity. From your measure of initial velocity, plot enzyme activity v. concentration of enzyme on a separate graph.

Part E; Substrate Concentration:  basically same as for enzyme concentration, part C. time course, velocity v. substrate and Lineweaver – Burke plot:  use substrate data to determine 1/v and 1/[S].  Make a table including these values.  Plot 1/v vs 1/[S].  Use this plot to determine Km and Vmax (See notes).

5. Discussion: Follow the outline in the manual for the usual format. Make it obvious, label each part, See part F of the lab manual for important points to cover.  Be sure to discuss all parts of the experiment. The parts may be considered as a whole in the first section of the discussion, part a.  Only a short summary was in the introduction since you had only a couple of sentences there. However each part is important. In the results section, part c, you will talk about each set of results for each part. However, economize on the numbers as in the pH lab. When can you use an individual number to describe a result or a line?  In the conclusion and analysis section, part d, you should discuss each part, but bring them together as a whole. What is you conclusion about enzyme activity from the data and some analysis of the mechanism of enzyme catalysis to explain it. Parts e (errors) and f (relevance and application) are the same. A Citation is required and must be integrated into the body of the lab report. Indicate where used.   When you are making statements of fact, they must attributed to an author in a reference, could be Campbell, lab or some outside resource wither hard copy or from the web.  Pay attention to how you cite a reference in the text and at the end of the report.

There are many styles of citation. Basically you need the information so that someone else could easily go to the library or web and find the source.  For example a journal might look like the example below. The initial number refers to where in the lab report it was used and that number appears there as well.  A book citation might have an editor as well.

1. Rak, Y. & Arensbure B. Linear measurements dental enamel. J. Physiol. 91, 34- 84 (2004)

A web citation is a bit different. You have to have the URL. It is usually easiest to copy the URL into the text. I is also a good idea to include a copy of the Home page which will have the URL at the top. We have to be able to find the page easily.

For your analysis, and our grading, the bottom line is: can you demonstrate an understanding of enzyme function that goes beyond the fancy pictures in Campbell. Can you demonstrate that you understand catalysis from the quantitative data in the graphs? The lab report, particularly discussion, should demonstrate understanding of the reaction catalyzed by the enzyme. You should be able to write and explain the simple reaction statement: E + S ? [ES] ? to E + P. Most of this would come from lecture, Blackboard or reference for the background in the intro and part a of discussion. Note that Campbell is not really a suitable reference since it is very weak on enzyme kinetics. There must be another reference. From the background you develop, you should be able for formulate a question or hypothesis that the exercise will answer.

Remember the MM formula. You are measuring; appearance of product. This is covered in the lab manual, lecture and Blackboard. What is the difference between the product and the substrate?” What is the material used in part A (std curve) as opposed to what you put into the tubes in B, C, D & E?

You should be able to describe the effect of increasing enzyme and substrate concentration on the rate of the reaction and understand that for the enzyme to reach Vmax, the substrate has to be in much greater concentration that the enzyme.

You should understand what a rate is, v₀ or the intial rate instead of the overall slope of the line. Why use the initial rate? Remember this is a chemical reaction. What is the relationship of enzyme concentration to rate of reaction? Why measure rate?  What is the importance of the units of rate?  How to both Km and Vmax (below) affect rate? What is the relationship between the two measures?  You should understand what Km represents, the affinity or catalytic efficiency, and what Vmax represents, the maximum rate of enzyme catalysis

Each table and plot should be discussed in detail, not every number, but the important trends and outcomes from the data. It should be quantitative though, not just higher or lower. Talking about rate of reaction is a good way to organize results part of discussion.

Controls should be discussed at some point. Why use a control? Look at the questions at the endo fo the lab chapter for help here. Answer them either in your discussion or at the end of the lab report.

From B: How does enzyme activity change with time? What is the difference between activity with and without enzyme?

From C: Does enzyme vary linearly with enzyme concentration (assuming [S] is present in saturating excess)?  What conditions might make for a non-linear graph?

From D: Why does enzyme activity saturate (stop increasing) although [S] continues to increase? What is the Michaelis constant (Km)?

The last section of the lab, F has details of the lab report. Be sure to read it carefully. There are a series of questions in the lab and at the end. These questions should be answered as you write the analysis and conclusion.  You do not have to answer them separately. Incorporate them into the conclusion. It would be a good idea to answer them to focus your thinking and then incorporate the ideas into your report. Look at the questions on buffer concentration and sample repeats for example.

 

 

 

 

How to approach the enzyme lab results (see section F of lab for details)

1. Organize your raw data.  What do you have from the lab in the protocol tables? Remember this is all in OD. You have to use the standard curve to turn it into a Concentration. Think about the units for rate at the end.

B. Standard Curve

Table of Molarity of p-Nitrophenol standard (0.05 mM) v. OD (5 dilutions)

Plot data points – use Excel and the trend line equqtion to get concentrations from OD.

Don’t confuse the standard solution with the substrate solution for B, C, D.

C. Rate of Reaction (enzyme v. no enzyme)

Table of  time points for OD, molarity (use std curve to fine) for both samples

Plot concentration of product v. time — 2 lines, control (water) and sample (enzyme)

Determine the “best fit” line (linear) using Excel (no need for least squares).

Determine enzyme activity from a 1 minute interval in linear area.

This is enzyme activity or initial velocity (Vo) in micromoles/ml/min.

Note that for the no enzyme trial (water) the data may bounce around a bit –smooth it out.

D. Enzyme Concentrations Note there are 2 graphs for this part.

Table of time points for OD, molarity  for 6 enzyme concentrations

Plot concentration of product v. time – 6 lines for different samples

Determine the “best fit” line (linear) using Excel.

Determine 6 enzyme activities as in B. Make a table of all activities somewhere

Plot enzyme activity (Vo) v. enzyme concentration (ml of serum) — one line

E. Substrate Concentrations Note there are 3 graphs for this part.

Table of time points for OD, molarity for 6 substrate concentrations

Plot concentration of product v. time – 6 lines for different samples

Determine the “best fit” line (linear). Where is it linear?

Determine 6 enzyme activities as in B. Put them in the activities table

Plot enzyme activity (Vo) v. substrate concentration (molarity) one line

You will have to find the substrate concentration (stock is 5 mM)

Use your old stand-by calculation: M1 X V1 = M2 X V2 (what is V2?)

Lineweaver Burke Plot – this is y=mx+b version of activity plot.

Table of 1/Vo (1-5) and 1/[S] (1-5)

Plot line 1/Vo v. 1/[S].  best fit line (approximately linear)

Determine Km and Vmax.  How do they compare to values from above?

If you can’t remember this graph, use the enzyme kinetics info from Blackboard.

Lab Report:  Remember this is a 30 point lab report, otherwise a standard format for report.

The major part is the tables of data and the graphs you produce form the tables.  Make sure they are all clearly labeled with a title and #s for easy reference. This is an enzyme kinetics lab. What are you trying to learn about enzymes from the graphs that pretty pictures in the book don’t tell you? How do enzymes work? What is the relationship between Km and Vmax? Campbell skips this area, so you will have to consult Blackboard, the lecture, or a web site for background and references. Look at section F about the write up for more details and further questions to ponder. What does precision mean here?  Does it relate to errors? You have replicate runs of one enzyme concentration. Be careful with discussion of controls.  There is a method to convert OD to conc. using Excel on Blackboard. Basically similar to what you did in the Spec lab.

 

Note: DO NOT submit the coversheet through Safe Assign along with the lab report. This skews your match percent. Also, DO NOT submit the group protocol either as this has the same effect of artificially upping the match percent.

CLICK BUTTON TO ORDER NOW