Download Amylase Experiment

Biology 20L
Amylase write-up
These are some notes to help with the amylase write-up. Unless your schedule of assignments says
otherwise, it will be a Data Set: Submit an abbreviated report, essentially Results/Discussion, followed by Figures
and Tables. The figures should be very clearly labeled and each should have a descriptive title. The Discussion
section should report any difficulties with the experiment and any conclusions. Was Vmax established for your
sample? How do you know? If not, what is your estimate of the true Vmax? Is your measured value of reaction
rate an over-estimate or an under-estimate? What is your final conclusion about the reaction rate of your original
sample (i.e., How much starch can your saliva break down?)
Vmax
-- Show plots of the time course (Abs vs. time), showing all dilutions for the samples. It is probably best to
put all dilutions for the sample on the same graph. That way, the slope of each dilution is visible right there with the
other dilutions--remember, you are trying to compare the various dilutions to each other.
-- Get initial slopes from each time course. Be sure to show on your graph where the slopes were taken -which points were used. Some reactions give curves whose slopes begin to change immediately, giving a
characteristic concave-upward curve. These reactions are from the samples with more concentrated enzyme. They
are curving because the reaction is slowing down as the substrate is consumed. The initial segment of the curve will
have the highest slope for this sample (“Measured Initial Rate” in the table below). This may not be Vmax, but it will
be the best you get from this particular dilution. Use that value for the velocity of the reaction for this sample. Other
samples may have roughly straight lines, with some “noise” in them. If they curve concave downward, this is noise.
(How could the reaction speed up, after all?) If you do not have at least three initial points in a line, the sample is
probably not at Vmax. If you do have three or more points in a line, this indicates that consumption of some
substrate is occurring, but the [S] has still not fallen below the threshold where the rate is decreasing. In other
words, you are using up the excess substrate, but it is still in excess. You are probably at Vmax. One way to prove
you are at Vmax is to run several dilutions and show that the dilution is accompanied by a concomitant drop in
reaction rate (proportionality). Another way is to plot the data as shown below. This graphical approach takes
some of the uncertainty out of relying on proportionality alone. The course website has a spreadsheet download for
curve-fitting.
-- Prepare a table showing the initial rate data for each dilution. Note that as less enzyme is added, the
conversion of substrate to product (reaction rate) decreases. Compute the rate of the reaction for totally undiluted
saliva (Adjusted rate, Undiluted Rate or Projected Rate, or Raw Saliva or some other title) as shown in the Table.
Think of this as what the rate of the enzyme would have been had you not diluted it at all. If you use the form of the
dilution factor that is less than 1 as shown, you get this undiluted rate by dividing the measured rate of reaction by the
dilution factor. (Some people prefer to think of it as multiplying by 50, for example, rather than dividing by 1/50.)
The “Undiluted Rate” will be greater than the measured rate. If it is not, you probably used the dilution factor upside
down.
Note in the example that the apparent rate (last column) continues to increase as the sample is diluted. This
means that each molecule of enzyme is becoming more efficient at turning over substrate as the enzyme gets more
dilute (despite the fact that the bulk rate is slower). This is consistent with your knowledge of the reaction on a micro
scale: As enzyme gets more scarce, each enzyme molecule has more access to substrate. Finally, in the last two
samples of the example below, proportionality is reached (undiluted rate is constant). This means that the enzyme
molecules are already overwhelmed by substrate and the last dilution does nothing more to allow the enzyme
molecules more access to substrate. This is proof that Vmax has been reached in samples of 1/1000 and all
samples more dilute.
Dilution
Measured Initial
Rate (A/min•ml)
100
50
25
6
3
1/10
1/50
1/200
1/1000
1/2000
Undiluted Rate
(A/min•ml)
1000
2500
5000
6000
6000
-- Plot the Undiluted Rate as a function of the dilution. This is essentially plotting the standard MichaelisMenton curve ([S] vs. rate). The x-axis can be thought of as really being [S]/[E]. For [E] constant (standard
presentation), the axis is [S]. For [S] constant (our experiment), the x-axis can be thought of as 1/[E]. If [E] is 1/10,
1/50, etc., then 1/[E] is 10, 50, etc. Did you reach Vmax? For the made-up data presented here, Vmax is clearly
reached at 6000 A/min•ml.
Rate vs. 1/[E]
7000
A/ml.min
6000
5000
4000
3000
2000
1000
0
0
500
1000
1500
2000
1/[E]
ENZYME ACTIVITY
-- Using the highest "Undiluted Rate" obtained -- which should derive from the most dilute sample(s) (see
last column in Table above) -- convert the Vmax rate sample to actual, physical starch amounts. Your starch-only,
no-enzyme control ("S") gave you an absorbance value. If the stock starch solution was 0.1% (or 1.0 mg/ml), or
some other known concentration, you can calculate the concentration of starch (in mg/ml) present in each reaction to
begin with. Remember that the reaction had other things in it besides the stock starch. The reaction will therefore be
at some starch concentration lower than the stock. Check your protocol and figure out what that concentration is (x
mg/ml). You measured the amount of color produced by this amount of starch (A600 ). This concentration and
absorbance, taken together, give a conversion factor which can be used to convert any A600 into actual starch units.
The conversion factor is in the form:
x mg/ml = 1.0
A600
“= 1.0” in this case means that the numerator and
denominator refer to the same sample
Any A600 value that comes from the iodine assay (or any value with complex units, such as A600 /min, etc)
can be multiplied by this conversion factor. This will convert the units to mg/ml starch but will not change the
magnitude of your number, because the conversion factor is equal to 1.0. All conversion factors must equal zero.
For example: 5280 ft/mile, 106 mm/km, 2.2 lbs/kg, etc. Because starch is not well-defined in terms of how many
glucose subunits there are in each starch molecule, we cannot convert to molar units, and therefore report the results
in mg/ml.