Transcript BSC 2010L TA meeting - ENZYME KINETICS

Quiz 1
•Grab a quiz sheet
•Write down section #
•Take everything off desk
•You have 10 minutes…
1. What organelle functions in energy
production in animal cells. What
about plants cells?
2. Which of the following is membrane
bound?
Nucleus or Nucleoid
3. Which are the two modes of
reproduction in Ciliates, such as
Paramecium?
_________________
_________________
4. Trypanosoma belongs to the Phylum
_________________
5. This organism belongs to the
Kingdom _________________ and
moves by means of ____________
6. What is a unique feature of a
parfocal microscope?
a) light is formed on the slide
b) can use both eyes to look at an
image
c) focus is retained as you move
between objective lens
d) one eye can be focused
independently of the other
7. How many structures of a protein are
there?
Spectrophotometry
. Use of spectrophotometers.
. Zero the machine.
. Composition and use of a
blank.
. How to adjust wavelength.
. Proper use of cuvettes.
ENZYME KINETICS
Proteins
• A protein's function is due entirely to its
overall shape = conformation.
• Conformation is determined by 1˚, 2˚, 3˚,
and perhaps 4˚ structure of the protein.
The primary
structure of a
protein
*sequence of
amino acids
The
secondary
structure of
a protein
*Hydrogen
bonding
between
C&N
The tertiary
structure of
a protein
*Interaction
of amino
acids and
H2O
The quaternary structure of proteins
*2 or more protein molecules joined together
Enzymes
• Enzymes are protein catalysts.
• Lower the activation energy necessary for a
chemical reaction to occur.
• Act on a substrate, form an enzyme-substrate
complex. Ultimately results in a product.
• We are using barley amylase enzyme on a
starch substrate.
Formation of an enzyme-substrate complex
Environmental factors affecting enzyme activity
Blanking spectrophotometer
-Set wavelength to 560 nm
-With chamber empty, set Transmittance
T=0 with left knob
-Place blank cuvette in spectrophotometer
-Set Absorbance A=0 with right knob
-Replace blank for experimental cuvette
-Take reading!!!
BASIC PROCEDURE
BLANK
35 ml
starch
35 ml
H 2O
(or buffer)
INITIAL
5 ml
H 2O
0.1 ml
I 2KI
TIMED SAMPLES
1 ml
enzyme
1. Prepare 2 erlenmeyers (one for pH and
one for temperature)
2. Set wavelength to 560 nm
3. Prepare a blank and blank your spec
4. Collect your initial reading (Time zero)
5. TA will put 1 ml of enzyme in your
erlenmeyer.
6. Start timing…
7. As the proper time interval approaches,
swirl the flask to mix and draw 5 ml of
starch/enzyme up into the pipette.
You should pipette up the solution
about 15 sec before the reading time.
8. At the reading time, release the
solution into the cuvette and mix - the
iodine stops the reaction immediately.
9. Readings should generally be taken
within about 5 minutes.
Experiments:
• You will work in groups of three.
• Each group will perform one run from the pH
experiment (4.0, 4.5, 5.0, 5.5, 6.0 and 6.5)
• Then, each group will do one of the
temperature variables (15º, 30º, 45º, 55º,
60º and 70º)
pH experiment
-Add 35 ml of buffer to 35 ml of starch solution.
-Blank is 1:1 dH2O:buffer (+ iodine)
Table 5-4. Absorbance readings over tim e of reactions at dif ferent pHs.
pH
4.0
4.5
5.0
5.5
6.0
6.5
0
2
4
6
TIME (min)
8
10
12
14
16
18
20
Temperature experiment
-Mix as in basic procedure. The reaction flask is
kept in a water bath.
-NOTE: Very cold and very hot solutions might
need to sit before being read.
Table 5-3. Absorbance readings over tim e of reactions at dif ferent temperature s.
Temp.(°C)
15
30
45
55
60
70
0
1
2
3
TIME (min)
4
5
6
7
8
10
20
I will come around and add
enzyme when your group has…
1.
Finished basic procedure
1. 35 mL pH buffer + 35 mL starch
2. Blank- 0.1 mL I + 2.5 mL pH + 2.5 mL DI H2O
3. Placed 0.1 mL I in each cuvette
2. Blanked Spectrophotometer
3. Taken time = 0
1.
Transfer 5 mL solution from flask into cuvette w/ I.
Make sure to mix!
All of the data will be shared
among the class
• You will record your data on the
computer.
• A data set will be printed out for each
student.
Reaction Rate
= 1/2 change in absorbance /
time for this change to occur
-There is a worksheet at the end of the chapter to help
calculate reaction rate.
- Additional copies of this worksheet can be printed off of
the homepage.
- Hand this in with your lab report, so that I can see your
calculations.
- There is another handout ("How to Calculate Reaction
Rates") that is available from the lab homepage that
details the calculation of these rates.
i
1.5
1.0
i 
0.5
f
A i-1/2
0.0
0
2
4
6
8
A
10 12
Time (min)
14
16 18
20
4 Figures
1.
x-axis: Time
y-axis: Absorbance
2. x-axis: Time
y-axis: Absorbance
3. x-axis: pH
y-axis: Rxn rate
4. x-axis: Temp.
y-axis: Rxn rate
*pH
*Temp.
ENZYME KINETICS
Reaction Rate Calculation Sheet
Temp.(°C)
15
30
45
55
60
70
Ai
Af
²A
*
1/2² A
†
Ai-1/2²A
TAi-1/2² A
R.r. (*/†)
Lab report 1 (50 points)
• Refer to Appendix A - pp. 191-195
guidelines for writing a core lab report.
• Ecology has all journal articles prior to
1996 on-line.
http://www.jstor.org/journals/00129658.html
Figures
• These take some time to get right.
• Pay close attention to format.
All lab reports will be
submitted to me AND
Turnitin.com ON TIME!!!
Attached to hard copy will
be 4 hand drawn figures
and signed academic
honesty form.
Chirp Rate (Hz)
21
20
19
18
17
16
15
14
20
25
30
35
Temperature ( C)
Figure 1. Chirp frequency (Hz) of the striped ground cricket (Nemobius allardi ) at
different ambient air temperatures. The data presented are those of Pierce (1949).
• Four graphs for this lab report:
2 figures (Absorbance vs. time)
• temperature vs. time
• pH vs. time
2 figures (RR vs. Tº/pH)
• Reaction rate vs temperature
• Reaction rate vs. pH
• Best fit curve for each set of data
Absorbance
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
Time (min)
14
16
18
20
For this first report, ALL FIGURES
MUST BE DONE BY HAND (not by
computer). In the future, you may
use computer generated figures
(5) English
• (3) Spelling, grammar, clarity
• (2) Format
(1) Title
(6) Abstract
(12) Introduction
• (8) Background info
• (4) Hypotheses
(6) Materials and Methods
• (4) Experimental design and materials
• (2) Data collection and analysis
(8) Results
• (4) Tables and figures
• (2) Stats
• (2) Text
(10) Discussion
• (8) Interpretation of data
• (2) Alternative hypothesis/sources of error
(2) Literature cited
• (1) All references cited in the text
• (1) All references in text are cited
For next class
Quiz
• Bring charts to class or my office hours
for me to look over!
• Read Ch 9 & 10 and “Animal Taxa”
worksheet
• Bring dissection kits!