Created by D. Norton 2009. Adapted from the AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast, D. Koehnen/Rushford
The Effect of Cigarette Smoke on Oxidative Phosphorylation in Yeast
I. Introduction
Cigarette toxins are documented to interrupt the activity of the cytochrome proteins in the
mitochondrial electron transport pathway. [Journal of Biological Chemistry vol 217, page 453] This
experiment looks at the effect of cigarette smoke on the activity of electron transport in yeast
cells. It is an adaptation from AP Biology Lab #4B [Measuring Photosynthetic Activity in Spinach Tissue,
Using Spectrophotometer Analysis].
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II. Materials and Equipment
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II.
Red Star yeast, prepared with warm water and 0.5 M sucrose starter.
Aspirator flask
Spectrophotometer
Cuvettes
DPIP Indicator
.1 M Phosphate Buffer
Procedure
*Yeast samples should be prepared with warm water and .5 M sugar prior to use in the
following procedure-About 5 minutes prior to class/Use Red Star Yeast: 2 tsp per 100 mL
sugar solution. Keep warm [a flood light works well.] Filter just prior to use.
*Smoke water samples are prepared using an aspirator flask to draw cigarette smoke into 200
mL samples of water.
1
Created by D. Norton 2009. Adapted from the AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast, D. Koehnen/Rushford
Launch Microsoft Office Outlook.lnk
1.Follow the DPIP lab procedure, but test for yeast respiration (MET e- flow) instead of chloroplast eflow: Don’t ADD the DPIP until just before the ZERO min. reading!
Blank Yeast Samples-no Smoke
Yeast Samples-Smoke
Phosphate Buffer:
1 mL 1 mL
1 mL
Distilled Water:
4 mL 3 mL
3 mL SMOKE Water
DPIP:
---1 mL
1 mL
*Filtered Yeast:
2.
4 drops 4 drops
4 drops
Warm up the spectrophotometer at 640. Zero %T with NO CUVETTE and the LEFT KNOB.
3.
Use the “blank” tube in the table above to set %T to 100%, using the RIGHT KNOB.
4.
Record
%T ofthe
each
tube and repeat
at 5 minute
intervals
for 20-30
minutes.
Objective:
Toinitial
compare
%transmittance
vs.readings
time standard
curve
for yeast
not exposed
to
4.1
Keep the yeast samples warm in between readings/flood light works fine.
cigarette
smoke
to
those
samples
exposed
to
both
filtered
and
non-filtered
cigarette
smoke.
5.
Graph %T vs. time to plot the baseline rate of respiration in yeast.
6.
Repeat the procedure for an introduced variable and collect and average data from multiple trials.
*May need to adjust drops. Be sure to run a test sample before class!
IV. Individual Group Data
Cuvette
0
5
10
15
20
25
30
Yeast-No
Smoke
Water
%T
YeastUnfiltered
Smoke
Water
YeastFiltered
Smoke
Water
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Created by D. Norton 2009. Adapted from the AP Biology Lab Series #4 and Effect of Cigarette Smoke on Oxidative Phosphorylation in
Yeast, D. Koehnen/Rushford
V. Class Data
Lab
Group
1
2
3
4
5
6
Average
%T No
Smoke
0 min
5 min
10 min
15 min
20 min
25 min
30 min
%T
Unfiltered
Smoke
0 min
5 min
10 min
15 min
20 min
25 min
30 min
%T
Filtered
Smoke
0 min
5 min
10 min
15 min
20 min
25 min
30 min
Processing Data
Graph the AVERAGE %T vs. Time for each of the samples. Plot all data on one
common graph.
VI.
Conclusion:
What is the effect of cigarette smoke on oxidative phosphorylation [electron
transport within the cytochrome complexes of the mitochondria] in yeast?
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