Purdue Bio 110 Lab Practical 2

In the oxygen production lab, what compensation was made for the heat generated by the lamps? A. A tank of water was placed in the light path to absorb heat. B. An amount was subtracted from each oxygen production rate to account for temperature effects. C. A heat-resistant strain of Chlorella was used. D. All of the above represent means that were used to compensate for the heat. E. Only A and B above were used to compensate for the heat.

A

Which of the following is done to determine the absorption spectrum of a substance? A. Measure its absorbance over a range of different wavelengths of light. B. Plot the rate of absorbance change versus time. C. Measure its absorbance at one wavelength over time as the substance is reduced. D. Measure its absorbance at one wavelength over time as the substance is oxidized. E. Subtract the absorbance of the substance when it is reduced from the absorbance of the substance when it is oxidized.

A

If the "no light control" rate was negative, what was happening to oxygen in the dark Chlorella tube?

If the "no light control" was negative, then Chlorella were consuming oxygen.

Which of the following is/are true of the solubilities of the pigments studied in lab? A. Carotene is more soluble in ethanol than in petroleum ether. B. Chlorophyll a is more soluble in methanol than in petroleum ether. C. Chlorophyll b is more soluble in petroleum ether than in methanol. D. All of the above are true concerning the pigments studied in lab. E. Only B and C are true of the pigment solubilities.

A

Why do you suppose a buffer was used for the isolation and function assessment of chloroplasts?

A buffer was used to control the pH. This is necessary to maintain a constant external environment for the chloroplasts, which helps them survive the experimental procedures. Also, many biological reactions are sensitive to pH; the buffer held the pH steady to eliminate changes in pH as an experimental variable.

Lab 2 - Oxidation of Water: What is DPIP?

-Lipid soluble so it can pass through the membrane -When oxidized it is blue -When reduced, not oxidized, it is clear -Intercepts electrons

Lab 1- Photosynthesis: Chlorophyll Extraction: Procedure

1. Extraction of pigments from spinach leaves 2. Separation of chlorophylls from carotenoids and then chlorophyll a from chlorophyll b 3. Determination of the absorption spectra of the chlorophylls and carotenoids

Lab 3- Production of Oxygen (Part TWO): What was the calibration procedure conversion?

1. Measure water with no dissolved oxygen 2. Add sodium sulfite and cobalt chloride -used to chemically deoxygenate water -The sulfite reacts with the oxygen to make sulfate which takes about 100 seconds to completely remove dissolved oxygen from the water sample 3. Stir water to catch the oxygen in the air

Which of the following wavelength settings is best for having a Spec 20 detect the absorbance of light by a substance that is blue in solution? A. 600. B. 425. C. 450. D. 475. E. 400.

A: Blue solutions strongly absorb light in the red region (~600nm) of the visible spectrum.

Lab 1- Photosynthesis: Chlorophyll Extraction: What wavelength corresponded to the blue color range on the spectrum?

About 400

Lab 1- Photosynthesis: Chlorophyll Extraction: What wavelength corresponded to the green color range on the spectrum?

About 500

Lab 1- Photosynthesis: Chlorophyll Extraction: What wavelength corresponded to the red color range on the spectrum?

About 600

Lab 1- Photosynthesis: Chlorophyll Extraction: What was the dependent variable (y-axis)?

Absorbance

Lab 1- Photosynthesis: Chlorophyll Extraction: Calibration of Spectrophotometer

Adjust your wavelength (big knob on the top right), filter control @600(switch on the bottom left) With nothing in chamber turn the left knob so readout is set on zero With blank(control) in the chamber turn right knob to 100

Lab 3- Production of Oxygen: The relationship between PFR and the rate of oxygen production is...

As PFR increases the rate of oxygen increases, linear positive slope

In the oxygen production lab, how was PFR varied? A. The PFR was varied by adjusting the offset control on the amplifier. B. The PFR was varied by adjusting the distance between the light source and the tube containing Chlorella. C. The PFR was varied by adjusting the color of the light source. D. The PFR was varied by moving the light source. E. The PFR was varied by adjusting the temperature of the light source.

B

Lab 1- Photosynthesis: Chlorophyll Extraction: What color photons pack the most energy?

Blue

The absorption spectrum of carotenes as determined in lab sometimes showed an absorption peak in the red region of the spectrum. However, the absorption spectrum of carotenes shown in the textbook did not show such a peak. What caused this difference? A. Hot ethanol was used to extract pigments in our lab; the extraction procedure outlined in the textbook used cold ethanol. B. The spinach used in lab was briefly boiled in water; the procedure outlined in the textbook did not use boiling water. C. The carotenes extracted in our lab were contaminated with chlorophyll; the carotenes used in the textbook were not contaminated with chlorophyll. D. The spectrophotometer used in our lab was able to emit light in the red region of the spectrum; the one used in the textbook could not. E. In our lab we used the proper amount of carotenes; in the textbook the amount of carotenes was inadequate to measure absorption in the red region.

C

Which of the following do chloroplasts and sodium bisulfite hold in common? A. Sodium bisulfite may break down into DPIP and chloroplasts may produce DPIP. In either case the DPIP produced may oxidize water. B. Sodium bisulfite may break down into DPIP and chloroplasts may produce DPIP. In either case the DPIP produced may reduce water. C. Sodium bisulfite and chloroplasts may cause DPIP that is initially blue in solution to become colorless. D. Sodium bisulfite and chloroplasts may cause DPIP that is initially colorless in solution to become blue. E. Sodium bisulfite and chloroplasts both may oxidize water by adding electrons to the water.

C: Both sodium bisulfite and chloroplasts may reduce DPIP. DPIP looses its color when it becomes reduced.

The reduction of DPIP by chloroplasts was coupled to which reaction below? A. The oxidation of carbon dioxide. B. The oxidation of oxygen. C. The oxidation of water. D. The oxidation of NADPox. E. The oxidation of sodium bisulfite.

C: When water is oxidized in photosynthesis it looses electrons. Those electrons are available to reduce DPIP

In the oxidation of water lab that utilized the blue dye DPIP, which of the following represents a measured (dependent) variable? A. Light intensity. B. pH. C. Change in absorbance over time. D. Wavelength of light. E. Temperature.

C: You measured the loss of blue color (i.e., the reduction of DPIP) by noting the change in absorbance over time.

Lab 3- Production of Oxygen: (In the dark) What consumes the oxygen and how is it consumed?

Chlorella. It is consumed through cellular respiration and the oxidiziation of glucose

What are the relative solubilities of chlorophylls a and b in petroleum ether and 92% methanol?

Chlorophyll a has a higher solubility in petroleum ether than in methanol. Chlorophyll b has a higher solubility in methanol than in petroleum ether

Note the electron carriers in the thylakoid membrane above. When chloroplasts are exposed to light, an electron acceptor, DPIP, is reduced by electrons from carrier #3. When a particular plant poison is added to illuminated chloroplasts, DPIP is not reduced. Which of the following explains that observation? A. The poison blocks the oxidation of water. B. The poison blocks the transfer of electrons from #1 to #2. C. The poison blocks the transfer of electrons from #2 to #3. D. All of the above are valid possible explanations for the lack of DPIP reduction. E. None of the above could possibly explain the lack of DPIP reduction.

D: A block at any of these points will prevent DPIP reduction.

Which of the following exercises that you performed in lab best shows that chloroplasts play a crucial role in the color change of DPIP? A. Determining the difference in the absorption spectra of chlorophylls a and b that were removed from chloroplasts by boiling ethanol. B. Comparing the change in absorbance of an illuminated tube completely covered with aluminum foil with the change in absorbance in an identical illuminated tube that was not covered with foil. C. Checking the absorption spectra of oxidized versus reduced DPIP. D. Comparing the absorbance changes in separate tubes in which increasing amounts of chloroplasts were added. E. Determining the difference in the absorption spectra of chlorophyll a and carotenoids that were removed from chloroplasts by boiling ethanol.

D: Showing that increasing amounts of chloroplasts increase the rate of DPIP reduction provides evidence that chloroplasts are the active ingredient in this experiment.

What was the purpose of using a buffer for the isolation and function assessment of chloroplasts? A. The buffer maintained a constant temperature in the chloroplast preparation; this was necessary because the lights used in the experiment produced heat. B. To maintain a constant amount of light (about 500 foot-candles) in the chloroplast preparation. C. To maintain a constant amount of carbon dioxide in the chloroplast preparation. D. To eliminate pH as a variable in the experiment. E. To maintain a constant amount of oxygen in the chloroplast preparation.

D: The buffer maintained constant pH.

DCMU is as an herbicide; it blocks the transfer of electrons out of photosystem II. How would DCMU affect the production of oxygen in plants?

DCMU should lower the rate of oxygen production when compared to the rate produced by the same PFR and no DCMU. Oxygen production will fall because it is dependent on an electron-deficient Photosystem II. If photosystem II can't pass electrons to the next electron carrier in the thylakoid membrane, it won't become electron-deficient; this means that water oxidation and oxygen production will slow or stop. (The DCMU blockade may not be 100% effective.)

If heat is necessary for the DPIP to change color, what should have happened in the "No light, heat" control?

DPIP should lose blue color and the absorbance should at 10 minutes should be less than at 0 minutes.

Lab 2 - Oxidation of Water: What is the proper name of DPIP?

Dichlorophenol indophenol

Which of the following are capable of reducing DPIPox? A. Sodium bisulfite. B. Illuminated chloroplasts. C. Potassium Chloride (KCl) D. All of the above. E. Only A and B above.

E

Which of the following is/are true of the absorption spectrum of chlorophyll b as studied in lab? A. Chlorophyll b had an absorption peak in the green region of the visible spectrum. B. Chlorophyll b had an absorption peak the red region of the visible spectrum. C. Chlorophyll b had an absorption peak the blue region of the visible spectrum. D. All of the above are true of the chlorophyll b absorption spectrum. E. Only B and C are true of the absorption spectrum of chlorophyll b.

E

Lab 1- Photosynthesis: Chlorophyll Extraction: What were the 3 different blanks used for calibration?

Ethanol layer- 95% ethanol Ether layer- petroleum ether Methanol layer- methanol

True or False: Cellular respiration cannot occur when the lights are off

FALSE: Cellular respiration occurs even when the lights are off is just not as noticeable in the dark

What would the consequence to photosynthesis be if the chlorophyll molecule were transparent to light?

For chlorophyll to be of any use in photosynthesis, it must absorb or capture light energy. The captured energy is used to excite electrons that are utilized to produce ATP and reduce NADPox. The ATP and reduced NADP (along with CO2 and other compounds) are used in the production of sugars. Transparent and colorless chlorophyll would not absorb light; it would simply transmit all the light that hit it. Chlorophyll that did not absorb light would be useless in photosynthesis.

What is the relationship between PFR and oxygen production rate in Chlorella?

Generally, the higher the PFR, the greater the oxygen production rate. Photons (essentially packets of light energy) strike Photosystem II and cause it to lose an electron that is 'caught' by a nearby electron acceptor. The electron-deficient Photosystem II then removes electrons from water bringing about the production of oxygen. The higher the PFR (photon fluence [flow] rate) the higher the rate at which photons strike Photosystem II and the higher the rate of water oxidation.

What wavelengths do you think would have been least effective for driving the reduction of DPIP? Why?

Green wavelengths would have been least effective because they are least well absorbed.

What would the absorption spectrum of a colorless, transparent substance look like?

If the substance were colorless and transparent, its absorbance at visible wavelengths would (ideally) be zero.

Lab 2 - Oxidation of Water: Adding more chloroplasts to the reaction does what?

Increases the rate of the reaction (speeds up the reaction) and raises the absorbance

If DPIP spontaneously loses its blue color, what should have happened in the "No light, no heat" control?

It should lose blue color and the absorbance at 10 minutes should be less than at 0 minutes.

Does light alone appear to be needed to drive the DPIP reduction? How can you tell?

Light alone appears to account for the great majority of the color change of DPIP because light alone causes the greatest loss of blue color.

Lab 3- Production of Oxygen (Part ONE): What is the independent variable (x-axis)?

Light quantity/PFR (photon fluence rate)

Lab 2 - Oxidation of Water: What happens to the thylakoid membrane?

Light strikes the thylakoid membrane of the chloroplasts exciting the electrons so they no longer function normally. The excited electrons reduced the DPIP from the chlorophyll

If we had been able to provide illumination of different wavelengths for the chloroplast preparation that all had equivalent energy levels, what wavelength(s) do you think would have been most effective for driving the reduction of DPIP in the reaction you studied today?

Maximum absorbance of photosynthetic pigments is in the blue and red regions of the visible spectrum. So, blue or red wavelengths should have been the most effective at driving the DPIP reduction reaction.

Lab 1- Photosynthesis: Chlorophyll Extraction: What did chlorophyll b have a high affinity for?

Methanol

How much photosynthesis would a plant carry out if it were illuminated by green light alone?

Not much, because chlorophyll cannot absorb much light energy in the green region

Lab 3- Production of Oxygen: In the dark, there is no _______ produced but there is _______ consumed

Oxygen, oxygen

Lab 1- Photosynthesis: Chlorophyll Extraction: What did chlorophyll a have a high affinity for?

Petroleum ether

Lab 2 - Oxidation of Water: What was the relationship on the graph?

Positive slope, linear, rate increases as the number of chloroplasts added increases

By blue light? By orange and red light?

Since chlorophylls absorb light in these regions, a plant should carry out photosynthesis (and therefore grow) considerably better under these lights than with green light alone.

Lab 3- Production of Oxygen: What was the relationship between millivolts on the computer read-out and the concentration of dissolved oxygen of dissolved oxygen in the water?

Take the computer (mV) and divided by the dissolved oxygen concentration (mg/L) 1mV=_______ mg/L of dissolved oxygen

For what do plants use oxygen?

The Chlorella were carrying out aerobic respiration, that is, they were using oxygen to oxidize sugars that they had made previously.

Why was bisulfite added to the "blank" tubes in your experiments with chloroplast function?

The bisulfite reduced the DPIP, so the blank had everything in it that the experimental tube had, except the blue color.

What are the relative solubilities of carotenoids and chlorophylls a and b in petroleum ether and 95% alcohol?

The carotenoids are more soluble in 95% ethanol than petroleum ether. On the other hand, the chlorophylls are more soluble in the petroleum ether than 95% ethanol

Where do these electrons normally end up when the full process of photosynthesis is occurring? How important is this for the overall process of photosynthesis?

The electrons are normally given to CO2, thereby reducing it. Reduction of CO2 to sugar is the essence of photosynthesis.

In the reaction you have been studying in this laboratory, where are the electrons that reduce the DPIP coming from?

The electrons originate from water

How might DCMU negatively affect the production of glucose in a plant?

The electrons that ultimately are used to help make sugar (which the plant needs to survive) come from water that photosystem II oxidizes. If photosystem II can't deliver electrons, sugar production will stop, and eventually the plant will die.

Lab 1- Photosynthesis: Chlorophyll Extraction: Spectrophotometer parts are?

The light source, monochromator, test tube, light detector, amplifier, and readout

In the experiment where the amount of chloroplasts was varied, what do your results show about chloroplast function?

The more chloroplasts, the faster the dye reduction, as shown by the data when graphed. (See graphs above.) The direct relationship between rate of DPIP reduction and amount of chloroplasts indicates the dependence of water oxidation/dye reduction on active chloroplasts.

Chlorophyll is green; how does this relate to its absorption spectrum?

The point here is that chlorophyll a and chlorophyll b have absorption peaks in the blue and red regions. This means that chlorophylls transmit green light and, therefore, appear green.

Lab 3- Production of Oxygen (Part ONE): What is the dependent variable (y-axis)?

The rate at which Chlorella produces oxygen

Lab 2 - Oxidation of Water: What was the dependent variable (y-axis)?

The rate of water oxidation/rate of DPIP reduction

Lab 3- Production of Oxygen (Part ONE): What is the relationship on the graph?

The relationship for the graph is linear, positive slope, up and to the right More energy = more oxygen

If light and heat are required for DPIP to lose its color, how should the change in absorbance be different between the "Light and heat" control and the "Light, no heat" condition?

There should be greater loss of blue color in the "light and heat" experiment than the "light" alone experiment.

Lab 3- Production of Oxygen (Part TWO): What is the independent variable (x-axis)?

Time

Lab 3- Production of Oxygen (Part TWO): What is the dependent variable (y-axis)?

Voltage (mV)

Lab 1- Photosynthesis: Chlorophyll Extraction: What was the independent variable (x-axis)?

Wavelength

What major property of chlorophyll a, chlorophyll b, and carotenoids did we take advantage of in order to separate them from one another?

We exploited the different solubilities of the pigments in petroleum ether, ethanol and methanol

Lab 2 - Oxidation of Water: What was the independent variable (x-axis)?

mL of Chloroplasts added (0.1mL,0.2mL,0.4mL)