A Note About Grades . . .

The second lab exam will only be worth 63 points. Instead, in each of the next 3 labs you can earn additional points by:

· Getting to class on or before 4 p.m. [1 pt]

· Completing all experiments and submitting a brief handwritten summary. A summary should only contain

o Complete results [1 pt]

o Explanations for any discrepancies between expectations and observations [1 pt]

· Fully taking part in the lab, and cleaning your working area and tools—to get this point, show me your working area before saying adios [1 pt]

Photosynthesis

In the manual, please do not read the sections: “The Reactions of Photosynthesis” (pp. 97-8) and “An Overview of Photosynthesis” (p. 98)

Literally: photo=light; synthesis=production

Without photosynthesis we’d suffocate (why?) and starve (why?). So, interdependence of photosynthetic and non-photosynthetic creatures.

Blue-green algae (cyanobacteria—you have seen some in an earlier lab) have no special organelles for photosynthesis. In protists (e.g., euglena), plants, photosynthesis occurs in chloroplasts. The photosynthetic reaction requires energy, like driving a car (the fuel here is light). Chlorophyll, which is green, absorbs red, blue portions of the visible spectrum and deflects green (that’s why chlorophyll is green, because it doesn’t absorb green). Today’s lab is consists of 3 experiments:

A. Chromatography of photosynthetic Pigments: Goal: Separate active and accessory pigments of photosynthesis

Before you come in, green leaves have been mixed with petroleum ether and acetone, then grounded into a dark green liquid.

Procedural changes: The chromatography strip should be cut straight (not to a point, as shown in figure of p. 99). Instead of a pipette, use a fine tip of a brush to “paint” a thin line of plant pigment across the strip, about 1 cm from the bottom. Allow the line to dry and repeat several times until the painted line is dark green in color.

Follow instructions on pp. 101.

Expectations:

Pigment type	pigment	color	Rf
Main	chlorophyll a	blue-green	0.65
Main	chlorophyll b	olive-green	0.45
Accessory (helps photosynthesis by catching more light)	xantophyll	yellow	0.71
Accessory	carotene	orange	0.95
(if algae is present in extract)	phaeophytin	grey	0.83

B. Light reaction in isolated chloroplasts

DCPIP is toxic

Before you came in, a chloroplast suspension had been prepared.

A portion of the photosynthetic reaction can take place in the dark, and doesn’t concern us here. In this lab, we are only working with the light phase of the reaction. In the living cell, this phase culminates in the reduction of NADP⁺.

In our experiment, we work with chloroplast suspension, and use an NADP⁺ substitute—DCPIP (2,6-dichlorophenolindophenol)—to observe the reaction. This is made possible because DCPIP is blue and becomes colorless when it is reduced. So, loss of blue color tells us that the chloroplasts are doing something. Note however that this reaction (=The Hill Reaction) does not measure photosynthesis itself, only photoreduction of DCPIP. So this is an indirect way of measuring photosynthesis.

Actually, the color situation is a bit more complicated. I add the blue DCPIP to the green chloroplast solution and get a blue-green solution. If photosynthesis takes place, after photoreduction I am back to the green color, instead of blue-green.

P. 102 provides 4 conditions:

Test Tube Abbrev	Test Tube	DCPIP present?	Light provided?	Chloroplasts	Expectation
L	Light	yes	yes	normal	+ DCIP reduced, return to green
D	Dark	yes	no	normal	- No source of energy to drive reaction, no color change (blue-green)
B	Boiled	yes	yes	boiled	-No enzymes to drive reaction (b-g)
C	Control	no	yes	normal	- photosynthesis occurs, but suspension remains green; No DCPIP, no way to gauge rxn

C. photosynthesis gives off oxygen

In photosynthesizing aquatic plants, you can see the oxygen bubbles forming near the leaves. Some of this oxygen is dissolved in the water, and can be measured.

Now, follow instructions on p. 102-3. We exhale into the deoxygenated water to increase the amount of CO² available to the plant. After this, oxygen level should be 7.4 ppm (despite the blowing of air, which still includes a lot of oxygen. Can you think of a better experimental design?). We place the plant in one cup, and no plant in the other (that’s the control). If photosynthesis is taking place, we should get higher dissolved oxygen in the experimental cup than in the control cup.