BIO 161 Lab Quiz #5

Which fraction contained the cleanest source of chloroplasts?

P2; free floating chloroplasts without cellular debris/large membranes or whole cells. This is the BEST fraction to use in order to study photosynthesis and ETC in chloroplasts!

What does the change in absorbance represent?

Electron transport activity

What was the purpose of sub-cellular fractionalization?

Isolate one sub cellular structure (chloroplasts) from the many other organelles in the cell. This is usually achieved by spinning the contents of the cells in a centrifuge

More dense organelles will form a pallet at the bottom.

Less dense organelles will remain suspended in the supernatant.

The Hill Reaction

Uses a molecule called DCIP. DCIP in its oxidized form is blue. In the presence of chloroplasts, DCIP will steal electrons from the light reaction. Specifically, the electrons are accepted by DCIP just before they reach photosystem I (PSI). When DCIP becomes reduced by these electrons it changes from blue to clear.

Standard Reaction Time

The SRT is the amount of time it takes for half of the DCIP in a chloroplast sample to become reduced when the sample is exposed to light.

In the week 1 lab you determined the absorption spectrum of chlorophyll a. Based on this spectrum, which colors would you expect to produce the LEAST reduction of DCIP?

Wavelengths that are not readily absorbed by chlorophyll are not expected to produce excited electrons and, therefore, are not expect to produce rapid reduction of DCIP.

Green and yellow

Effect of DCMU on light reaction

DCMU is thought to kill plants by preventing the transfer of electrons from photosystem II (PSII) to the electron carrier plastoquinone (PQ).

A goal of this laboratory was to determine the effect of different wavelengths of light on the light reaction of photosynthesis. Identify the positive and negative controls important to this investigation. Explain how each helped you determine the effect of the colored lights.

Positive control - white light, White light is actually a combination of light from the entire spectrum, so white light is partially absorbed by chlorophyll A. This is due to the fact that chlorophyll A absorbs in the red and blue wavelengths, which are included in white light. This is why white light, when wavelengths are separated due to a disturbance (change of medium like light reflecting through water drops) splits into a rainbow. It is a positive control since we know that there must be some wavelength of light within white that chlorophyll A absorbs.

Negative control - dark light

Would you expect DCIP to easily cross membranes? Explain your answer, using what you know about chloroplasts structure and the Hill reaction.

The Hill reaction is the transfer of electrons to an electron acceptor with the presence of light in

chloroplasts. Since the Hill reagent, DCIP, was used only within isolated chloroplasts, it will be

most likely that DCIP would not easily cross membranes.