Chapter 8: Photosynthesis\Respiration - Endnotes

  1. Chloroplasts are extremely light labile. They should be kept cool and in reduced light at all times, except when actually being used for analysis of photosynthesis. Once diluted, the chloroplasts will be reasonably unstable and you should work as rapidly as possible while keeping all reactants as cool as possible.

  2. Be careful to not confuse the pigment CHLOROPHYLL with the organelle CHLOROPLAST.

  3. DCMU is a powerful electron acceptor and will inhibit System II electron transfers in intact chloroplasts.

  4. May substitute 10 mM Tetrazolium Blue @ 580 nm, 20 mM Methyl Red @ 440 nm, or .001 M C_2 ,C_3 ,C_5 ,C_6 -Tetramethyl-p-pheynlenediamine (DAD) if System I is to be specifically measured. DAD will reverse the uncoupling of System I induced by DCMU.

  5. Sunlight is best, but photofloods will do. If photoflood lights are used, a heat filter must be used. This can be accomplished by placing a small aquarium full of water between the light source and the beaker containing your chloroplast suspensions.

  6. Fresh cauliflower can be substituted if the homogenization is done with a mortar and pestle. There will be some plastid contamination, but it should not affect respiration measurements with normal room light exposure. Weigh cauliflower and begin with step 3. There will be significantly fewer mitochondria isolated per gram of tissue than with liver.

  7. May be omitted. It works to protect the mitochondria from rapid degradation.

  8. Prepare just before use. A stock solution of 0.33% may be stored frozen in an amber bottle. Thaw and dilute just before use.

  9. Oxidation of p-phenylenediamine oxalate (PPDO) changes the dye from its colorless form (reduced) to purple (oxidized).

  10. This is easier to do if the data are entered directly into a computer spreadsheet program. Refer to Appendix D.

  11. The isolation of intact chloroplasts is a significant factor in the success of this exercise. Many of the structures and functions of chloroplasts are altered during the extraction process. Refer to Table 8.1.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --