..... In the early days of biochemistry, when there were no "properly" trained biochemists, all advances were made by workers scattered all throughout the sciences. Thus it might be appropriate to subtitle this exercise as:

The Discovery of Helium on the Sun AND the Order of Cytochrome Utilization

This is because physicists played an important role in opening this important door into the land of biochemistry and the transport of electrons from NADH to oxygen gas.

..... It had long been known that prisms "break up" white light into the rainbow of colors. ..Then it was found that when using the sun's light there were a multitude of very thin black lines seemingly spaced randomly upon the rainbow. ..These were soon shown to be "absorption lines" caused by intervening gases, most of which were not the atmosphere of the earth, but rather the much more abundant gases in the solar atmosphere. ..It was known then that each gas would cause very specific lines to form, and that all the lines but a few could be accounted for by the known elements. ..What was the nature of the gas that absorbed the wavelengths causing those few mysterious lines? ..Physicists quickly determined that atoms of the unknown element consisted of two protons and probably two neutrons and attendant electrons. ..Since this element was first detected on the sun (Greek: helios), it was named helium.

..... So what does this have to do with cytochrome biochemistry? ..If a suspension of cytochromes is held up to a pure white light (such as an incandescent lamp, which give light without absorption bands), three major absorption bands are seen. ..With typical great imagination using a convention proposed by a Dr. Soret, they were called "alpha," "beta", and you can guess the third! ..(The convention is that the band having the shortest wavelength is "alpha".) ..This gave Drs. Warburg and Keilin a great idea - IF each band represented a different cytochrome. ..They knew that just because the spectral properties lined them up alpha to beta to gamma, that didn't mean that the electrons would be passed from one to another in that order.

..... So how did they do it? ..Just like you will! ..What Warburg and his friends found out first was that if they purposefully oxidized all the cytochromes, the absorption bands were intense, and if the reduced them, then they all but disappeared. .. Now suppose you start with oxidized cytochromes, and then you add a poison that blocks the passage of electrons from one particular one to another (or you add something like DPIP that steals the electrons away from the pathway - as you did in the photosynthesis experiment #4). .. What would you expect to see after adding some reducing agent? ..Suppose you saw two bands disappear. .. That would mean the remaining one was the last one in the pathway!.. Get the drift? ..There are many poisons and short-circuit compounds known to affect the cytochrome pathway: .. DPIP, methylene blue (MB), cyanide, and a number of antibiotics such as antimycin. ..There are even reducing agents that will send their electrons into the pathway partway down the chain: ascorbate (vitamin C) is a popular one. ..Thus by using various combinations of poisons, electron-traps (DPIP and MB), and electron donors, you ought to be able to figure out the "map" of the pathway. It will be one of these:
.....

MATERIALS

..... In general you will need:
.....

• A spectroscope or some sort of spectrophotometer
  (the former is highly preferable and if used will cut the length of the exercise greatly)
• A source of mitochondria
• A bright incandescent light source
• A collection of agents that target the cytochrome pathway
  • poisons
  • electron acceptors
  • electron donors

.....

1. The SPECTROSCOPE. ..This is something that many chemistry and physics labs have locked away. ..Once you have one, FIRST ask for instructions on how to use it. ..Once you have learned how to use it, have a little fun and scout out the world around you. .. Here is a short list of things to look at:
   .....
   • FIRST! ..Look at the fluorescent lights overhead. ..They are very handy almost omnipresent lights that will help you get into proper focus: ..focus on the mercury vapor emission lines, which are brightly superimposed on a background rainbow. .. DO NOT TOUCH THE FOCUS HEREAFTER!
     .....
   • Your incandescent light source (you should see a smooth rainbow with neither bright or dark lines superimposed on it).
     .....
   • Look at the sky during the day. .. Even on cloudy days! ..All the light you will "scope" will be that of the sun - even if diffused through clouds. ..Do you see dozens of very thin dark lines? ..Those are the solar absorption lines mentioned earlier.
     .....
   • Look around for any neon, argon or xenon lamps. These are usually very small on/off indicator lights on some items of equipment. ..Hold the spectroscope right up to the light and take a peek at the emission lines.
     ..
   • Obtain a Meeker burner, which is a Bunsen burner with a little grill on top, which breaks up the Bunsen's one flame into dozens of closely packed little flames. ..One at a time place on the grill chuncks of various salts your chemistry teacher can give you. ..Light the burner and "scope" the colors. .. Now you see several different sets of emission lines.
   
   .....
2. MITOCHONDRIA. ..Yeast have been used long ago (Journal of Biological Chemistry vol 217, page 453 (1955)), but a more concentrated source would be preferable - afterall cytochromes are usually RED, and yeast is usually not seen as being red. ..Think: RED meat! ..Liver is even REDDER! ..Get some fresh meat/liver/heart(best of all!), and treat the meat just as you would the spinach leaves in the isolation of chloroplasts in your AP Biology Lab Manual Expt #4. ..Your final suspension of mitochondria should be cloudy, cold and ready to use.
   .....
3. A bright INCANDESCENT LIGHT is required. ..A good one might be simple a 100 W bulb in a lamp fixture. ..To help shield your eyes, between you and the lamp set up a large piece of cardboard that has an appropriately placed 1 cm diameter hole in it. ..Put sample tubes up to the hole when looking at them.
   .....
4. A collection of agents that target the cytochrome pathway
   1. Oxidizer: oxygen as when swirled in air.
   2. poisons
      1. 0.1 M KCN
      2. 1.8 mg Antimycin A in 10 ml of 95% ethanol
   3. electron acceptors
      1. 0.01 M methylene blue (187 mg/50 ml water)
      2. 27 mg DPIP per 100 ml of water
         (2,6-dichlorophenolindophenol)
   4. electron donors
      1. 0.001 M NAD⁺
      2. sodium hydrosulfite (solid) (Na₂S₂O₄)
      3. 0.5 M sodium succinate (6.8 g/50 ml water)

METHODS

..... Set up a series of 18 or a few more test tubes, and put 2 ml of the mitochondria suspension into each tube. ..Then as you do each of the steps in the following list, "scope" them! ..Make sure that the SSS is placed up against the test tube while viewing.
.....

1. Swirl this "control-1" tube to oxygenate it.
   .....
2. Add 0.1 ml of succinate and swirl this "control-2" tube; view it
   allow to stand ten minutes with occasional swirling
   view it again.
   .....
3. Add a few crystals of sodium hydrosulfite to this "control-3" and compare with "control-1".
   .....
4. Add 0.1 ml of KCN to each of three tubes and do the above three "controls" again.
   .....
5. Add 0.1 ml of DPIP for another set of three tubes.
   .....
6. Add 0.1 ml of ascorbate for another three tubes.
   .....
7. Add 0.1 ml of methylene blue to another three tubes.
   .....
8. Add 0.1 ml of ascorbate and 0.1 ml of DPIP to a tube
   .....
9. Add 0.1 ml of ascorbate and 0.1 ml of KCN to a tube
   .....
10. Add 0.1 ml of ascorbate and 0.1 ml of Antimycin A to a tube
    .....
11. Add 0.1 ml of 0.001 M NAD⁺ AND 0.1 ml of KCN to a tube AND 0.1 ml of methylene blue
    .....

RESULTS