Radio-Isotopic Labelling

..... Suppose that your kitchen larder is being raided by pesky little ants and you want to find out where they are coming from. So you gather several hundred of them and spray them with a dye that is a highly fluorescent fuchia when observed under ultraviolet light. Later that night, under the cloak of darkness you use your UV lamp to inspect all the known ant-beds surrounding your house. At one of them there are a number of glowing ants. So you found the culprit colony and set to work with your chemical arsenal.

.....In this anti-ant campaign you used a few tagged ants to find out what happened to them. Similarly you can tag atoms, but not with a dye, but rather with substitutes that are chemically identical but they are radioactive. So were you to "feed" a plant some ₁₄CO₂ you would expect that the starch being produced would rapidly begin to become radioactive. If on the other hand you had also fed the plant some ₃₂P-phosphate, you would not expect that the starch would become radioactive with the high energy ₃₂P because starch doesn't contain any phosphorous.

..... Thus biochemists have very effectively employed radioisotopes as tracers: to trace atoms through the metabolic pathways. Another use for them is to make proteins radioactive while not labelling anything else. Or nucleic acids, or DNA but not RNA or vice versa. What you need to do for this is to determine which elements are found only - ONLY! - in the molecule of interest and nowhere else. ₁₄C is thus used to label almost everything; ₃₅S is used to label proteins because no other major bulk constitutent of cells contains sulfur in any appreciable amount. Sometimes elements are shared by several different biochemical groups, so then what is done is to feed the "mother" culture not elements but rather multi-atomic building blocks. Were you to feed ₁₄C-uracil to an E. coli that was busy making T4 phage, you'd see very little radioactivity in the phage that came from this infection. Why? Because T4 is made up of only protein and DNA, neither of which contains the building block uracil (an RNA component!). However, were we to have feed the infected E. coli some ₁₄C-thymidine ("T"), we'd see the progeny viruses very radioactive. Got it?

..... Interestingly you can feed several different radioisotopes to a single culture and be able to discern which isotope is which, even though they are all radioactive. The trick is to use a scintillation spectrometer. The substance, cell, or whatever that has several different isotopes in it is immersed in a liquid mostly of toluene with a small amount of other "bathroom tile" molecules. When a beta-particle blasts out of a radioactive nucleus and bumps into this toluene mix, some electrons are bumped out of orbit, and then flop back towards the orbit from which they were ejected. As they fall from orbit down to next orbit in going back home, each drop causes a flash of light (fluorescence). Each "twinkle" or scintillation is seen and counted by the device you are using.

.....If a very powerful beta bumps out an electron, it is likely to have socked that electron very far out from its nucleus. When that electron bumbles its way back home, the first step is a big one - a very bright twinkle in the high energy end of the spectrum (blue). Weaker betas don't have so much bang, so the returning electrons can only give out dimmer twinkles that are redder. Thus the "spectrometer" part of the name of your device: it is able to discern different colors as well as keep track of the counts twinkling of each color. Thus biochemists really like using ₃H, ₁₄C, ₃₅S, and ₃₂P because their ejected betas have energies of the ratio of 1:10:10:100. So it is easy to discern ₃H from the others, and ₃₂P from the others.

..... Now look back at the quiz, and see if this clarifies anything for you.

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