FISHERY BULLETIN; VOL. 73, NO. 2 



much the same way as do the nonradical natural 

 fatty acids present in membranes (Libertini et al. 

 1969; Hubbell and McConnell 1969; Schreier-Muc- 

 cillo et al. 1973). By using appropriate instrumen- 

 tation, it was found that these radicals could be 

 used as submicroscopic probes (or labels) for 

 investigating membrane structure. This has 

 become known as spin-labeling, and forms the 

 underlying theme of this paper. This report 

 describes our work on contaminant-host interac- 

 tion in fingerling salmon. To show how these 

 studies were performed, we need to arm ourselves 

 with some basic background information. Let us 

 review briefly some very important points con- 

 cerning radicals, electrons, and nuclei. 



THE FOUNDATION OF 

 SPIN-LABELING 



Free Radicals 



Many free radicals are known or have been 

 isolated. Most, as we know, are quite reactive 

 chemically, and unless conditions conducive to 

 their formation and stabilization (trapping) are 

 maintained, radicals normally disappear once 

 formed. Radical reactivity stems from the fact 



MAGNET ON, 

 ELECTRONS FREE IN SPACE 



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MAGNET OFF 



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ENERGY 



LEVEL 



DIFFERENCE 



Figure 1. -Resonance condition for isolated electrons. In the 

 absence of external field, energy levels are indistinguishable. 

 When a magnetic field is applied, two levels (and only two levels, 

 by quantum mechanical restrictions) are populated by electrons. 

 By employing X-band microwaves (9 GHz) with the proper 

 energy {hv = gpH; see for instance, Roubal 1972), flipping 

 between levels occur and the absorption of microwave energy is 

 observed. 



that radicals, by definition, are molecules which 

 contain one or more unpaired electrons. Two 

 radical partners normally pair together to yield 

 end products with the normal complement of two 

 electrons per chemical bond. This is the usual 

 covalent bond and is characteristic of organic 

 compounds. 



There is one class of free radicals, the nitroxides, 

 which are stable under many of the usual labora- 

 tory conditions. Nitroxide stability derives from 

 resonance and other contributing factors, but we 

 need not discuss these here. Many nitroxides are 

 relatively easy to synthesize, provided the neces- 

 sary starting intermediates (some of which are 

 rare) are at hand. 



The important point to be made is the fact that 

 nitroxides can be used to characterize biological 

 systems. Nitroxides so used are called spin-labels; 

 spin from the fact that it is the unpaired elec- 

 tron(s) (which is/are spinning) which forms the 

 basis for the label or probe. Spin-labeling might 

 just as conveniently be called spin-probing. 



Spinning Electrons and 

 Their Magnetic Properties 



All electrons are in a state of motion; they all 

 spin about on their axis. Spinning electrons are 

 therefore moving charges of electricity. Thus 

 electrons are magnetic. Spinning electrons 

 therefore are influenced by an external magnetic 

 field such as produced by a solenoid or elec- 

 tromagnet. When a sample of free radicals is 

 placed between the poles of an electromagnet, the 

 spin of the electron is described as clockwise or 

 counterclockwise and is depicted in Figure 1. 



Of immediate consequence is the fact that one 

 spin condition is more stable than the other, and is 

 so indicated by the reference to the energy of the 

 system as shown. Although one population level is 

 more stable than the other, the temperature of the 

 system is always great enough to insure that the 

 higher level contains just about as many electrons 

 as the lower level. This is something akin to plac- 

 ing two bar magnets end to end. If they are 

 aligned N-S N-S, we know from everyday 

 experience that the interaction will be attractive 

 and stable. If, on the other hand, we try to force 

 them N-S S-N, we know again from experience 

 that this is an unstable situation and requires an 

 expenditure of energy (heat in the case of elec- 

 trons, physical in the case of magnets) to maintain 



300 



