ROUBAL and COLLIER: SPIN-LABELING TECHNIQUES 



them aligned in this fashion. Apart from this, 

 however, the analogy extends no further, for in 

 the realm of electrons and nuclear phenomena, 

 quantum mechanical postulates hold and the com- 

 mon experience of our everyday world does not 

 pertain. 



The important point we want to make here is 

 that we can induce an electron with one spin rota- 

 tion to flip over and assume the alternate rotation. 

 Energy is required to do this. To achieve flipping, 

 the sample is irradiated (while between the poles 

 of the electromagnet) by microwaves with a 

 frequency of 9 gigahertz (GHz). Thus we see that 

 what we are really talking about is just another 

 type of spectroscopic method. The presence of free 

 radicals is detected by measuring the loss of 

 microwave energy. The actual instrument used for 

 such studies is called the electron paramagnetic 

 resonance (EPR) spectrometer (also called the 

 electron spin resonance/ESR/spectrometer). 



At this point we must consider other factors 

 which contribute to the total magnetism of the 

 system. Remember that the unpaired electron is 

 not merely floating freely about in space. It 

 belongs to a molecule. In fact it is coupled to a 

 nucleus, and in the case of nitroxides, to a nitrogen 

 nucleus, which is itself a magnetic entity. Thus in 

 the presence of an external field, the nuclear 

 magnetism can couple with the external field and 

 alter the magnetism immediate to the electron. 

 We must now consider this situation, called 

 hyperfine splitting (hfs). 



Hyperfine Splitting (hfs) 



Simply stated, it is found that the nitrogen 

 magnetism can add to, subtract from, or be 

 orthogonal (no interaction) to the external field. 

 This is depicted in Figure 2. 



Note that the situation of Figure 1 is now 

 modified. The original resonance (flipping) condi- 

 tion is broken down into three resonances (hfs). 

 The flipping from one level to the other is depicted 

 by the double ended arrows A, B, and C. Certain 

 restrictions are placed on flipping, and we find that 

 only those shown are allowed. All levels are equally 

 populated, and an actual EPR spectrum of a ni- 

 troxide in dilute solution is shown in Figure 3. 



Spin-Label Spectra 



While the spectrum of Figure 3 tells us most 

 conclusively that we are dealing with a nitroxide. 



MMNET ON  NITNOeEN NUCLEUS 



CONTRItUTON 



MABNET ON 



HA6NET OFF/ 



-MAGNETIC FIELDS ADD 



-NO PARTICIPATION 8Y NITROOEN 

 -MAGNETIC FIELDS SUBTRACT 



Figure 2.— Resonance condition for the case of one electron 

 interacting with a nitrogen-N" nucleus. Due to quantum 

 mechanical restrictions, only those transitions shown are 

 allowed. 



it serves no further purpose other than a possible 

 quantitation of the amount of radical (number of 

 spins) present. If all we ever measured were three 

 sharp, hyperfine lines, we could not use nitroxides 

 as spin-labels. 



Fortunately, when a nitroxide is placed in an 

 actual biological system, the hyperfine lines are 

 modified both in shape, intensity (relative height), 

 and in spacing. These spectral modifications are 

 environment dependent, and it is this dependency 

 which makes nitroxides valuable as probes for 

 characterizing biological systems. 





-13 Ogauss — 



-13 Ogauss - 



J 



Center line at 3379 gauss 



MAGNITIC FIELD STKEnGTH - 



Figure 3.-EPR spectrum of the nitroxide compound potassium 

 peroxylamine disulfonate (Freemy's salt) (lO-'M) in water at 

 room temperature. Three hyperfine lines of equal intensity, and 

 spaced 13 gauss apart, distinguish nitroxides in water. 



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