28 : 5/ Magnetic Measurements 535 



volume used could be reduced to the active volume. Likewise, the high 

 minimum-concentration limit on the electron spin resonance apparatus 

 can be reduced by compensating for the water absorption. 



The Gouy balance has been used to investigate heme proteins 

 including hemoglobin, catalase, peroxidase, and cytochrome c. In 

 several cases, more than one stable form can be prepared. It has 

 also been used to observe the cuprous-cupric change in the enzyme 

 laccase. 



The Rankine balance at the Johnson Foundation has been used to 

 observe the reactions of metmyoglobin, catalase, and peroxidase. It 

 has also been used to observe free radicals in the conversion of xanthine 

 to uric acid catalyzed by xanthine oxidase. These free radicals were 

 not detected by spectrophotometric methods. 



Electron-spin resonance apparatus has been used to study metmyo- 

 globin and methemoglobin reactions in the frozen state. It has also 

 been used to demonstrate free radicals in dried chlorophyll and in protein 

 pastes. ESR data have been interpreted to indicate that certain flavo- 

 protein-enzyme intermediates are free radicals. Some of these experi- 

 ments are discussed in papers cited in References 5 through 9. The use 

 of electron-spin resonance in locating the iron atoms in heme-protein 

 crystals is referred to in Chapter 15. 



A striking example of the use of electron-spin resonance techniques 

 occurred in studies of the peroxidation of ascorbic acid. Absorption 

 spectrophotometry showed that the enzyme, peroxidase, reacted with 

 hydrogen peroxide to form a first intermediate complex. This then 

 reacted with ascorbic acid to form a second intermediate complex and 

 then the free enzyme. However, the enzyme complexes change in 

 steps involving one electron, whereas the oxidation of ascorbic acid and 

 reduction of hydrogen peroxide involve two electron changes. Measure- 

 ments of electron-spin resonance showed the existence of a free radical. 

 It was postulated that the following scheme could account for all the 

 observations 



E + H 2 2 -± E-Sj. 



E-Sj + AH 2 ^E-S U + AH- 



E-S n + AH 2 -± E + AH- + 2H 2 



2AH- -^A + AH 2 



where 



E = peroxidase 

 AH 2 = ascorbic acid 

 AH - = ascorbic acid free radical 

 E-S = enzyme-substrate intermediate complex 



A = dehydroascorbic acid 



