133 



Helmut Beinert and Bessel Kok 



radical species are fully detectable by EPR and that no 

 interactions interfere with this detectability . 



The concentration of unpaired electrons was measured by 

 EPR spectroscopy at ambient and low (-50" to -70") tempera- 

 ture as will be described in detail elsewhere (12). For the 

 experiments at room temperature a double cavity was used, 

 which held both sample and standard in matched flat cells. 

 A benzene solution of diphenylpicrylhydrazyl (DPPH) was used 

 as a steindard at room temperature and a pitch sample in KCl 

 and nitrosyldisulfonate in KOH at low temperature. The 

 standards as well as the integration procedure used were 

 cross checked. Conditions were chosen that saturation of the 

 EPR signals with microwave power did not occur or was suf- 

 ficiently small that it could be corrected. In order to 

 ensure saturation of the pigment suspension witn light, i.e. 

 maximal signal development, the signal amplitude of serial 

 dilutions of these suspensions was measured until a linear 

 relationship between signal amplitude and concentration was 

 observed. The double integration of the derivative signals, 

 which resulted in the quantitative estimate of unpaired spins, 

 was based on signals obtained in this linear region. Values 

 were also observed in the dark and after addition of ferri- 

 cyanide. The value obtained with ferricyanide can in many 

 cases serve as a control for light saturation and maximal 

 signal development, as the concentration of unpaired elec- 

 trons produced by an excess of this oxidant is either very 

 similar to that obtained on illumination under saturating 

 conditions or larger. Only materials were selected for study 

 in which overlapping signals (broad signal, 20 gauss, slow 

 decaying or "S" signal, and Mn (II) signal) were absent or 

 small. Overlap, when occurring, was corrected. 



Lumn 



Results obtained under satisfactory experimental condi- 

 tions are summarized in the table. The last vertical col\ 

 gives the calculated ratio of unpaired spins per P700, Most 

 values cluster around a ratio of 2, although the low tempera- 

 ture experiments on the TX 27 preparations and the experi- 

 ments on Anacystis yielded higher ratios. We have no 

 explanation for the high values obtained at low temperature. 

 Although a different standard (pitch) was used at low temper- 

 ature than at room temperature, a comparison of both stand- 

 ards gave excellent agreement. It is likely that the high 

 values obtained for whole Anacystis and the same preparation 

 after sound treatment are due to overlap of the narrow EPR 

 signal with the broad light induced EPR signal in such 



