1834 SPECTROSCOPY AND FLUORESCENCE OF PIGMENTS CHAP. 37C 



one of the low quantum yield of photoxidation of chlorophyll (cf. chapter 

 18). 



The quenching effect of quinone (and other oxidants) on chlorophyll 

 fluorescence is shown by these experiments to be unrelated to the presence 

 of the two extra ring hydrogens in the chlorin system (since it is equally 

 strong in porphyrins, which contain no such hydrogen atoms). 



In continuation of the work of Livingston and Ke on quenching of 

 chlorophyll fluorescence (Vol. II, Part 1, chapter 23, pp. 781, 787), and of 

 Livingston, Watson and McArdle on its activation (chapter 23, p. 766), 

 Watson (1952) described in more detail the effect of phenylhydrazine. 

 With chlorophyll a in a polar solvent (such as methanol), a quenching was 

 found that followed the Stern-Volmer equation, with a constant Ki = 3.2 

 liter/mole (table 23.IIIC gave A'l = 3.7 l./mole). Phenylhydrazine has no 

 noticeable effect on the absorption spectrum of chlorophyll a in methanol; 

 nevertheless, quenching seems to be due to complex formation (rather than 

 to kinetic encounters), because it develops only several minutes after the 

 addition of the quencher. This complex formation is reversible; at least, 

 a gradual return of fluorescence with time was noted when acetone was used 

 as solvent (instead of methanol), and this could be attributed to disappear- 

 ance of phenylhydrazine by the formation of hydrazone. 



With chlorophyll b in methanol, the previously described change in 

 absorption spectrum upon addition of phenylhydrazine (cf. pp. 649, 786) 

 was confirmed. The absorption peak moves from 652 to 672 m^u. 



When chlorophyll a is dissolved in a rionpolar solvent (benzene, or n- 

 pentane), small quantities of phenylhydrazine (up to 0.05 mole/1.) activate 

 fluorescence (cf. table 23.IIIA), while larger quantities produce quenching 

 (as stated on p. 768); the absorption spectrum is changed significantly 

 only in the activating stage. An expression was proposed for the intensity 

 of fluorescence as a function of phenylhydrazine concentration that assumed 

 two independent associations of chlorophyll with phenylhydrazine (P) — 

 one causing activation and the other quenching : 



(37C.5) <p/<Pma.. = A',[P] + A'3[A]/|1 + A', [A] + (A', + A'.,)[P] + AiA^PJ^} 



where Ki and Ko are the two association constants for Chi and P, and Ks is 

 the association constant for Chi and an "adventitious" activator. A, which 

 is supposed to account for residual fluorescence in nonpolar solvents. The 

 empirical data fit this equation with A'l = 1900 (for benzene or n-heptane), 

 K2 = 16 (benzene) or 58 (n-pentane), and AsfA] = 0.27 (benzene) or 0.16 

 (n-heptane). 



(For application of these results to photosensitization of the phenyl- 

 hydrazine-methyl red reaction, see chapter 35, section 5.) 



Evstigneev, Gavrilova and Krasnovsky (1950) pointed out that the 



