818 FLUORESCENCE OF PIGMENTS IN VIVO CHAP. 24 



cence of some plants is highly sensitive even to minor changes in humidity : 

 for example the fluorescence of Pleurococcus colonies on wood bark van- 

 ished after one or two hours in an atmosphere of less than 80% relative 

 humidity (at 25° C.) ; while the fluorescence of Mnium pundatum disap- 

 peared when the humidity declined below 85%. The fluorescence of the 

 leaves of Adiatum and Paretaria was found to be somewhat less sensitive, 

 but it, too, ceased to be visible after one or two days in an atmosphere of 

 75% relative humidity. 



The fluorescence of sharply dried leaves cannot be restored by simple 

 wetting, but returns upon immersion into boiling water. A similar trans- 

 formation of the "sensitive" fluorescence of live cells into the "stable" 

 fluorescence of dead cells can be achieved by freezing or immersion into 

 ether. In the latter case, the fluorescence after the treatment is consider- 

 ably stronger than it was in the living state. 



Seybold and Egle interpreted these results as indication that practically 

 all chlorophyll in leaves is present in a nonfluorescent (probably, protein- 

 bound) state, but that a small fraction of the pigment is dissolved in a 

 lipide phase, and therefore capable of fluorescence. They suggested that, 

 upon drying, the fraction of chlorophyll normally present in the lipide 

 phase is transferred into the colloidal aqueous phase, while, upon heating, 

 chlorophyll is first extracted from the lipide phase into the colloidal pro- 

 teinaceous phase (thus causing the fluorescence to disappear), but later 

 returns into the lipophilic material (concomitantly with the denatura- 

 tion of the proteins and melting of lipides) , and thus again becomes fluores- 

 cent. (Metzner 1937 also had attributed the "burst" of fluorescence 

 caused by heating to the melting of the lipides.) Underlying this "two- 

 phase" hypothesis of Seybold and Egle was the conviction that all chloro- 

 phyll-protein complexes are nonfluorescent. However, while this seems 

 to be true enough of pure chlorophyll-protein precipitates (cf. page 775), 

 it does not apply to complexes which contain both proteins and lipides 

 (e. g., to "coacervates" of the type described by Hubert and Frey-Wyssling ; 

 cf. chapter 23, page 777). Seybold and Egle's argument is therefore not 

 convincing. The effects of heating and drying on chlorophyll fluorescence 

 in vivo can be explained in a much simpler way than suggested by Seybold 

 and Egle : by assuming that the pigments normally contained in a weakly 

 fluorescent protein-chlorophyll-lipide complex lose the protection against 

 self-quenching (and therefore become nonfluorescent), when the lipides 

 melt in the heat and form a separate phase, but diffuse into this new phase 

 if the pigment-protein link is broken by denaturation {e. g., by somewhat 

 more prolonged heating) and thus again become fluorescent. The dis- 

 placement of the fluorescence bands of chlorophyll in living cells (by 5-15 

 mjLi toward longer waves from their position in organic solvents) agrees 

 with the assumption that fluorescence is emitted by the same chlorophyll 

 molecules responsible for the (similarly displaced) absorption bands. 



