328 JAMES H. C. SMITH 



18 for a glycine buffer extract (pH 9-5). Because of the irradiation 

 necessary for measuring fluorescence polarization, the protochlorophyll is 

 largely converted into chlorophyll, consequently, the fluorescence obtained 

 is mostly irom chlorophyll. These values, 15 and 18, are lower than the 

 value for chlorophyll in castor oil, 28-9. Three possibilities suggest them- 

 selves to account for the lowered polarization: one, that the holochrome 

 rotates more freely than chlorophyll immobilized in castor oil; two, that 

 it transfers its energy to other chlorophyll molecules; or three, that the 

 pigment is free to rotate within the holochrome. 



Because the holochrome is so large, it cannot conceivably rotate fast 

 enough to depolarize its fluorescence. The second suggestion of energy 



0-4 08 1-2 



Chlorophyll absorbance 



Fig. 2. The reciprocal of the fluorescence polarization of the protochlorophyll- 

 chlorophyll holochrome plotted against the optical density of the chlorophyll 

 maximum ( ~ 670 m^u) at different stages of greening. 



transfer between chlorophyll molecules also seems improbable in view of 

 the small number of pigment molecules per holochromatic particle. This 

 leaves only the third alternative as likely. 



An estimate of the limiting fluorescence polarization value when no 

 energy transfer exists can be obtained by extrapolating the fluorescence to 

 zero pigment concentration. This was done by extracting the holochrome 

 from leaves at different stages of greening, and by relating the fluorescence 

 polarization with chlorophyll content through the expression 



i/P = iIPo + ACt (8) 



in which P is the polarization of fluorescence measured ; A is a constant ; 

 C is the optical density of the chlorophyll peak at about 670 rufx, which is 

 proportional to the chlorophyll content; r is the lifetime of the activated 

 state; and Pq is the polarization when C is zero. A plot of ijP against 



