PHYSICAL AND CHEMICAL PROPERTIES — PROTOCHLOROPHYLL HOLOCHROME 329 



chlorophyll absorbance, Fig. 2, gave a straight line within the experi- 

 mental error which extrapolated to a value of 15-4 for Pq. This is in good 

 agreement with the \alue of 15% obtained by direct measurement on the 

 protochlorophvll holochrome. This indicates that the lower value of 

 fluorescence polarization in the original protochlorophyll holochromes is 

 not due to energy transfer between chlorophyllous pigments. 



The third alternative put forward to account for the definite but sub- 

 maximal polarization of the holochromatic pigment is that the pigment 

 exists in the holochrome in such a way as to have partial freedom of 

 rotation. This could be accomplished if the pigment were attached to the 

 amino-acid "tails" of the holochrome protein similarly to haem in haemo- 

 globin and myoglobin [8, 10]. 



Alkaline inhibition of protochlorophyll transformation 



If the binding of haem in haemoglobin and of protochlorophyll in its 

 holochrome are analogous, then the bonding of protochlorophyll to 

 protein in the holochrome should be influenced by treatment with alkali 

 at specific pH values [n]. The eftectiveness of the various pH values for 

 disrupting the pigment-protein complex should depend upon the acid 

 dissociation constants of the amino-acid groups binding the pigment. 



Inasmuch as the transformation of protochlorophyll to chlorophyll is 

 stopped when the protochlorophyll is separated from the protein, a dis- 

 sociation by treatment with alkali should stop the transformation. The pH 

 at which the transformation is stopped should be characteristic of the 

 ionization constant of the protochlorophyll-amino acid complex involved. 

 Conversely, the pH values at which the transformation is stopped should 

 indicate what amino acid groups hold the pigment. For this reason, a 

 detailed studv of the efi"ect of alkali on the protochlorophyll holochrome 

 and on the inhibition of the transformation has been undertaken. 



Effect of pH on the protochlorophyll-chlorophyll transformation 



The various degrees of inhibition of the protochlorophyll-chlorophyll 

 conversion caused by treatment of the protochlorophyll holochrome at 

 diflFerent alkalinities are shown in Fig. 3. The protochlorophyll holochrome 

 was suspended in solutions of various pH values for different lengths of 

 time. At stated intervals, samples were removed, neutralized with glycine, 

 and spectrophotometered before and after being illuminated for 3 min. 

 The optical densities of the chlorophyll formed were measured at the 

 chlorophyll absorption maximum, ~ 678 m/t. In Fig. 3, thev are plotted 

 as ordinate against the time of standing in the alkaline medium. 



As is evident from Fig. 3, pH values between 7-20 and 9- 16 have little 



