PHYSICAL AND CHEMICAL PROPERTIKS — PROTOCHLOROPHYLL HOLOCHROME 33 1 



the pigment attaches itself to protein through a bonding that is sensitive 

 to hydroxy! ions, such as amino groups that form ammonium compounds, 

 or phenohc groups that act through hydrogen bonding. This may be 

 iUustrated as follows: 



R— NH2 + HOR' = R^NHa H+ OR' 



When the acidic hydrogen ion is neutralized the addition compound 

 dissociates 



R— NH., H+ -0R'+ OH = R— NH0 + HOH+ -OR' 



In the case of the protochlorophyll-protein complex, when it is dis- 

 sociated by hydroxyl ions, the protochlorophyll could no longer be 

 transformed to chlorophyll by light. This is the proposed explanation for 

 the inhibition of the transformation by action of hydroxyl ions. 



The pH values that inhibit the photochemical transformation corre- 

 spond to the dissociation constants of certain amino acids which may be 

 implicated in the bonding of protochlorophyll. These amino acids with 

 their approximate pK values [12] are e-amino of lysine, 9 -410 10 -6, phenolic 

 hydroxyl of tyrosine, 9-8 to 10-4, and the sulphydryl of cysteine, 9-1 to 

 10 -8. These values may differ considerably from one protein to another, 

 and even in the same protein. For example, Stracher [13] found in the 

 spectrophotometric titration of myosin two groups ol tyrosine residues 

 with pK values of 10-5 and 12-2 respectively. For this reason, no precise 

 values for the pK values of the amino acids in the protochlorophyll 

 holochrome can be assigned a priori. 



IONIZATION CONSTANTS FROM TITRATION CURVES 



'Fhe titration cur\e of protochlorophyll holochrome is shown in Fig. 4. 

 From this curve it is obvious that two titration steps exist within the pH 

 range effectively inhibiting transformation of protochlorophyll. The 

 inflection points are at pH values of about 10-2 and 11 "3. For groups with 

 these pK values, ionization would be about 10",, complete at pH 9-2 and 

 10-3. Thus it appears that the coincidence of the pK values from the 

 titration curve and the pH values efl'ectively inhibiting protochlorophyll 

 conversion makes the assumption reasonable that the pigment is bound 

 to protein through the amino acid residues — the most likely candidates 

 being lysine, cysteine, and tyrosine. 



IONIZATION CONSTANTS FROM SPECTROSCOPIC MEASUREMENTS 



Changes in pH profoundly modify the protein part of the proto- 

 chlorophyll holochrome as variations of the ultraviolet absorption spectrum 

 show. In Fig. 5 is pictured the absorption of protochlorophyll holochrome 



