76 MACROMOLECULAR COMPLEXES 



TABLE 6 



Reversible Blocking of the e-NH:. Groups of Reconstituted Collagen 



(Ichthyocol) by Carbobenzoxychloride and the Concomitant 



Change in Its Recalcification in vitro 



fi mole e-NHo Groups Mg Ca/g Mg P/g 



Specimen Available/g Collagen Collagen Collagen 



Calcified Approximately 60 Hours at 25° C 



Control 198 68.2 34.7 



Treated with carbobenzoxy- 

 chloride : 39 3.5 2.3 



After catalytic removal 



of carbobenzoxy groups 180 40.4 23.8 



Calcified Approximately 36 Hours at 25° C 



Control 242 26.5 14.9 



Treated with carbobenzoxy- 

 chloride 42 1.3 0.65 



After catalytic removal of 



carbobenzoxy groups 97 5.5 1.8 



lently bound to the mineral ions ( presumably phosphate ) , and sub- 

 ject to the misgivings about the steric or electrostatic hindrance as 

 noted earlier in interpreting the e-NH., blocking experiments which 

 showed the importance of such groups in in vitro calcification, we 

 may interpret this to mean the interaction between the vast majority 

 (95 per cent or more) of the e-NH^ groups and mineral ions is 

 electrostatic. This does not rule out the possibility that the initial 

 event, particularly in vivo, is a phosphorylation of collagen to form 

 either -N-P-alkyl amides or -O-P-ester linkages. Only a small num- 

 ber of such phosphate esters or amides need be formed to start the 

 nucleation process, and the remaining phosphate ions as well as 

 calcium ions could then simply be electrostatically attracted to the 

 rest of the polar amino acid side chain groups of the collagen and to 

 each other. 



Further experiments are now in progress in which a number of 

 other amino acid side-chain groups are being blocked singly and 

 in combination, and in which not only the final result of nucleation 

 (i.e., the amount of mineral formed) but also the binding of the 

 various constituent mineral ions is being studied. These experi- 

 ments should lead to a clearer understanding of both the exact roles 

 of specific amino acids and mineral ions and the sequence of events 

 leading up to nuclei formation. 



