MACROMOLECULAR AGGREGATES IN CALCIFICATION 75 



enough and sufficienth' suggestive to warrant discussion here. Our 

 attention has been primarily concentrated on the e-NH^ groups of 

 h'sine and hvdroxvlysine partly because this group is quite easily 

 l)locked without altering either the molecular structure of collagen 

 or its macromolecular aggregation state and partly because of the 

 likelihood that this group would be involved in phosphate-binding, 

 which we feel is an important step in nucleation. Experiments in 

 which the e-NH. groups were specifically blocked in varying 

 amounts by several different reagents showed a definite correlation 

 between the number of e-NHo groups available and the total amount 

 of mineral deposited. Fig. 12 and Table 5 give examples of the re- 



TABLE 5 



Effect of Blocking the e-NH_. Groups of Deminerolized Fish Bone 



(Collagen) with l:Fluoro:2:4 Dinitrobenzene (FDNB) 



on Its Subsequent Recolcificotion in vitro 



( Recalcified at 25° C for approximately 48 hours) 



* A sham control which was exposed to the same reagents under 

 identical conditions of pH, etc., but without the addition of FDNB. 



suits of such experiments. One of the most convincing pieces of 

 evidence thus far was the demonstration that the e-NH^ groups 

 could be reversibly blocked, and that there was a concomitant 

 change in the amount of mineral deposited (Table 6). 



While such experiments indicate that the group substitution has 

 been carried out without destroying the molecular structure of the 

 collagen, they do not rule out the possibility that the observed re- 

 sults are, in fact, due to the shielding ( either electrostatic or steric ) 

 of groups adjacent to the e-NH. groups, rather than to the e-NH^ 

 groups themselves. 



Since availability studies of fully demineralized bone and dentin 

 indicate at least 95 per cent of the e-NH;, groups are not cova- 



