MACROMOLECULAR AGGREGATES IN CALCIFICATION 69 



though there are a host of such enzymes for phosphate. Further- 

 more, if Ca++ were transferred enzymaticallv to the collagen, any 

 covalent chelating or co-ordinating structure formed would be very 

 unreactive, i.e., would not be very accessible to reaction with the 

 other ions making up the crystal lattice; such bonds would tend to 

 demineralize (similarly to other chelates like ethylenediaminetetra- 

 cetic acid, EDTA), rather than to initiate nuclei formation. On the 

 other hand, covalent phosphate compounds such as -N-P or -O-P 

 alkyl amides or esters would still be very reactive, with Ca + + for 

 example. Since the phosphate groups are not only the struc- 

 tural "backbone" of the apatite lattice, but are also more Hkely to 

 be involved from the biological standpoint, their role in the forma- 

 tion of the initial fragments of the crystal lattice or nucleus would 

 appear to be equally as important as that of the calcium ions, and 

 probably more important. Indeed, the proposal that phosphoryla- 

 tion of free amino groups constitutes the initial step in calcification 

 has been made in the past (Polonovski and Cartier, 1951; Cartier, 

 1951, 1952; Cartier and Picard, 1955; Zambotti, 1957), and it has 

 been suggested that pyrophosphate radicals are the initially bound 

 phosphate moiety (Cartier, 1959). 



Two general methods of attack are possible in an effort to ascer- 

 tain the reactive amino acids involved in the nucleation phenomena. 

 In the first, which we may term the "availability" method, one may 

 determine the difference ( if any ) between the number of side-chain 

 groups of a particular amino acid available in the fully mineralized 

 matrix and the number available in demineralized matrix, the as- 

 sumption being that groups which are not reactive in the mineral- 

 ized tissue are not available because of interaction with the mineral 

 ions. In the second method, specific amino acid side chains of de- 

 mineralized collagens are blocked or altered, and the effect of such 

 alterations evaluated by comparing the amount of mineral deposited 

 during in vitro calcification with untreated controls. 



Solomons and Irving ( 1958 ) have studied the availability of the 

 epsilon amino groups (e-NH^) of lysine and hvdroxylysine in bone 

 and dentin by reacting such tissues with l:fluoro:2:4 dinitrobenzene 

 (FDNB). They noted that, as bone and dentin were demineral- 

 ized in stages by a mild chelating agent (EDTA), there was a direct 

 relation between the availability of such groups to FDNB and the 

 amount of total mineral present in the sample (calcium and phos- 

 phate) (Fig. 11). 



