82 MACROMOLECULAR COMPLEXES 



a relatively small amount of the phosphorus is bound by the collagen, making 

 quantitative determinations difficult. Secondly, the amount of phosphorus- 

 binding is so small that the correction for the amount of phosphorus in the 

 solvent water of the fibrils is proportionately large. Since there is no way as 

 yet of determining accurately the amount of "bound" or "free" water in the 

 fibrils, we have been unable to correct for this factor quantitatively. Thirdly, 

 part of the phosphorus appears to be irreversibly bound to the collagen; in 

 fact, there may be a spectrum of phosphorus-binding with different binding 

 energies, and when this is coupled with diftusion phenomena, the final results 

 are as yet impossible to interpret. Similar difficulties have been encountered 

 by workers using S-^-^ to study the sulfate-binding of solid keratin structures 

 such as hair (Underwood and White, 1954). 



In relation to the mechanism of nucleation, when phosphate-binding was 

 measured in solutions the concentrations of which were similar to those used 

 in calcifying solutions (calcium and phosphate solutions), the amount of phos- 

 phate bound was very small compared to the amount of phosphorus bound or 

 otherwise adsorbed in the initial stages of nucleation before detectable amounts 

 of crystallization had occurred. This has suggested that the "building up" of 

 the nucleus is not a simple addition of phosphate and calcium ions independent 

 of each other, but rather a cooperative phenomenon in which the calcium ions, 

 phosphate ions, and certain amino acids interact collectively to influence the 

 state of aggregation of the mineral ions. To this must be added the possibility 

 that specific clusters of calcium and phosphate ions are the initially bound 

 moiety. 



Dr. Green (Rutgers University): Have you examined the collagens in 

 elasmobranch fishes? Have you any information bearing on the failure of these 

 collagens to be mineralized? 



Dr. Glimcher: We have onlv done a few experiments with the so-called 

 elastoidin of shark fins. As you know, this collagen has some of the low-angle, 

 as well as the wide-angle, x-ray diffraction properties of other native collagens, 

 but is cross-linked and very insoluble. The tissues also contain another as-yet- 

 unidentified protein (Gross and Dumsha, 1958). Since we were not able to 

 get the elastoidin free of other constituents, or to solubilize and reconstitute 

 the fibrils, we were unable to investigate this system adequately. 



I can only speculate as to why such tissues are not normally mineralized. 

 We do not know whether, in fact, the collagen in elastoidin has the same 

 molecular structure and macromolecular aggregation state as other native 

 collagens. According to the concept which I presented today, however, if 

 the structural characteristics were the same, one would have to investigate: 

 (1) the metastability of the extracellular fluids; (2) the possibilitv of the 

 presence of inhibitory substances; and (3) the lack of regulatory mechanisms, 

 presumably enzyme-controlled, which are probably needed to initiate the 

 nucleation process in vivo. We could presumably get some useful information 

 about these questions if it were possible in vitro to test the mineralizing 

 capacity of both the native tissue and the purified elastoidin in a fashion similar 

 to the native collagen-rich tissues, and of the purified collagen from these 

 tissues in a fashion similar to the experiments which I have already described. 



