62 MACROMOLECULAR COMPLEXES 



nective tissues it is possible to reconstitute in vitro not only the 

 native-type (640-700- A axial repeat) collagen fibrils, but also a 

 number of other fibrillar forms (Schmitt et al, 1942; Highberger 

 et al, 1951; Gross et al, 1954, 1955; Schmitt et al, 1955a, 1955b). 

 These various fibrillar forms represent different aggregation states 

 of the same macromolecules and therefore reflect differences be- 

 tween the stereochemical relations of the amino acid sidechain 

 groups of adjacent macromolecules. Since it is also possible 

 to isolate the macromolecules and linear aggregates of the macro- 

 molecules (protofibrils), we were thus provided with a unique sys- 

 tem which would not only test the nucleation hypothesis, but which 

 also could distinguish whether this property (if such existed) was 

 a function of the structure of the macromolecule itself or was de- 

 pendent on steric factors and a characteristic electrical charge dis- 

 tribution provided by the aggregation of the macromolecules into 

 fibrils (Fig. 6). 



The experiments were conducted by exposing the various types 

 of fibrils, macromolecules, and protofibrils, as well as previously 

 demineralized bone collagen (shown by low-angle x-ray diffraction 

 to have maintained its 640-A long-range fibrillar order) to meta- 

 stable calcium phosphate solutions. 



Under identical physicochemical conditions, only the native-type 

 (640-700- A axial repeat) reconstituted collagen fibrils and the de- 

 mineralized bone collagen were able to nucleate apatite crystals 

 from metastable calcium phosphate solutions. The failure of the 

 various other fibrils, representing different aggregation states of the 

 same macromolecules, to initiate this phase change demonstrated 

 the rather remarkable steric specificity required in the nucleation 

 phenomenon. Since it was also demonstrated that the individual 

 tropocollagen macromolecules and the protofibrils were unable to 

 initiate crystallization, it was evident that the specificity did not lie 

 in the macromolecule itself, but in the specific manner in which the 

 collagen macromolecules were packed to make native fibrils, i.e., 

 their macromolecular aggregation state. The structural matching 

 in the case of collagen depends in the first instance upon the integ- 

 rity of its molecular structure, which assures a specific linear array 

 of amino acid residues in the covalent polypeptide chains. Yet even 

 when this condition is met it is not sufficient to provide a nucleation 

 site. Nucleation occurs only when the tropocollagen particles are 

 aligned both laterally and longitudinally in the specific array char- 



