COLLAGEN IN FOSSIL BONE

Amino acids have frequently been demonstrated1 in acid solutions of well-preserved fossils, some of great age. They are undoubtedly often fragments of proteins that originally were present but whose inherent instability is such that they do not persist over geologically significant periods of time. Nevertheless, there are reports of collagen found in mummified human remains2 several thousand years old, and in mastodon bones3 preserved under permafrost conditions; and recently it has been stated4 that this protein has been identified in the fossilized antlers of a deer-like animal of Miocene age (4 30,000,000 years). Evidently it is of fundamental importance to determine if any protein, considered as the basic material of all living organisms, can endure, for its detailed examination and analysis could make substantial contributions to our knowledge of evolutionary processes. The search for such ancient proteins is one aspect of the broader study that can now be made of the microstructure and composition of the fossilized remains of earlier forms of life. MIicroradiography using X-rays of appropriate wavelengths can establish at the microscopic level whether original structures have been preserved or have been replaced through mineralization; X-ray spectroscopy and diffraction can determine the nature of whatever replacement has occurred. In the case of fossilized teeth, bones, and shells, the calcium phosphate and carbonate that were their original minerals can be selectively removed by chemical means, and microanalyses made of both the organic and inorganic components of the residues thus obtained. The present paper is a preliminary account of the nature of carbonaceous residues from Early Pleistocene bones collected from the Mendivil Ranch locality (Benson fauna) in Southern Arizona. The specimen analyzed in detail was the heel bone (calcaneum) from an animal of the genus Equus5. All samples chosen for study were ones whose fragments were positive for carbon when charred in the absence of oxygen, and whose residues showed no optical or electron microscopic evidence of recent microorganismal invasion. This examination for invasion has been found essential because experience shows that in a fossil the presence of actively growing molds may not be macroscopically apparent. Obviously, subsequent analysis for protein or amino acids is meaningless if such organisms are present. Thick sections of cleaned bone were decalcified by prolonged treatment at room temperature in 1-2.5% trichloroacetic acid, sometimes in the presence of 4% formaldehyde to prevent attack by airborne bacteria. For electron microscopy the residues from this acid treatment, with pulverization either before or after decalcification, were washed repeatedly by centrifugation. A final suspension in water was dried down on the usual collodion-covered grids, shadowed with chromium, and strengthened for microscopy by the vertical evaporation of a thin layer of carbon. Examination was carried out with a small Philips EM\75 electron microscope. Fields rich in fibrous material were common, and many of these