Survival of Histological Structure and Biochemical Constituents in an Ancient Mummified Weddell Seal. Part III. Survival of Biochemical Constituents

  1. William Henry Burt
  1. Lung-Hsiung Hsu and
  2. Elmon L. Coe

Published Online: 3 APR 2013

DOI: 10.1029/AR018p0207

Antarctic Pinnipedia

Antarctic Pinnipedia

How to Cite

Hsu, L.-H. and Coe, E. L. (1971) Survival of Histological Structure and Biochemical Constituents in an Ancient Mummified Weddell Seal. Part III. Survival of Biochemical Constituents, in Antarctic Pinnipedia (ed W. H. Burt), American Geophysical Union, Washington, D. C.. doi: 10.1029/AR018p0207

Author Information

  1. Department of Biochemistry, Medical School Northwestern University, chicago, Illinois 60611

Publication History

  1. Published Online: 3 APR 2013
  2. Published Print: 1 JAN 1971

ISBN Information

Print ISBN: 9780875901183

Online ISBN: 9781118664773

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Keywords:

  • Deae cellulose chromatography;
  • Lipid constituents;
  • Liver fractions;
  • Macromolecules and mucopolysaccharides;
  • Mummified Weddell seal;
  • Soluble RNA (S-RNA);
  • Triose phosphate isomerase;
  • Water content

Summary

The biochemical constituents in the skin and liver of a recently dead frozen baby Weddell seal (frozen and maintained at −20°C for 2 or more years) were compared with the constituents in corresponding organs of a mummified carcass of a young Weddell seal found in the Taylor Valley of Antarctica and radiocarbon-dated as being about 1400 years old. The seal mummy was severely weathered on one side and was highly dehydrated, containing only 12% water in the skin and liver, as compared with 54% in the skin and 72% in the liver of the recent seal. The ancient seal tissues showed signs of extensive chemical decomposition. One of the most striking features of the ancient seal tissues was the presence of an ubiquitous yellow-brown contaminant with a relative absorption maximum around 300 nm; it was readily water soluble, appeared in most protein fractions, and interfered with many standard spectro photometric analyses. The much higher quantity of acid-soluble nitrogen in the ancient seal tissue also indicated extensive degradation of polymers to small molecules. The total nitrogen content per gram dry weight was identical in the two livers, however; thus all the original material from the ancient liver was still present in some form.

Of the usual tissue constituents examined, the neutral lipids appear to have survived the best; the cholesterol and triglyceride content of the ancient liver was 50–100% of the content in the recent liver. Phospholipids seem somewhat less stable, amounting to 10–23%. The poly peptide fraction of ancient seal liver contains about 10% of the equivalent recent liver fraction, although much of this may have been larger fragments of the partially decomposed original protein. Several techniques for detection of RNA were tried, but no RNA could be demonstrated in the ancient liver or skin; the most sensitive test implied that the RNA content would have to be less than 1% of the content in the recent liver to escape detection. Hexosamine determination on a crude mucopolysaccharide preparation from skin indicated that as much as 6% may have survived, but uronic acid determination gave values of less than 1%. Assays for several enzyme activities demonstrated the presence of triose phosphate isomerase and catalase in the ancient liver at levels of, respectively, 0.005% and 0.03% of the activity in the recent liver. Lactate dehydrogenase, malate dehydrogenase, myokinase, monoamine oxidase, and esterase were all easily measurable in recent seal tissues, but were not detected in the ancient seal. The activities of the two enzymes found are sufficiently low to raise the possibility that they were derived from some more recent contaminant rather than the seal itself, although circumstantial evidence favors the seal as the source.

Comparisons of the catalytic properties of the triose phosphate isomerases from the two seals revealed several significant differences. The Michaelis constant K m of the enzyme extracted from the ancient seal liver, with glyceraldehyde-3-phosphate as a substrate, was much higher (1.5 mM) than the constant of the enzyme from recent liver (0.33 mM ), which corresponded to values reported for the triose phosphate isomerase from other mammalian sources (0.3–0.5 mM). To determine whether such a change in K m could be produced by spontaneous alterations in the protein, preparations of recent seal enzyme were exposed to mild heat treatment and then assayed; exposure to 62°C for 40 min inactivated about 70% of the enzyme and increased the K m of the surviving enzyme from 0.3 to 0.8. A comparison of heat inactivation of the enzymes from the two seals showed that the enzyme from the ancient seal is actually somewhat more sensitive to heat than the enzyme from the recent seal and therefore ruled out the possibility that the enzyme was originally a minor, but much more stable, isozyme in a population of isomerases. The pH optima of the two enzymes are indistinguishable (between 7.0 and 7.5), although the isomerase from the ancient seal exhibits a greater instability in the high pH range. Qualitative comparisons of electrophoretic mobilities on cellulose acetate in pH 8.6 barbital buffer, with an enzyme specific stain, indicated that the enzyme from the ancient seal liver has a slightly higher mobility toward the cathode than does the enzyme from the recent seal liver or crystalline rabbit muscle triose phosphate isomerase, which moves at the same rate as the recent seal enzyme.

A study of the relative abundances of amino acids in the cold-acid-soluble fraction of ancient seal liver led to some tentative conclusions about the relative stabilities of these amino acids. The free amino acid content of the ancient liver is about 15 times greater than the content of the recent liver, which indicates that most of this quantity has been derived from degradation of protein; the amount accounts for about 67% of the nitrogen lost from the alkali-soluble polypeptide fraction. Since not all amino acids are equally abundant in tissue protein, the ratio of the free amino acid content in ancient seal liver to the content in the bulk protein of rat pancreas was calculated for each amino acid, and these ratios were compared. The ratio for valine was arbitrarily set at 1.00, since the valine peak obtained on ion exchange chroma tography was large, sharp, and well separated from the other components, and other ratios were adjusted accordingly. Of the 18 amino acids estimated, 8 gave abundance ratios ranging from 0.6 to 1.1 and therefore probably represent the relatively stable molecules that survived in amounts approximating the original distribution; these amino acids included cystine, valine, lysine, glycine, isoleucine, leucine, phenylalanine, and proline. Two amino acids, tyrosine and alanine, gave much higher ratios (2–3), which possibly indicates an unusual abundance in seal liver, but which more probably resulted from a formation of these amino acids from other amino acids. Both aspartic and glutamic acids yielded low ratios (0.3–0.4), which suggests some instability in these molecules, and 5 amino acids, arginine, histidine, methionine, serine, and threonine, gave ratios of less than 0.1, which indicates that these amino acids were highly unstable. How much of the instability was due to enzymatic factors rather than inherent chemical instability is difficult to assess. The abundance of the eighteenth amino acid, tryptophan, was estimated roughly from paper chromatograms to be about 0.5. Although less accurate, this estimate indicates that tryptophan belongs in the relatively stable group.