Ancient proteins in fossils from

Venta Micena and Cueva Victoria

Enrique GARCÍA OLIVARES, Concepción BORJA

Unidad de Inmunología, Facultad de Medicina
Universidad de Granada

Proteins in fossils from Venta Micena and Cueva Victoria viere analyzed by Lowenstein at the University of
California, San Francisco with a radioimmunoassay (RIA), and by Borja and García-Olivares at the Immunology
Unit of the University of Granada with an enzyme-linked immunosorbent assav (ELISA). These independent
studies involved the use of polyclonal and monoclonal antibodies against albumin from different species.The
results from San Francisco (RIA) and from Granada (ELISA) viere compared at the International Conference on
Human Palaeontology, held in Orce in September 1995 (Borja and García-Olivares, 1995 and Borja in this
volume; Lowenstein, 1995). Both sets of data showed that the albumin detected in fossils VM-0 and VM-1960
was immunologically more similar to human albumin than to albumin from other species. Lowenstein also found
collagen and transferrin in VM-0, which were more similar to human proteins, and Borja and García-Olivares
detected IgG in VM-1960 that was more similar to human IgG. Neither group detected human albumin or IgG
in fossils CV-1 and CV-2 from Cueva Victoria, assigned to hominids, but both found the reactions for albumin
that would have been predicted from the morphological assignments of the three equid fossils and two bovid
fossils, and for IgG in four equid fossils. Not surprisingly, some fossils (two equid fossils and three fossils assigned
to hominids) failed to yield proteins. An unexpected result was the finding of human albumin in an equid fossil,
probably as a result of external human contamination. This has been used by Palmqvist as the basis for his
criticism of thc rest of the concordant results from the two groups (Palmqvist, 1997).

PALMQVIST'S CRITICISM OF VENTA MICENA FOSSIL PROTEINS

Palmqvist has recently praised the immunological work done by one of us on the proteins in the Venta
Micena fossils (Borja, 1995) in the Bulletin of the Spanish Society of Palaeoanthropology: "...Esta Tesis constituye
un trabajo de investigación modélico, que marcará un punto obligado de referencia en Paleontología Molecular..."
(This Ph D thesis constitutes a model piece of rescarch work that will be an obligatory reference in Molecular
Palaeontology) (Palmqvist, 1996). This contrasts sharply with his current opinion: "...The immunologic analysis
of this (VM-0) and other presumably hominid remains froni Venta Micena are far froni being conclusive..."
(Palmqvist, 1997) and more sharply with one of lis articles in a local newspaper (Ideal, 15.11.96) "...in view
of his (García Olivares) scant, contradictory results and proven lack of scientific method..." ("...a tenor de los
escasos y contradictorios resultados obtenidos por el momento en el curso de sus trabajos, salvo contrastar, aún
más si cabe, su demostrada carencia de método científico..."). His criticism can be summarized in the following
points:

1. The detection of human albumin in a equid fossil from Venta Micena.

2. A review paper noting the contradictory results obtained in analyses of blood residues on archaeological
artefacts (Fiedel, 1996).
3. The detection of very high amounts of proteins in such very old bones (Venta Micena).
4. The absence of IgG in bones buried for 2 months (Cattaneo et al., 1993), which appears incompatible
with the findings of Borja and García-Olivares (1995) of this protein in fossils from Venta Micena.

Let us examine these arguments.

AN UNEXPECTED IMMUNOLOGICAL REACTION IN THE FOSSILS FROM VENTA MICENA

The groups in Granada and San Francisco have studied the presence of albumin or IgG in a total of 12
morphologically well-matched equine and bovid fossils from Venta Micena (this sample does not include the
fossils assigned to hominids), and have detected only one unexpected result: an equid fossil in which a high

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 GARCÍA-OLIVARES, E.

reaction to human albumin was found.This result has been cited as a possible example of accidental contamination
(Lowenstein, 1995), an event which is not unsual when dealing with fossil biomolecules, particularly ancient
DNA rather than with proteins. Our conclusion is that great care must be taken not to contaminate the
specimens by human contact. Samples, especially the controversial ones, should be tested and retested in different
centers if possible, and very strict controls must be used. Palmqvist's conclusion is categorical: this single
discrepant finding invalidates the rest of concordant results.
Contamination is very unlikely to have occurred in the equid and bovid fossils in which a horse and bovid
protein respectively were detected, and in which unexpected reactivity for a human protein might be considered
a sign of contamination. But, how can we know whether the reactivity for human albumin or IgG detected in
a fossil assigned to a hominid was due to external contamination or tu an actual fossil protein still able to provide
genetic information? To detect modern contamination in a possibly human bone we have developed the following
procedure (Borja et al., 1997):
The specimen is washed with phosphate buffered salive (PBS) and then treated with a decalcifying solution
of EDTA. Any contaminant protein "attached" to the bone should appear in the PBS extracts. In fossils VM-
O,VM-1960 and VM-3691, assigned to hominids, PBS extracts were, however, negative, whereas their corresponding
EDTA extracts gave positive reactions for human protein (Borja and García-Olivares, 1995 and Borja in this
volume; Lowenstein et al., submitted). This demonstrated that the protein detected was indeed integrated in the
mineral structure of the bone, and was not superficial, as would be expected if this protein had originated from
exogenous contamination.

A BONE IS A BONE, NOT A STONE

Another source Palmqvist cites in support of his criticism about our results with fossil bones is a review
article reporting the discrepant results obtained in immunological studies of blood residues of prehistoric stone
tools (Fiedel, 1996). Although Palmqvist claims and erroneously reported that Fiedel's paper deals with "...studies
of proteins recovered from fossil bones and blood residues from prehistoric artefacts...", he does not seem to have
read the article carefully, since the paper deals only with prehistoric artefact. We have hardly searched for fossil
bonos in Fiedel's article and we have found any.
Discrepant results from archaeological stones cannot be extrapolated to fossil bones, in contrast with what
Palmqvist appears to be irnplying. Downs and Lowenstein, 1995 have carried out a series of blind tests of blood
residues archaeological stones, and in fact, these results are commented in Fiedel's paper: "...Downs' study is, to
the author's knowledge, the only truly objective test that has been conducted to date..." Downs and Lowenstein
found that archaeological blood residues generally could not be identified reliably. Nevertheless, whereas proteins
cannot be clearly identified on artefacts only a thousand years old, these biomolecules can survive under the right
conditions and be characterized, in much more ancient fossil bones. Lowenstein detected species-specific collagen
and serum factors in fossils as old as a 0.5 Myr-old Horno erectus, a 1.9 Myr-old Australopíthecus robustus (Lowenstein,
1981) and 8 Myr-old Rarnapíthecus and Sívapithecus (Lowenstein, 1983). Furthermore, collagen has been found
in a 10-Myr-old unidentified bone (Rowley et al., 1986); osteocalcin has been extracted from 13-Myr-old fossil
bovid bones and from 30-Myr-old fossil rodent teeth (Ulrich et al., 1987) and collagen fibrils were observed with
electron microscopy in a 200-Myr-old dinosaur bone (Wykoff, 1972). Moreover, osteocalcin from a bovid fossil
retained functionally active gammacarboxyglutamic acid residues after 13 Myrs (Ulrich et al., 1987). These results
clearly show that proteins from some fossil bones, although fragmentary, can persist and retain enough of their
immunological properties to provide usable genetic information for periods of milions years.
Preservation seems to be favored by the fact that proteins are embedded in the mineral phase of the bone,
and this provides considerable protection from degradation by environmental conditions. The mineral phase also
appears to preserve, by diffusion, even the cartilage of some fossil bones (Franc et al., 1995). Obviously this
protection does not work for proteins of blood residues on archaeological artefacts, since m this case the
biomolecules are not integrated in the specimen, but simply "stuck" to its surface, where they are probably more
vulnerable to the effect of environmental agents. For this reason, despite the number of recent reports dealing
with blood residues on prehistoric artefacts, many of them contain conflicting or non-reproducible results. By
confusing bones and stones Palmqvist commits one of the most common errors in science: the fallacy of
inappropriate extrapolation (Skrabanek and McCormick, 1989).

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 ANCIENT PROTEINS IN FOSSILS FROM VENTA MICENA AND CUEVA VICTORIA

THE NANOGRAM, RATHER A SMALL UNIT

Palmqvist also bases his criticism on "...the detection of verv high amounts of proteins (unexpected in such
old fossils)..." However, Palmqvist provide any background information that would allow the reader to judge
whether the amounts detected in the fossils from Venta Micena are abnormally high. To prove that something
is abnormally high or low, the figures need to be compared \vith other established measures.
Palmqvist's claim makes reference to two parameters: the amount of proteins and the age of the fossils. Is
nanogram range too high for 1.6 Myr fossils? Palmqvist seems surprised that 4 of human albumin was
detected with RIA in fossils VM-0 andVM-1960 (Lowenstein. 1995). Much lower amounts of albumin in fossils,
in the range that Palmqvist would apparently expect to find, would have been below the limits of detection of
the methods we used to search for proteins. According to Palmqvist's reasoning, any detectable quantity in fossils
from Venta Micena would appear to be suspiciously high.
We performed quantitative experiments to compare VM-0 and fresh human bone. We found 595 ng of
albumin/mg of bone in this later bone and 9.8 ng/mg in the fossil (Lowenstein et al., submitted). Moreover,
osteocalcin was detected at 5 ng/mg in a 13 Myr old fossil bone (approximately 5 times less than in a recent
bone) (Ulrich et al., 1987).
Although immunological reactions with fossils give acceptable information about the genetic similarity with
modern species, the amounts of protein found cannot be taken at a face value, since we are obliged to compare
fossil proteins that are denatured and broken up, with intact modern proteins. Thus, we have used the terco "ng-
equivalents" to denote that the amounts reported are not absolute values (Borja et al., 1997).
Many authors agree that there is not strict relation between the biomolecules content and the age of the
fossils or ancient bones. In many instances, older bones contain more biomolecules than younger ones (Ulrich
et al., 1987; Hagelber et al., 1991). Fossils therefore cannot be judged solely by their age. Other factors such
as the physicochemical enviroment, that have affected the fossil over the time appear to be more important.
Nevertheless, these factors are not well established and no general rule for molecular preservation can be applied
to fossils.

No ALL BONES ARE THE SAME

The last element of Palmqvist's criticism, and the one that gives rise to "...the most severe reservations on
the published reports about protein survival...", is based on a study by Cattaneo et al., 1993. These authors
reported that IgG did not survive in a bone buried for two months. Does Palmqvist conclude that IgG cannot
be preserved for longer than two months in any buried bone, and therefore that it is impossible to detect this
protein in any fossil? Tuross and Stathoplos (1993) have clearly shown that IgG from fossil bones can be separated
by gel electrophoresis and identified with Western blotting.
Is the ELISA used by Cattaneo et al. sensitive enough to detect proteins at very low concentrations?
Cattaneo et al. (1990) reported that they detected "...extremely small amounts (10 ng) of protein..." They needed
grams of bone for protein extraction and reported their results in semiquantitative tercos (-,+,++,+++). In our
hands, RIA, ELISA and dot-blotting were able to detect as little as 1 ng or less of protein; moreover, we needed
only few milligrams of fossil bone, and reported results of a more quantitative nature.
More importantly, a fossil bone is a unique specimen that becomes a fossil only under very particular
conditions. These conditions are responsible not only for the existente of the fossil, but also, probably, for the
preservation of biomolecules. 1.6 Myr-old fossil bones and recent bones buried under garden conditions for two
months are by no means comparable. Nevertheless, we have also studied buried bones (collected 10 years after
burial a cemetery) and found amounts of albumin not much higher than those detected in fossils (32.6 ng/mg).
This apparent contradiction can be explained if we consider that after death, under normal (garden/cemetery)
conditions, proteins tends to disappear. However, under the singular conditions that lead to fossilization, as at
Venta Micena, proteins are probably "frozen" into the mineral phase of the bone so that they are preserved for
millions of years. Palmqvist's attempt to apply to fossil bones conclusions obtained from recent bones is another
example of inappropriate extrapolation (Skrabanek and McCormick, 1989). As Poinar pointed out in Nature:
"...There are many types of fossilization processes, and to assume that the breakdown of DNA is similar in all
of them, or is equivalent to that in non-fossilized material, is not scientific..." (Poinar, 1993)

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