Journal of Archaeological Science 1992,19,237-241

Blood Residues on Ancient Cautionary Note

P. R. Smith” and M. T. Wilson”

(Received 6 December 1990, revised manuscript accepted 25 April 1991)

Evidence is reviewed which suggests that proteins in general, and haemoglobins in particular, are unlikely to survive burial for many centuries in their native homogeneous state required for crystallization or for identification of species by isoelectric point determination. Crystal morphology is, in any case, an ambiguous way to determine the species of origin.

Keywords: HAEMOGLOBIN, CRYSTALLIZATION, ISOELECTRIC POINT.

Introduction The archaeological world was excited several years ago by the claim that haemoglobin crystals could be grown from blood residues recovered from the surfaces of prehistoric tools and that the animal species from which the haemoglobin originated could be identified from both the shape of the crystals (Loy, 1983; Bahn, 1987; Gurfinkel & Franklin, 1988) and from the isoelectric point of the haemoglobin (Nelson et al., 1986; Bahn, 1987). If substantiated, these techniques would be of great use to archaeologists. For example, it would be possible to know the type of animal hunted in a given region at a specific time and hence also to monitor the movement of species across continents. The validity of these claims remains, however, open to question.

Two aspects of these studies demand consideration. Firstly, what is the evidence that protein molecules can survive the passage of time in a homogeneous and unchanged state that allows crystals, isomorphous with the active protein, to form? Secondly, given crystallization of a protein, under what conditions can the crystal morphology be used diagnostically to identify the source animal species?

It has been suggested that burial in the soil may provide protection for haemoglobin present on the prehistoric tools (Loy, 1983; Hyland, 1990; Gurfinkel, 1988). However, it has been shown using immunological methods (Ascenzi et al., 1985; Smith, 1990; Smith & Wilson, 1990) that even when haemoglobin is protected within ancient vertebrae and femurs, it is the density of the bone that determines the degree to which the haemoglobin survives; the more exposed to the soil the haemoglobin is, the less well it survives the passage of time. It is possible that haemoglobin may, through for example, electrostatic interactions, bind tightly to clay surrounding the stone surface. This process may prevent leaching of haemoglobin into the surrounding soil but, in our view, is unlikely to prevent

“Department of Chemistry and Biological Chemistry, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.

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238 P. R. SMITH AND M. T. WILSON

degradative processes (oxidation, bacteria etc.) modifying the immobilized protein. It has also been suggested (Sensabaugh, 1971) that dried blood proteins are likely to aggregate to form a single insoluble proteineous mass which resists the leaching process. In this insoluble form, however, the protein will not be capable of forming crystals. We have also shown by ELISA, affinity chromatography and absorbance spectroscopy (Smith, 1990; Smith & Wilson, 1990) that the haemoglobin survives in its Apo form, i.e. in the absence of the haem moiety, and as a family of proteins degraded to different extents. As such, the identical molecules necessary for crystal formation are not available. It is not even necessary for the protein to have fragmented into several chains to prevent the formation of isomorphous crystals. An absence of the haem group, unfolding of the protein, or oxidation of the aromatic amino acid residues would all be sufficient to prevent crystallization.

Species Identification Through Crystal Morphology Let us consider what we believe is the highly unlikely possibility that haemoglobin molecules survive in their original native state suitable for crystallization. From the work of Reichert & Brown (1909) and Washino & Else (1972) there is little doubt that the morphology of the crystals of haemoglobin obtained from different animal species varies considerably. In order to use these differences diagnostically, however, more than just the appearance of the crystals is required. The lengths of the crystal axes and the angles between them must be determined (Reichert & Brown, 1909). It is also necessary to catalogue large numbers of haemoglobin crystals with which to compare the shapes of the haemoglobin crystals obtained from unknown sources. In some cases the animals killed with the tools may now be extinct, making identification with reference to the modern protein impossible. There is a limited number of symmetrically different arrangements which the crystals can take, and so some animal haemoglobins will have the same or very similar morphology. This is always assuming, however, that the crystals are in perfect condition.

Crystals of haemoglobin can be formed with relative ease using well-established techniques (Adachi & Asakura, 198 1). It is clear from Figure 1, however, that even with the use of analytical reagent grade chemicals and pure haemoglobin solutions imperfectly formed crystals are obtained as a result of residual impurities. Imperfections in crystals can be either chemical, due to foreign atoms or vacancies in the crystals; or physical, due to strain, dislocations, grain boundaries, twin planes and stacking faults (Laudise, 1970). It seems likely that any crystals obtained from haemoglobin which had been in a soil environment for thousands of years would have many more imperfections due to impurities, which would disguise the true morphology of the crystals. When comparing the crystals obtained from the blood meals of mosquitos, Washino (1977) found that the salt concentration affected the crystal morphology. In addition, it is common for proteins to crystallize in two or more different forms, depending on the arrangement of the molecules within the crystal (Buhn, 1964).

It is also the case that different animal haemoglobins have different solubilities and so require different buffer concentrations for crystallization. Washino (1977) found it necessary to divide samples into two and treat the two halves with very different concentrations of buffer in order to crystallize many of the haemoglobins.

Loy (1983) has claimed that four different animal species could be identified from one crystal aggregate. However, when Washino (1977) attempted to identify the multiple hosts comprising a blood meal, he found that the results varied with different combi- nations of hosts. Some blood mixtures gave no crystals, some produced one or both of the individual crystal forms, and still others formed hybrid crystals.

BLOOD ON ANCIENT TOOLS

Figure 1. Crystals of human deoxyhaemoglobin A ( x 6.3 magnification) prepared according to Smith (1990).

240 P. R. SMITH AND M. T. WILSON

Another point to consider is the survival of other proteins besides haemoglobin. There are many plasma proteins in blood, the major ones being albumin, fibrinogen and the a, /3 and y globulins. Although these are not as highly concentrated in the blood as haemoglobin, they are still present in considerable concentrations. Albumin has been detected immunologically in several archaeological samples and been shown to survive well over the centuries (Prager et al., 1980; De Jong et al., 1974; Wilson et aE., 1977). Thus, it would seem reasonable that if haemoglobin survived on the tools then some albumin, or the other proteins mentioned, would have survived also and have appeared in the crystallization process.

Species Identification By p1 Value It has also been suggested (Nelson et al., 1986; Bahn, 1987) that the p1 of ancient haemoglobin can be used to determine its animal origins. The p1 of a protein is defined as that pH at which the protein bears no net charge and therefore reflects the number and nature (pKas) of the charged amino acid residues comprising the polypeptide sequence. A protein will have a different p1 when in the denatured than when in its native states as the pKa value of amino acids depends on their local environment. Similarly, a change in a surface amino acid on a protein would result in a change in the measured p1 of that protein. For example, haemoglobin A has a pI of 6.87 whereas haemoglobin S (with one amino acid difference, PGlu,-+val) has a pI of 7.09. Therefore, for pI measurements to be of any value in determining the origin of ancient haemoglobin, it would be necessary for that protein to have remained in its original native state over the passage of thousands of years. This has been shown by immunochemical methods not to be the case (Ascenzi et al., 1985; Smith, 1990; Smith &Wilson, 1990).

Conclusions It is clear from the foregoing that we are highly sceptical of the claims that proteins survive in a form suitable for crystallization. However, we cannot merely dismiss such reports on the basis of marshalling arguments which may themselves be flawed in ways unknown to us. Nevertheless, when claims are made which run counter to current scientific intuition regarding the biochemistry of protein we feel that the onus of proof weighs heavily on the claimant rather than on those who are doubtful. To be convinced that haemoglobin can be crystallized from stone tools, we would like to see: (i) That the alleged crystals were birefringent; (ii) That the material comprising the birefringent crystal exhibited the spectrum of haemoglobin. The ferric spectrum may be confused with other iron compounds and thus the deoxy and CO spectra should be reported. Ideally the oxy/deoxy optical transition under vacuum should be evident. (iii) The concentration of haemoglobin, measured easily from the above spectra, should be compatible with the amount of Hb present as calculated from the crystal weight. This is important to ensure that the crystal is haemoglobin and not of some other compound with traces of haemoglobin contaminant. (iv) At least a partial amino acid sequence analysis as a protein present in sufficient quantities to be crystallized is abundant enough for this. (v) Ideally, an X-ray diffraction pattern.

References Adachi, K. & Asakura, Y. (1981). Aggregation and crystallization of haemoglobins A.S.C.

Journal of Biological Chemistry 256,1824-l 830.

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Ascenzi, A., Brunori, M., Citro, G. & Zito, R. (1985). Immunological detection of haemoglobin in bondes of ancient Roman times and of iron. Enealithic Ages-Proceedings of the National Academy of Science, U.S.A. 82,717&7172.

Bahn, P. G. (1987). Getting blood from stone tools. Nature 350, 14. Buhn, C. (1964). Crystals: Their Role in Nature and in Science. New York: Academic Press. De Jong, E. W., Westbroek, P. & Westbroek, J. J. (1974). Preservation of antigenic properties of

macromolecules over 70 million years. Nature 252,63-64. Gurfinkel, D. M. & Franklin, U. M. (1988). A study of the feasibility of detecting blood residues

on artifacts. Journal of Archaeological Science l&83-97. Hyland, D. C., Tersak, J. M., Adovasio, J. M. & Siegel, N. I. (1990). American Antiquity 55, 104. Laudise, R. A. (1970). The Growth of Single Crystals. New Jersey: Prentice-Hall Inc. Loy, T. H. (1983). Prehistoric blood residues: detection on tool surfaces and identification of

species of origin. Science 220, 1269-1271. Nelson, D. E., Loy, T. H., Vogel, J. S. & Southon, J. R. (1986). Radiocarbon dating of blood

residues on prehistoric stone tools. Radiocarbon 28, 170-l 74. Prager, E. M., Wilson, A. C. & Lowenstein, J. M. (1980). Mammouth albumin. Science 209,

287-289. Reichert, E. & Brown, A. (1909). The CrystaIlography of the Haemoglobins. Publication 16.

Washington: Carnegie Institute of Washington. Sensabaugh, G. F., Wilson, A. C. &Kirk, P. L. (1971). International Journalof Biochemistry 2,

545-549. Smith, P. R. (1990). The detection of haemoglobin in ancient human skeletal remains. Ph.D.

thesis, University of Essex, Colchester, U.K. Smith, P. R. &Wilson, M. T. (1990). Detection of haemoglobin in human skeletal remains by

ELISA. Journal of Archaeological Science 17,255-268. Washino, R. K. (1977). Identljication of host bloodmeals in arthopods. U.S. Army Medical Res.

and Develop. Correspondence, Washington, DC. Washino, R. K. & Else, J. G. (1972). American Tropic&Medicine, 21, 120-122. Wilson, A. C., Carlson, S. S. &White, T. J. (1977). Annual Rview of Biochemistry 46,573-639.