Yoka Kaup

Ancient materials analysis of a pharaonic embalming tar

Details of mummification techniques used in dynastic Egypt have emerged from writings in subsequent ancient texts, in which the application of oils (kedros, cedrium) derived from the cedar tree have been described by Herodotus (490–425 bc) and by Pliny the Elder (ad 23/24–79). But scholars have since argued that these products were prepared from juniper trees and not from cedar — an assertion that is widely accepted by Egyptologists but which has never been verified by chemical analysis. Here we use gas chromatography to analyse the constituents of a sample of unused entombed embalming material from 1500 bc at a site in Deir el-Bahari, Egypt, and find that its components probably originated from the cedar tree. We also identify one component, guaiacol, as having notable preservative properties.

Embalming was used in Old Kingdom

Our knowledge of the conservation techniques used in the Old Kingdom of ancient Egypt is limited. Examinations of a clavicle fragment of the mummy of Idu II, secretary general of the pine wood trade office (2150 ± 50 BC), revealed saturation with a wealth of sodium and wood tar compounds, many of which were highly antiseptic as no microbial contamination was noticed. This pretreatment had ensured the preservation of bone alkaline phosphatase in an enzymically and immunologically active form. This extends the use of embalming to one thousand years earlier than previously thought.

Borate in mummification salts and bones from Pharaonic Egypt.

Mummification processes in Pharaonic Egypt were successful using sodium salts. Quite frequently sodium concentrations in mummified bones ranged from 300 to 4000 micromol/g. In the search for an effective inorganic conservation compound our choice fell on boric acid. The possible presence of borate in mummification salts used in Pharaonic Egypt was of special interest both historically and biochemically. In two salt samples, one from the embalming material of Tutankhamen (18th dynasty, 1336-1327 BC) and the second from Deir el-Bahari (25th dynasty, 700-600 BC) borate was found, amounting to 2.1+/-0.2 and 3.9+/-0.1 micromol/g, respectively. In five of the examined bone fragments from the Junker excavation at Giza (Old Kingdom) similar borate concentrations i.e., 1.2 micromol borate/g bone were seen. It must be emphasized that the usual borate content of contemporary autopsy is far below the detection limit. The elevated borate content in both mummification salt and ancient bone samples support the suggestion that borate-containing salt had been used. There is a striking correlation of both borate concentration and alkaline phosphatase activity. When both sodium salts and borate were essentially absent no activity at all was detectable. With increasing borate concentrations the enzyme activity rises significantly. Attributable to the distinct biochemistry of the tetrahydroxyborate anion it was of interest whether or not borate may stabilize alkaline phosphatase, an important and richly abundant bone enzyme. This enzyme was chosen, as it is known to survive more than 4000 years of mummification. In the presence of borate oligomeric species of this zinc-magnesium-glycoprotein at 400,000 Da became detectable. Attributable to this borate-dependent stabilization of the enzyme molecule a significant temperature resistant increase of the enzymic activity was measured in the presence of up to 2.5 mM borate.

Peer Reviewed: Embalming In The Old Kingdom Of Pharaonic Egypt

Controversy exists over whether embalming was already being performed in the Old Kingdom in the course of mummifying the deceased.

Zn2Mg alkaline phosphatase in an early ptolemeic mummy.

Bone samples of a ptolemeic mummy have been employed to study the mode of conservation on the intactness of Zn2Mg alkaline phosphatase in both structure and catalytic activity. A protein of Mr = 190 ± 10 kDa being identical to the 200 kDa enzyme of fresh human bones was successfully isolated. Regardless of age 200 kDa protein bands and a distinct sub unit at 60 kDa were seen in SDS-PAGE electrophoresis. The 200 kDa band was also monitored by activity staining. The specific activity was 120 mU/mg and 65% of the respective activity obtained in the identical preparation using fresh human tibia or rib. The enzymic activity was inhibited in the presence of 1,10-phenanthroline and ʟ-homoarginine. Radiocarbon dating supported the assignment of the mummy to the early ptolemeic period. Among the many bactericidal and fungicidal components employed for mummification were aromatic alcohols, mono-and sesquiterpenes. Pistachio resin was the major balm resin used. The microbiological sterility of the bone surface was ascertained by independent bacterial and fungal examinations

MONOCLONAL ANTIBODIES RECOGNIZE 2300 YEARS AGED ALKALINE PHOSPHATASE

It was attempted to monitor the immunological response of monoclonal antibodies directed to human alkaline phosphatase in ancient Egyptian bones from the ptolemeic period. The intactness of the respective epitopes of the bone enzyme was successfully demonstrated in an ELISA. Fortunately, the mummified bone was not contaminated by fungi and bacteria due to the fungicidal and bactericidal reactivity of the ancient pretreatment employing resins of pistachio for mummification. The enzyme was enriched using gel chromatography, anion exchange and affinity chromatography to yield 310 +/- 7 mU/mg. The enzymically active fractions of the wheat-germ lectin affinity chromatography were subjected to ELISA. The best binding affinity was detected using the monoclonal antibody BAP A while the reactions of all the other four antibodies BAP B, BAP G, BAP 4A5 and BAP 5D4 were substantially diminished.

Enzymatic and immunological activity of 4000 years aged bone alkaline phosphatase

Structurally intact and functionally active human bone alkaline phosphatase was isolated from clavicle fragments of IDU, an Egyptian mummy of the Old Kingdom (2150 ± 50 BC). Both anion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme resulting in a specific activity of 180 ± 30 mU/mg. The intactness of the bone enzyme fractions of the wheat‐germ lectin affinity chromatography was successfully demonstrated in an ELISA using the monoclonal antibody BAP A. Fortunately, the mummified bone was not contaminated by fungi or bacteria.

Ancient metalloenzymes as possible markers in molecular archaeology

The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.

Effective Mummification Compounds Used in Pharaonic Egypt: Reactivity on Bone Alkaline Phosphatase

In Pharaonic Egypt from the Old Kingdom up to the Ptolemaic Period the deceased were pre-treated in the course of the mummification process using a wealth of embalming components including resins and many different wood tars. GC/MS studies allowed the elucidation of a great number of clearly separated compounds found in the ancient embalming material. Phenols, guaiacols, naphthalenes, monoterpenes, sesquiterpenoids, oxidised diterpene resin acids and triterpenoids were noticed. These results and particularly the detection of an unused embalming material shed some new light on the possible way as to how the different embalming materials might have been prepared and applied. It was striking to see the accordance of the present data with the well-known treatises by Herodotus (490-425 B. C.) and by Pliny the Elder (23/24-79 A. C.). The impact of the historical observations on modern science and in return the dramatic promotion of ancient texts stimulated by the present study is intriguing. An enzyme, alkaline phosphatase, bound inside mummified bones was a useful tool to reveal the efficacy of the embalming materials. Initial data showed that alkaline phosphatase isolated from embalmed bones from the Old Kingdom and the Ptolemaic Period was significantly more abundant and displayed a higher activity compared to the properties of the corresponding enzyme from non-treated mummified bones. Additionally, in a model study porcine ribs were pre-treated with four selected embalming compounds - guaiacol, limonene, α-pinene and p-cymene - and subsequently air-dried. Among the four selected compounds guaiacol was the most reactive species in the course of the preservation process. The specific activity of bone alkaline phosphatase rose 12-fold compared to that of the control. The enzyme itself remained unharmed as the observed relative molecular mass was surprisingly identical with the contemporary enzyme. It was again striking that the guaiacol derivatives were richly abundant in the unused embalming material mentioned above.

Intact mummified bone alkaline phosphatase

Our knowledge to the mode of conservation of mummified structurally and functionally intact biopolymers is limited. Rib samples of a well-preserved 2300-year old ptolemeic mummy were examined whether or not functionally active Zn2Mg alkaline phosphatase could be detected. A protein of M(r) 170 +/- 20 kDa being close to 200 kDa of the enzyme of fresh bones was successfully isolated. Both a 200 kDa protein and a distinct subunit of 60 kDa were seen in SDS-PAGE electrophoresis which was identical to those of fresh bone alkaline phosphatase. There was a significant enzymic activity of 17 mU/mg protein which could be inhibited in the presence of L-homoarginine and 1,10-phenanthroline.