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.