Does 13C-or 15N-labeling affect Cu(I)-thiolate cluster arrangement in yeast copper-metallothionein?

It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast 13C-and 15N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the 13C- and 15N- labeled Cu7-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.

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.