Electroanalytical determination of tungsten and molybdenum in proteins

Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.     

Polarographic determination of superoxide dismutase.

A polarographic procedure is described which allows determination of the catalytic constants for superoxide dismutase-catalyzed reactions. The method presents a single and rapid evaluation of the enzyme concentrations as well as determination of its activity under different conditions; e.g., pH between 9 and 13, presence of urea, guanidine, sodium dodecyl sulphate and inhibitors such as CN^? and N₃^?.The results fit very well with data previously obtained with other methods and show that this polarographic procedure can be used under conditions that render the other methods unsuitable for the measurement of the enzyme activity.     

Orphan SelD proteins and selenium-dependent molybdenum hydroxylases

   

Bacterial and Archaeal cells use selenium structurally in selenouridine-modified tRNAs, in proteins translated with selenocysteine, and in the selenium-dependent molybdenum hydroxylases (SDMH). The first two uses both require the selenophosphate synthetase gene, selD. Examining over 500 complete prokaryotic genomes finds selD in exactly two species lacking both the selenocysteine and selenouridine systems, Enterococcus faecalis and Haloarcula marismortui. Surrounding these orphan selD genes, forming bidirectional best hits between species, and detectable by Partial Phylogenetic Profiling vs. selD, are several candidate molybdenum hydroxylase subunits and accessory proteins. We propose that certain accessory proteins, and orphan selD itself, are markers through which new selenium-dependent molybdenum hydroxylases can be found.