Alkaline earth metals are not required for the restoration of the apoform of anthrax lethal factor to its holoenzyme state.

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase previously shown to require calcium and magnesium for the restoration of its catalytic function upon exposure of the apoprotein (apoLF) to Zn(2+). Since concrete Ca(2+)/Mg(2+) binding sites have not been identified in LF, the effects of alkaline earth metals on the enzymatic function of holoLF (ZnLF) and on the reconstitution of apoLF were reinvestigated. The current study reveals alkaline earth metals to be inhibitory at concentrations higher than 1mM. A combination of activity/inhibition assays and Tb(3+) luminescence spectroscopy was employed to unequivocally establish the presence of at least one inhibitory low-affinity Ca(2+)-site in LF. A comparative analysis of apoLF preparations obtained by dialysis and centrifugal filtration (following treatment of ZnLF with chelators) revealed the exposure of apoLF to low equimolar amounts of Zn(2+) to be sufficient for the full restoration of the protein's catalytic competence, a finding constistent with the picomolar dissociation constant of ZnLF determined in this study. The previously documented requirement of Ca(2+) and Mg(2+) in apoLF reconstitution may be explicable on the basis of contamination of dialyzed apoprotein preparations with residual chelator, a phenomenon not encountered with apoLF obtained by centrifugal filtration.

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon.

Zincon (2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator's versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn(2+), Cu(2+), and Co(2+) were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn(2+), Cu(2+), and Co(2+) with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn(2+) and Cu(2+) within the same sample is described. Using the prototypical Cu(2+)/Zn(2+)-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.