Nomenclature and possible evolutionary pathways of metallothionein and related proteins.

In dealing with metal-binding proteins from nonmam-malian species, nomenclature and possible evolutionary relationships between these molecules and metallothi-oneins in mammals are ofclear scientific importance. The workshop summarized below was oriented towards a discussion of these topics based on the currently known physical and chemical characteristics of these molecules. Since metal clusters are a central feature of metallothi-oneins, the workshop began with a detailed discussion of the protein preparative techniques used prior to nuclear magnetic resonance spectroscopy ofmammalian and crab metallothioneins. 113Cd Reconstitution of Metallothionein The techniques used for "13Cd reconstitution of me-tallothioneins from mammals and the crab Scylla serrata metallothionein have been extensively described elsewhere (1-6). This summary will attempt to briefly review the techniques involved and point out the major potential problem areas. In order to remove bound metals, the pH of the me-tallothionein solution is lowered to approximately 1.3 and the metals separated from the protein by either dialysis or gel filtration chromatography to yield the apoprotein. The apometallothionein concentration is calculated by measuring the UV absorbance at 220 nm by using the known extinction coefficient for this protein (7). In a glove box, under argon or nitrogen atmosphere, 1"3Cd in slight excess is added and the pH of the protein solution slowly raised by dropwise addition of Trizyma base. Phosphate buffer is not used for this procedure to avoid precipitation of "3Cd. Excess 1"3Cd is removed from the reconstituted protein by dialysis, ge filtration, or use of Chelex 100 resin. The criterion for success of this procedure is a calculated metal: protein ratio of 7-atoms of 113Cd/mole of protein. Partially oxidized protein samples at the outset are a problem in this technique due to presence of S-S bonds which prevent normal reconstitution. Use of reducing agents such as dithiothreitol to break these bonds prior to acid dialysis of the sample is a successful technique if the protein sample is not extensively oxidized. Nomenclature A number of potentially useful criteria for distinguishing metallothioneins from nonmetallothionein metal-binding proteins are listed below: Presence of metal clusters with bridging ligand; inducibility; high cysteine content/ aromatics (may or may not be present); high metal content ; operational characteristics, such as 50% loss of Zn at pH 4.5 and 50% loss of Cd at pH 3.0, and ultraviolet absorption spectra characterized by high 250 nm/280 nm ratios indicative of metal-thiolate bonds. There is a clear need for determining the function of metallothionein to …