Electrospray ionization (ESI) mass spectra of both
well-characterized and novel metallothioneins (MTs) from
various species were recorded to explore their metal-ion-binding
modes and stoichiometries. The ESI mass spectra of the
zinc- and cadmium-binding MTs showed a single main peak
corresponding to metal-to-protein ratios of 4, 6, or 7.
These findings combined with data obtained by other methods
suggest that these MTs bind zinc or cadmium in a single
predominant form and are consistent with the presence of
three- and four-metal clusters. An unstable copper-specific
MT isoform from Roman snails (Helix pomatia) could
be isolated intact and was shown to preferentially bind
12 copper ions. To obtain additional information on the
formation and relative stability of metal-thiolate clusters
in MTs, a mass spectrometric titration study was conducted.
One to seven molar equivalents of zinc or of cadmium were
added to metal-free human MT-2 at neutral pH, and the resulting
complexes were measured by ESI mass spectrometry. These
experiments revealed that the formation of the four-metal
cluster and of the thermodynamically less stable three-metal
cluster is sequential and largely cooperative for both
zinc and cadmium. Minor intermediate forms between metal-free
MT, Me4MT, and fully reconstituted Me7MT
were also observed. The addition of increasing amounts
of cadmium to metal-free blue crab MT-I resulted in prominent
peaks whose masses were consistent with apoMT, Cd3MT,
and Cd6MT, reflecting the known structure of
this MT with two Me3Cys9 centers.
In a similar reconstitution experiment performed with Caenorhabditits
elegans MT-II, a series of signals corresponding to
apoMT and Cd3MT to Cd6MT species
were observed.