Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn2+ or Cd2+ ions into two distinct domains: an N-terminal beta-domain that binds 3 Zn2+ or Cd2+, and a C-terminal alpha-domain that binds 4 Zn2+ or Cd2+. Although stepwise metalation to the saturated M-7-MT (where M = Zn2+ or Cd2+) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M-n-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K-1 < K-2 < K-3 < ... < K-7), weakly cooperative (where K-1 = K-2 = K-3 = ... = K-7), and noncooperative, (where K-1 > K-2 > K-3 > ... > K-7). Detailed ESI-MS metalation data of human recombinant MT la by Zn2+ and Cd2+ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn2+ and Cd2+ binding to rhMT 1a. (C) 2012 Elsevier Inc. All rights reserved.