Metallothioneins (MTs) are ubiquitous proteins vital for essential metal homeostasis and heavy metal detoxification. The twenty-cysteinyl mammalian metallothioneins protect the proteome by sequestering heavy metals into thermodynamically stable metal thiolate structures when metalated with seven Cd²⁺. At physiological pH, the first metal (M) thiolate (S_Cys) structures formed involve M(S_Cys)₄ terminal thiolates. With higher metal loading, M₄(S_Cys)₁₁ and M₃(S_Cys)₉ clusters form. As a regulator of the metallome, it is necessary to understand metal sequestration properties of MT in solution with other metalloproteins. We report that the association between apo-MT and apo-carbonic anhydrase (CA) enhances the formation of the protective mode of MT, in which Cd₄(S_Cys)₁₁-clusters form at much lower concentration levels than for the free apo-MT at physiological pH. Using stopped-flow kinetics and electrospray ionization mass spectrometry, we quantified this protective effect, determining that it is significant at pH 7.4, but the effect diminishes at pH 5.0. We report for the first time, the absolute stepwise binding constants of Cd²⁺ binding to human MT1a both in the presence and absence of CA through calibration by the known binding constant of Cd²⁺ to bovine CA. We report that this protein association affects the Cd²⁺ metalation rates of MT. The data support the physiological role of MTs as protectors of the metalloproteome from the toxic effects of Cd²⁺.