Abstract

The binding of cisplatin to metallothionein (MT) was investigated at 37°C in 10 mM Tris-NO₃ (pH ∼7.4) and 4.62 mM NaCl. The conditions were chosen to mimic passage of clinical concentrations of cisplatin through the cytosol. The reactions were monitored by high-performance liquid chromatography (HPLC), atomic absorption spectroscopy, and ultraviolet (UV) absorption spectroscopy. The UV data showed that several reactions occur, the first of which does not affect the absorbance (no Pt-sulfur bond formation). They also suggested that if [cisplatin] is large compared with [MT], the rate of subsequent reaction is between first and second order in [cisplatin] and between zeroth and first order in [MT]. HPLC eluates with 24 < retention time (t_R) < 27 min contained undialyzable Pt, which increased with reaction time and corresponded to Pt-thionein product. Eluates with 3 < t_R < 7 min corresponded to unbound cisplatin and allowed determination of second-order rate constants (k), using the second-order rate equation. The k value for cisplatin reacting with apo-MT was ∼0.14 M^–1 s^–1, Cd/Zn-MT ∼0.75 M^–1 s^–1, Cd₇-MT ∼0.53 M^–1 s^–1, and Zn₇-MT ∼0.65 M^–1 s^–1. Thus, cisplatin displaced Cd and Zn equally well. Leukocyte MT concentration was ∼1.0 mM, so that the kinetics of cisplatin binding to cellular MT is pseudo-first order (pseudo-first-order rate constant, ∼0.63 × 10^–3 s^–1; half-life, ∼18 min). With [cisplatin] = 10 μM, the rate of cisplatin reaction with MT is ∼6.3 μmol s^–1 cm^–3. We conclude that cellular MT can trap significant amounts of cisplatin and may efficiently contribute to cisplatin resistance.