Ultracentrifugation and inductively coupled plasma mass spectrometry for metal–protein equilibrium studies

Abstract

The coupling of separation by preparative ultracentrifugation and metal detection by inductively coupled plasma mass spectrometry (ICP-MS) has been explored for metal–protein equilibrium determinations. This study characterizes the stoichiometry as well as apparent (Kapp) and intrinsic (Kint) binding affinities of the metal–protein association for a model protein. In particular, the affinity of Cu2+ for the high affinity binding site in bovine serum albumin (BSA) is determined. Once equilibrium is established between Cu2+ and BSA, preparative ultracentrifugation moves the metalloprotein away from the meniscus, leaving unbound equilibrium copper in the protein free solution. Since the initial (total) concentrations of purified BSA and Cu2+ can be determined, the free copper concentration at equilibrium can also be determined by taking a small aliquot above the sedimenting boundary for analysis using ICP-MS. This analysis allows for the determination of free Cu2+ ion, which is identical to the equilibrium concentration prior to ultracentrifugation. From these data Kapp and Kint were determined at two different conditions, 100mM Tris(hydroxymethyl)aminomethane (Tris) at pH 9.53 and pH 7.93. log Kapp values of 17.6 and 14.6 were determined at pH 9.53 and pH 7.93, respectively. Furthermore, pH-independent log Kint values of −1.43 and −1.04 were determined at pH 9.53 and 7.93, respectively. While the log Kint at pH 9.53 was in good agreement with literature values obtained from alternative methods, Kint at pH 7.93 was about 2.5× larger than previously reported. BSA undergoes a structural rearrangement between pH 7–9, and the generally accepted pH-dependency of protein tertiary structure may be responsible for the variations in the “intrinsic” binding constant. The Cu–BSA binding affinity was also monitored in 100mM Tris 0.1% sodium dodecyl sulfate (SDS) solution at pH 7.93 in order to determine the effect of a denaturant on metal binding. Results for both log Kapp and log Kint were similar to those obtained in the absence of 0.1% SDS at pH 7.93. Overall, this study validates and shows the efficacy of combining preparative ultracentrifugation with ICP-MS detection for interrogating metal–protein associations while causing minimal equilibrium perturbations as a result of the separation and measurement processes. Advantages and disadvantages of this methodology are discussed as it relates to alternative methods for metal–protein studies.