Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol

Abstract

Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.