A rapid and precise method of determining the vanadium content of protein solutions by electron paramagnetic resonance spectroscopy is reported The method is based on the linear relationship between the signal intensity of the first-derivative electron paramagnetic resonance signal and the concentration of the VO(&O)t+ species formed in acid solution. Six different proteins were investigated in the concentration range IO-"-IO6 M. A precision of kl%, an accuracy of ?3%, and a detection limit of 25ppb for vanadium was obtained. This analytical method was developed to aid in investigations of metal binding sites in proteins by vanadyl ion (WY') electron paramagnetic resonance spectroscopy.
Recently, the vanadyl ion (V02+) has received considerable attention in this laboratory as a physicochemical probe of metal sites in the metalloenaymes bovine carbonic anhydrase (BCA) and carboxypeptidase (CPA), and the hormone insulin as well as other proteins (l-3). The need for a rapid and precise method for determining the total vanadium content of protein solutions prompted this study.
Present analytical methods to determine vanadium have been recently reviewed (4) and include : colormetric, volumetric, and spectroscopic methods such as emission, atomic absorption, and neutron activation analysis. Since much of our work with V02' binding to proteins employs electron paramagnetic resonance (EPR) spectroscopy, it was desirable to develop a method of determining the vanadium content of protein solutions by EPR. In this way, a vanadium analysis could be readily performed on the same sample used during the normal course of an EPR investigation.
The work of Guilbault and coworkers (5-7) on the EPR determinations of the amounts of vanadyl ion and other selected transition metal ions in solution suggested that a similar approach might be applied to proteins. 170