Metalloprotein investigations using capillary electrophoresis with X -ray fluorescence detection.

Abstract

X-ray fluorescence (XRF) detection was established as a viable detection method for capillary electrophoresis (CE) separations for the first time. Initial separations were performed on chelated metal cations. This confirmed that CE could be used with XRF for metal specific detection, and established detection limits in the 50 muM range for iron, cobalt, copper and zinc. When combined with ultraviolet detection for ligand (or protein) detection and quantification, the metal to ligand ratios were calculated. CE-XRF was then used to separate and detect the commercially available metalloproteins hemoglobin (iron), carbonic anhydrase I (zinc), carbonic anhydrase II (zinc) and superoxide dismutase (copper and zinc). The zinc and copper metalloproteins were successfully separated and detected. By combining UV and XRF detection, it was confirmed that the metal : protein ratios were 0.99 +/- 0.15 for zinc to carbonic anhydrase I, 0.87 +/- 0.23 for zinc to carbonic anhydrase II, and 0.98 +/- 0.25 for zinc to superoxide dismutase and 1.13 +/- 0.27 for copper to superoxide dismutase. It was also confirmed that the Cu to Zn ratio in superoxide dismutase was 0.87 +/- 0.26. All of these numbers were in good agreement with established values. Samples of wild-type superoxide dismutase (SOD) and mutant SOD were analyzed for their zinc and copper content. When combined with ultraviolet detection, CE-XRF was able to show that remetallated wild-type SOD had 3 forms of protein in solution, with a zinc to SOD ratio of 2 to 1 for the major component. It was also shown that when excess zinc is available to SOD, it is capable of binding more than 4 zinc atoms. Four mutant forms of SOD (A4V, D101G, G93A, S134N) were shown to exist in heterogeneous mixtures, and the S134N mutant was shown to have zinc and copper to protein ratios less than 2 to 1.