Two-Dimensional Polyacrylamide Gel Electrophoresis for Metalloprotein Analysis Based on Differential Chemical Structure Recognition by CBB Dye

Junko Ishikawa,Daichi Higo,Akinori Maeshima,Allyson Mellinger,Christa L Colyer,Shingo Saito,Soufian Ouchane,Masami Shibukawa,Marie-Line Bourbon,Anne Durand

doi:10.1038/s41598-019-46955-6

Abstract

In an effort to develop an analytical method capable of finding new metalloproteins, this is the first report of a new diagonal gel electrophoresis method to isolate and identify metalloproteins, based on the molecular recognition of holo- and apo-metalloproteins (metalbound and -free forms, respectively) by CBB G-250 dye and employing metal ion contaminant sweeping-blue native-polyacrylamide gel electrophoresis (MICS-BN-PAGE). The difference in electrophoretic mobilities between holo- and apo-forms was exaggerated as a result of interactions between the metalloproteins and the dye with no metal ion dissociation. The different binding modes of proteins with CBB G-250 dye, primarily related to hydrogen bonding, were confirmed by capillary zone electrophoresis (CZE) and molecular docking simulations. Due to in-gel holo/apo conversion between the first and second dimensions of PAGE, holo-metalloproteins in the original sample were completely isolated as spots off the diagonal line in the second dimension of PAGE. To prove the high efficiency of this method for metalloprotein analysis, we successfully identified a copper-binding protein from a total bacterial soluble extract for the first time.

Highlights

These metalloprotein methods can suffer from the dissociation of metal ions from holo- to apo-metalloprotein upon the addition of denaturing agents[14,15], along with serious contamination of metal ions in the separation field[3,11,15,16,17]
While no separation of holo- (Fe2-transferrin (Tf)) and apo-Tf was observed in conventional 1D native (CBB G-250 free)-PAGE (Fig. 1a) and SDS-PAGE (Fig. 1b), interestingly, we found that holo- and apo-Tf are completely separated by means of MICS-blue native (BN)-PAGE (Fig. 1c)
Different migration behaviours for holo- and apo-forms were observed for ceruloplasmin (Cp) bound with Cu2+ (Cu-Cp) and superoxide dismutase (SOD) bound with Cu2+ and Zn2+ (Cu/Zn-SOD) by the MICS-BN-PAGE method (Fig. 1d,e)

Summary

Introduction

These metalloprotein methods can suffer from the dissociation of metal ions from holo (metal-bound)- to apo (metal-free)-metalloprotein upon the addition of denaturing agents[14,15], along with serious contamination of metal ions in the separation field[3,11,15,16,17]. To address the issue of contaminant metal ions, we have previously studied thermodynamically and kinetically stable metal chelates to exhaustively remove trace contaminant metal ions from the separation field in PAGE21 In this method, which we have called metal ion contaminant sweeping-blue native-PAGE (MICS-BN-PAGE), the cationic TPEN (N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylene-diamine) complexes and anionic EDTA complexes formed with contaminant metal ions migrate towards the cathodic and anodic directions, respectively. By this method, the electrophoretic separation of biological samples is possible without their proteins encountering any doubly- and triply-charged contaminant metal ions (since the concentrations of such contaminants are decreased to lower than ppt levels). We present the concept of HAC-2D MICS-BN-PAGE for identification of metalloproteins, and the mechanism of electrophoretic molecular recognition of holo-/apo-protein forms, and using this novel technology we successfully isolated and identified a bacterial copper binding protein from a total soluble protein sample

Methods

Results

Conclusion