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

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.

Advanced Gel Electrophoresis Techniques Reveal Heterogeneity of Humic Acids Based on Molecular Weight Distributions of Kinetically Inert Cu2+-Humate Complexes

“…In addition, the formation of rather small supramolecular aggregates (<20 kDa) via complexation at low concentrations of Cu 2+ was directly observed in this study, which also provides deeper insight into the formation process of HA aggregates and their structure. The investigation of small HA components comprising the aggregates is now underway using a new PAGE system, called holo/apo conversion (HAC)-two-dimensional MICS-PAGE, which we successfully developed to selectively isolate matalloproteins …”

“…The investigation of small HA components comprising the aggregates is now underway using a new PAGE system, called holo/apo conversion (HAC)-two-dimensional MICS-PAGE, which we successfully developed to selectively isolate matalloproteins. 57 ■ ASSOCIATED CONTENT * S Supporting Information…”

Unusually Kinetically Inert Monocationic Neptunyl Complex with a Fluorescein-Modified 1,10-Phenanthroline-2,9-dicarboxylate Ligand: Specific Separation and Detection in Gel Electrophoresis

“…Polyacrylamide slab gel was prepared in the same manner as detailed in earlier reports. − Briefly, the gel was composed of a separation gel (AA/Bis 30%T/2.7%C, [Tris-Gly] = 94–48 mmol dm –3 at pH 9.4, the gel thickness of 0.5 mm, the gel length of 15 cm) and a stacking gel (AA/Bis 17%T/2.7%C, [Tris–HCl] = 94 mmol –3 at pH 8.8, the gel length of 1.0 cm) overlaid on the separation gel. The gel was polymerized in a glass chamber by mixing the gel monomer solution and buffer solution with APS and TEMED.…”

“…The gel was polymerized in a glass chamber by mixing the gel monomer solution and buffer solution with APS and TEMED. Under these gel conditions, it has been reported that metal–probe complexes are effectively concentrated into a narrow band based on the isotachophoresis principle in the stacking gel and are then separated into complex and probe bands in the separation gel. − A Tris-glycine buffer solution ([Tris-Gly] = 6.3–48 mmol dm –3 ) was used for the upper and lower migration buffer solutions, while 0.01 mmol dm –3 CyDTA was exclusively added to the lower migration buffer. The sample was loaded (typically 1.0 × 10 –5 –1.2 × 10 –4 dm 3 ) and then a voltage (1000–1300 V) was applied for 3 h for electrophoresis at a constant temperature of 288 K using a cooling core apparatus equipped with a low-temperature circulator unit (LTB-125, AS ONE Co., Osaka, Japan) for 3 h. After electrophoresis, the gel was removed from the glass plate.…”

“…A selection of fluorescent ligands (probes) suitable for polyacrylamide gel electrophoresis (native PAGE with no addition of detergent) with fluorescence detection were fabricated using a small chemical library (a collection of chemical substances) of probes with different chelating moieties (the chemical structures are shown in Figure ). During the PAGE separation of various L1 – L8 probes complexed with An ions in a probe-free separation buffer solution, the free probes are completely removed from the complex molecules, i.e., a concentration jump occurs to drive the dissociation reaction of the complex. − Thus, kinetic selection was conducted to find kinetically inert An–probe complexes from the library of L1 – L8 probes.…”