Reversible Immobilization of a Chimeric Protein Obtained by Genetic Fusion of a Choline Binding Domain With Galactosidase on a Choline Self Assembled Monolayer

3.5.1. Coupling of a Hydrophilic Spacer

For proper attachment of the protein the setting of a spacer molecule between the SAM and the protein is needed to facilitate the accessibility of biotin to the binding pocket on the protein.

1. The amidation of NHS-esters previously synthesized on the SAM is effected by the overnight immersion of the electrode into undiluted DADOO.

2. The unreacted diamine is removed by washing with ethanol followed by dipping in 0.1 M KOH.

3. As an alternative, the synthesis of a mixed SAM of DADOO and ethanolamine (EA) can be useful to prepare electrodes in which the DADOO spacers are diluted with shorter hydroxy-ending molecules. In this case the gold electrode is not immersed in DADOO but in DADOO-EA mixtures of both liquids. The DADOO-EA molar-ratio can be chosen as desired, but 1:5 is preferred. For this mixed monolayer, the electrode is washed in the same way as that for the DADOO-only one.

3.5.2. Activation of Terminal Amino Groups of TOA-DADOO Gold Electrodes

1. Activate the end free-amine group of DADOO by dipping the gold electrode in undiluted epichlorohydrin for 3 h.

2. Eliminate the unreacted epichlorohydrin by washing with ethanol-water solution (2:1 [v/v]) for 5 min.

3.5.3. Thiocholine Coupling to the Electrode Activated With a Self-Assembled Monolayer TOA-DADOO-Epi

1. The terminal epoxy groups generated in the previous step are dipped for 18 h with a 10 mM solution of ThC in 50 mM phosphate buffer, pH 8.0.

2. Wash the wire for 5 min with the same buffer.

3.5.4. Binding of a Chimera Protein Made of Fused $-Galactosidase and Choline-Receptor

1. To minimize the nonspecific adsorption of the chimeric protein dip the modified gold electrode for 10 min in a 0.5 mg/mL lactoalbumin solution.

2. Wash the wire with 50 mM sodium phosphate buffer, pH 7.3, for 10 min.

3. Incubate the washed wire in a 0.1 mg/mL chimeric protein solution in the same buffer.

4. Wash the electrode with the same buffer, as in step 2, for 2 to 3 min.

3.5.5. Measurement of the Enzymatic Activity

The course of the enzymatic reaction was followed by cyclic voltammetry (CV) analysis of the reaction product 4-aminophenol (20) (see Note 1). In this experi mental set up the gold wire serves as both enzyme support and electrode reporter of catalytic activity.

1. Insert the enzyme-SAM-modified wire in a conventional electrochemical cell with 2 mL of 50 mMphosphate buffer, pH 7.3, and 0.1 MKCl. Insert a platinum auxiliary electrode and an Ag/AgCl, 3 MNaCl reference electrode, connected to the cell by a salt bridge filled with the electrolyte solution.

2. Connect the cell to a electrochemical analyzer such as the CV50, BAS analyzer.

3. Add the substrate (p-aminophenyl)-D-galactopyranoside (PAPG) to a final concentration of 4 mM and immediately start to record cyclic voltammograms at 0.1 V/s in the potential interval from -0.2 V to +0.35 V, at intervals from 0 to 12 min.

a. Plot the oxidation peak current at 0.14 V against time.

b. Calibrate the electrochemical system by recording cyclic voltammograms at 0.1 V/s in the potential interval from -0.2V to +0.35V of 4-aminophenol hydrochloride solutions in a range from 0 to 5 mM. The current intensities of the aminophenol oxidation peak are plotted against concentration. Slopes about 45 |A/|mol/cm are obtained under the above experimental conditions.

3.5.6. Reversibility of the Chimera Protein Binding

1. Removal of the chimeric protein from the electrode is obtained by incubation of the enzymatic electrode in a 3 M choline solution; after 10 min incubation, no activity is detected.

2. Wash the electrode with 50 mM phoshate buffer, pH 7.3, 0.1 MKCl, and the electrode is ready for immobilization of a new chimeric protein sample.

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