Ethanolamine blocking carboxylates

PGIP, purified fi om P.vulgaris hypocotyls [11], was immobilized to the sensor ch via amine coupling. A continuous flow of HBS buffer (5 pl/min) was mantained over the sensor surface. The carboxylated dextran matrix of the sensor surface was first activated by a 6-min injection of a mixture of N-hydroxy-succinimide and N-ethyl-N - (3-diethylaminopropyl) carbodiimide, followed by a 7-min injection of PGIP (lOng/pl in 10 mM acetate, pH 5.0). Hie immobilization procedure was con leted by a 7-min injection of 1 M ethanolamine hydrochloride to block the remaining ester groups. 

Figure 1.125 Carboxylate groups may be blocked with Tris or ethanolamine using a carbodiimide-mediated process.

Dissolve the macromolecule containing carboxylate groups to be blocked at a concentration of l-10mg/ml in 0.1 M MES, pH 4.7, containing 0.1 M Tris or ethanolamine. Other conditions may be used to perform this reaction. See Chapter 3, Section 1 for further details. 

The immobilization of anti-P-endorphin to the carboxymethylated dextran (which is on the surface of the resonant layer) was via NHS/EDC chemistry. Prior to antibody coupling, the carboxymethylated dextran was activated twice with 0.4 M EDC/0.1 M NHS for 10 min. Anti-P-endorphin was coupled twice to the dextran layer in 10 mM sodium acetate buffer, pH = 5.0, at 25 pg/ml to ensure maximum loading. After coupling, the free activated carboxyl group was blocked with 200 pi of 1 M ethanolamine for two minutes. Finally, the cuvette with immobilized anti-p-endorphin was washed twice with 20 mM HCl and twice with PBS/0.05% tween 20, to eliminate the non-covalently bound antibody. The binding of the peptides to the antibody was carried out in PBS, pH=7.4, at 25°C. After the baseline was established for 150 pi of PBS, 50 pi of 3 mg/ml (0.1 mg/ml/peptide) crude peptides in water was added to the cuvette and the binding was monitored. When equilibrium was achieved (approximately 10 min), the unbound peptides were flushed away. 

Coordination interaction is also a factor that needs to be considered. Because a trigonal uncharged boronate contains a boron atom with an empty orbital, this can serve as an electron receptor for a coordination interaction. In addition to the previous report that unprotonated amines and carboxyl groups can serve as electron donors and thus can coordinate with boronic acids, the coordination of Lewis bases [e.g., fluoride ion) with boron can also occur, which can enhance the complexation between cis-diol-containing compounds and boronic acids. However, if there is a hydroxy group adjacent to the amine [e.g., ethanolamine), this hydroxy group can also interact with the boronate, which would block esterification between the boronate ligand... 

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