ELECTROSYNTHETIC IMMOBILIZATION OF PROTEINS FOR BIOANALYSIS

Use of electrosynthetic methodology allows the production of hen egg-w hite lysozyme (HEWL) either mononitrated at tyrosine 23 or bisnitrated at tyrosines 20 and 23, but never nitrated at tyrosine 53, This is a different sequence from that obtained by the chemical nitrating agent tetranitromethane, and when reduced by dithionite, the selectively mod ified enzyme can be anchored at pH 5 via the unique aromatic amino gro up to magnetic beads or other suitable matrices. HEWL so immobilised l oses less than 10% of cell-wall lytic activity compared with the appro ximately 50% loss of activity when immobilised by conventional methodo logy at pH 9 via essentially random reaction at lysine residues and ot her functionalities which are nucleophilic at this pH, This result off ers promise as a general method for selective protein immobilisation i n biosensors and similar applications. (C) 1998 John Wiley & Sons, Ltd .