REAGENTS FOR THE CROSS-LINKING OF PROTEINS BY EQUILIBRIUM TRANSFER ALKYLATION

Reagents (11 and 12) have been synthesized which can interact with nucleophilic groups on proteins and biopolymers by a sequence of consecutive Michael reactions yielding cross-linked and multi-cross-linked structures. Consecutive Michael reactions occur because Michael addition to the reagent then allows elimination of the trimethylammonium or mercaptonitrobenzoate function unmasking the latent double bond. A second Michael is then possible. These consecutive Michael reactions potentially allow the cross-link(s) to circumambulate the protein framework until the most thermodynamically stable crosslink equilibrium is established. Intermolecular cross-links can be established between protein subunits and multienzyme complexes. These reagents are designed so that addition and sequential cross-linking may be monitored by spectroscopy, and the cross-link may be subsequently fixed. This fixing process may be accomplished at variable times so as to provide a spectrum of cross-linked derivatives representing the sequence of steps. The chemistry of the process was established using cysteine, acetylcysteine, lysine, alkyl mercaptans, and alkylamines as models of protein residues. The intra- and intermolecular cross-linking character of these reagents was demonstrated using ribonuclease. Multiple cross-links could be introduced. In one of the modified ribonucleases, two links were introduced-one link was established between lysine residues 7 and 37; the other is probably between lysine residues 31 and 41. These links were determined by tryptic peptide mapping. A modified ribonuclease having three cross-links and intermolecularly cross-linked ribonuclease dimers and trimers were also produced. © 1979, American Chemical Society. All rights reserved.