Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of leu¹²⁹ or asp¹¹⁹

In the cross-linking reaction of lysozyme between Leu¹²⁹ (α-COO^-) and Lys¹³ (ε-NH₃⁺ using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from α to β between A and Gly¹⁰² was greatly reduced by addition of β-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)₃] When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the α-carboxyl group of Leu¹²⁹ was modified with the amine was newly obtained. On the other hand, when ammonia was added, the β-carboxyl group of Asp¹¹⁹ instead of the α-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp¹¹⁹ and Arg¹²⁵ besides that between Lys¹³ and Leu¹²⁹ is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the α-carboxyl group reduced the activity to ̃ 90% of that of native lysozyme. Des-Leu¹²⁹ lysozyme, which lacks Leu¹²⁹ also showed ̃ 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys¹³ and Leu¹²⁹ may play some role in maintaining the active conformation of lysozyine.