Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle

Catecholamines play an important role in cuticular sclerotization, an extracellular process used by insects at various stages of their life cycle to stabilize their exoskeletons. Analysis of products derived from the acid hydrolysis of insect cuticle provide for the first time direct structural evidence for the actual cross-links between catecholamines and histidyl residues of the cuticular proteins, Four adducts were purified by HPLC from a 6 M HCl hydrolysate of tobacco hornworm, Manduca sexta, sclerotized pupal cuticle, They were identified as 7-N-tau-, 6-N-tau-, 7-N-pi-, and 6-N-pi-histidyldopamine adducts using matrix-assisted laser desorption ionization mass spectrometry, 1D H-1 NMR, and 2D homonuclear nuclear Overhauser effect NMR spectroscopy in combination with molecular modeling, The molar ratio of these adducts in the hydrolysate was approximately 6:3:2:1, respectively, These adducts apparently are formed from Michael 1,4- and 1,6-addition reactions of the putative quinonoid sclerotizing agents, N-beta-alanyldopamine quinone and N-beta-alanyldopamine quinone methide, with both imidazole nitrogens of histidyl residues of proteins.