The relative contribution of calcium, zinc and oxidation-based cross-links to the stiffness of Arion subfuscus glue

Metal ions are present in many different biological materials, and are capable of forming strong cross-links in aqueous environments. The relative contribution of different metal-based cross-links was measured in the defensive glue produced by the terrestrial slug Arion subfuscus. This glue contains calcium, magnesium, zinc, manganese, iron and copper. These metals are essential to the integrity of the glue and to gel stiffening. Removal of all metals caused at least a 15-fold decrease in the storage modulus of the glue. Selectively disrupting cross-links involving hard Lewis acids such as calcium reduced the stiffness of the glue, while disrupting cross-links involving borderline Lewis acids such as zinc did not. Calcium is the most common cation bound to the glue (40. mmol. l(-1)), and its charge is balanced primarily by sulphate at 82-84. mmol. l(-1). Thus these ions probably play a primary role in bringing polymers together directly. Imine bonds formed as a result of protein oxidation also contribute substantially to the stiffness of the glue. Disrupting these bonds with hydroxylamine caused a 33% decrease in storage modulus of the glue, while stabilizing them by reduction with sodium borohydride increased the storage modulus by 40%. Thus a combination of metal-based bonds operates in this glue. Most likely, cross-links directly involving calcium play a primary role in bringing together and stabilizing the polymer network, followed by imine bond formation and possible iron coordination.