Hagfish slime exudate stabilization and its effect on slime formation and functionality.

Hagfish produce vast amounts of slime when under attack. The slime is the most dilute hydrogel known to date, and is a highly interesting material for biomaterial research. It forms from a glandular secrete, called exudate, which deploys upon contact with seawater. To study slime formation ex vivo and to characterize its material properties, stabilization of the sensitive slime exudate is crucial. In this study, we compared the two main stabilization methods, dispersion in high osmolarity citrate/PIPES (CP) buffer and immersion in oil, and tested the influence of time, temperature and pH on the stability of the exudate and functionality of the slime. Using water retention measurements to assess slime functionality, we found that CP buffer and oil preserved the exudate within the first 5 hours without loss of functionality. For longer storage times, slime functionality decreased for both stabilization methods, for which the breakdown mechanisms differed. Stabilization in oil likely favored temperature-sensitive osmotic-driven swelling and rupture of the mucin vesicles, causing the exudate to gel and clump. Extended storage in CP buffer resulted in an inhibited unraveling of skeins. We suggest that a water soluble protein glue, which mediates skein unraveling in functional skeins, denatures and gradually becomes insoluble during storage in CP buffer. The breakdown was accentuated when the pH of the CP buffer was raised from pH 6.7 to pH 8.5, probably caused by increased denaturation of the protein glue or by inferior vesicle stabilization. However, when fresh exudate was mixed into seawater or phosphate buffer at pH 6-9, slime functionality was not affected, showing pH insensitivity of the slime formation around a neutral pH. These insights on hagfish exudate stabilization mechanisms will support hagfish slime research at a fundamental level, and contribute to resolve the complex mechanisms of skein unraveling and slime formation.