Biochemical analysis of hyalin gelation: an essential step in the assembly of the sea urchin extraembryonic matrix, the hyaline layer

We have examined the effects of calcium and magnesium on both the structural characteristics and the self-association reaction of hyalin, a major protein component of the sea urchin extraembryonic matrix, the hyaline layer. In the absence of calcium, the circular dichroic spectrum revealed a protein possessing a high β sheet content. The presence of increasing concentrations of calcium resulted in an increase in β sheet content and a coincidental decrease in α helix. This effect occurred with an apparent dissociation constant (calcium) of 1.5 mM. The calcium-induced structural change was potentiated by magnesium. Similar concentrations of calcium protected hyalin from digestion by trypsin and this effect was potentiated by magnesium. The thermal denaturation profile of hyalin was modulated by calcium. At a concentration of 3 mM, calcium protected hyalin from thermal denaturation, an effect partially mimicked, but not potentiated, by magnesium. Calcium was also found to modulate both the intensity and the wavelength of maximal, endogenous tryptophan fluorescence. The effect of calcium on hyalin tertiary structure had a concentration dependence decidedly different from those reported above with an apparent dissociation constant of 0.18 mM. Collectively, these results delineate two distinct roles for calcium in modulating hyalin structure and allow us to define the pathway leading to hyalin-gel formation.