Thesis

Physical Characterization of Embryo Hyalin from the Sea Urchin, Strongylocentrotus purpuratus

Hyalin is a major glycoprotein of the hyaline layer, the extracellular matrix surrounding sea urchin embryos and is required for normal morphogenesis. It is an extremely large molecule consisting of three species ranging from 2.1x10^5-6.3x10^4. The aim of this thesis is to use separation and analytical methods to further characterize the components of hyalin with special reference to its carbohydrate moieties. Hyalin was purified according to the procedures of Gray J, Justice R, Nagel GM, Carroll EJ Jr. 1986. Resolution and Characterization of a Major Protein of the Sea Urchin. Journal of Biological Chemistry, 261(20), pp. 9282-9288. Three major species of hyalin with sizes of 228kDa, 147kDa, and 63kDa were observed using analytical size exclusion chromatography on a Sephacryl S500 (10/300) column. The pI of the hyalin was also characterized to be pH6.3, pH4.9, and pH4.8 for three components. To examine the nature of the carbohydrate content of hyalin beta-elimination with sodium hydroxide was done and was followed kinetically at 240nm. Based on these results hyalin was determined to contain o-linked carbohydrates. The sugar moieties on hyalin were further characterized using lectins. Hyalin was found to bind to concanavalin A, soybean agglutinin, wheat germ agglutinin, and Ricinus communis agglutinin. Of the components detected by size exclusion chromatography, only the 63kDa component of hyalin was shown to consist of mannose or glucose moieties that bound to ConcanavalinA and suggest the heterogeneity of sugar moieties for hyalin. Further physical characterization of hyalin is important in understanding the structure-function relationships and how its individual components and glycans are involved in embryonic development.

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