Fluorescent morphological probe for hyaluronate.

Hyaluronate levels change dramatically during morphogenesis of various tissues and organs. Morphological detection of the exact temporal and spatial distribution patterns of hyaluronate may help to elucidate its role in morphogenesis. Since no specific direct method for visualizing hyaluronate with the light or electron microscope is currently available, we have developed a morphological probe by exploiting the high-affinity interaction of cartilage proteoglycan with hyaluronate. The core protein of this proteoglycan consists of a region that binds specifically to hyaluronate with a high association constant, and a region to which the majority of sulfated polysaccharide chains are covalently attached. The polysaccharide chains were removed by treatment with chondroitinase ABC, and the core protein, labeled with rhodamine, was used as the probe. This fluorescent probe binds reversibly and specifically to [3H]hyaluronate in a binding assay using ammonium sulfate precipitation of the core protein. The probe has been used to visualize the cell surface hyaluronate of rat fibrosarcoma cells, 3T3 cells, and SV-40 transformed 3T3 cells, three cell types with significantly different amounts of cell surface-associated hyaluronate.