Purification of prostaglandin D synthase by ceramic- and size exclusion chromatography.

Prostaglandin D synthase (L-PGDS) is a major glycosylated polypeptide in cerebrospinal fluid (CSF). The overexpression of L-PGDS in inflamed bovine mammary glands indicates its role as biomarker. No diagnostic tool for the quantitative detection of L-PGDS in cows has been reported. Immunometric ELISA tests might help to identify inflamed bovine tissue. The isolation of pure bovine L-PGDS, which is required for the generation of monoclonal antibodies, is an important prerequisite for a diagnostic ELISA test. Our goal was to identify a suitable technique to generate pure L-PGDS from bovine substrates. In the present study a two-step method for the purification of bovine CSF using ceramic hydroxyapatite chromatography followed by size exclusion chromatography is described. Subsequently, the identification of bovine L-PGDS was demonstrated by Western blot analysis and the high grade of the pure product was shown by 2-D PAGE. The yield of purified L-PGDS was 6.8 mg/l bovine CSF. L-PGDS from bovine CSF is shown to consist of multiple isoforms identical in molecular mass and pI values to those in previously described secretions of inflamed bovine mammary glands. In addition, the method was successfully applied to the purification of L-PGDS from human CSF.

NSET molecular beacon analysis of hammerhead RNA substrate binding and catalysis.

Nanometal surface energy transfer (NSET), which describes an energy transfer event from optically excited organic fluorophores to small metal nanoparticles, may be used as a molecular beacon/ruler similar to FRET, but with advantages over this classical technique. Here we use NSET to measure Mg(2+)-induced conformational changes for a hammerhead ribozyme and confirm these measurements using FRET. These optical experiments enhance our understanding of the different kinetic pathways for this ribozyme.