This chapter focuses on the purification process of neutral sphingomyelinase from human urine. About 2–3 liters of normal urine from male subjects is collected daily. Male volunteers are preferred over females because of lack of contamination by blood cells, excessive number of uroepithelial cells, and urogenital secretions. The urine sample is immediately centrifuged at 16,270 g (30 min at 4°) in a Sorvall RC-2B refrigerated centrifuge. The cell pellet, a transparent layer at the bottom, is carefully removed, the supernatant is concentrated in a Millipore Minitan ultrafiltration device, using a filter (PTGC-OMT-05) with a nominal cutoff of Mr 10,000. Then, the urine concentrate is further concentrated to 5–10 ml in an Amicon YM10 membrane filter with a nominal cutoff of Mr 10,000. The filters are washed with 1% NaOH and water and reused up to several weeks. Fresh filters are used daily for ultrafiltration purposes. The urine concentrate is applied to a Sephadex G-75 column preequilibrated with the acetate buffer and eluted. First, absorbance at 280 nm is measured in all the fractions and the positive samples are used for protein and enzyme activity determinations. The concentrate (30 mg protein) obtained following Sephadex G-75 column chromatography is dialyzed against glycine and placed in a Biolyte gel bed containing carrier ampholytes of pH range 4–10 using a sample applicator provided by the manufacturer.
ASJC Scopus subject areas
- Molecular Biology