Purification of acid sphingomyelinase from human placenta: Characterization and N‐terminal sequence

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Abstract
Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A‐Sepharose, octyl‐Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti‐ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110 000‐fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent‐containing assay system. Analysis of the final preparation by SDS‐PAGE resulted in a single protein band with a molecular mass of ∼75 kDa, which was reduced to ∼60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N‐terminal amino acid sequence of the mature placental enzyme.