Chromatofocusing of purified placental sphingomyelinase

To establish the profile of the multiple forms of placental lysosomal sphingomyelinase, we have developed in a preliminary step, a new sequence of purification. This protocol has a good yield such that the final preparation is representative of the whole enzyme. Using chromatofocusing, purified sphingomyelinase is resolved into 4 major forms whose pI's are 7.2, 6.5, 5.9 and 5.2. Chromatofocusing is preferable to isoelectric focusing because it is more rapid, more reproducible and above all it provides a constant profile of activity which is not influenced by experimental conditions, in particular, detergent concentration.