Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography.
Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.
D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D010455:Peptides / Q000032:analysis; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D012685:Sepharose / Q000031:analogs & derivatives; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification*; D013379:Substrate Specificity
D010455:Peptides; D012685:Sepharose; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase