Abstract
Arylsulfatase A (cerebroside sulfate sulfohydrolase) was purified 3500-fold at a 7% yield from human urine. A crude urinary protein concentrate was prepared by treating pooled urine with ammonium sulfate and subsequently drying the precipitate with acetone. The powder thus obtained was extracted with buffer and was subjected to chromatographic and electrophoretic procedures as follows: (a) ammonium sulfate reverse gradient solubilization chromatography; (b) DEAE-cellulose chromatography; (c) Sephadex G-200 gel filtration; (d) preparative polyacrylamide gel electrophoresis; (e) SP-Sephadex chromatography; and (f) antialbumin-Sepharose chromatography. The enzyme was judged to be essentially homogeneous by: (a) a single band on polyacrylamide gel electrophoresis at two pH values; (b) formation of a single precipitin line on immunodiffusion against its antiserum: (c) complete freedom from albumin, the major contaminating protein; and (d) a single band on sodium dodecyl sulfate gel electrophoresis.
Highlights
Arylsulfatase A was purified 3500-fold at a 7% yield from human urine
Arylsulfatase A appears to be composed of two subunits of identical size
The kinetic constants K, V, pH optimum, and the energy of activation were determined for the substrates, 4nitrocatechol sulfate, 4-methylumbelliferyl sulfate, and cerebroside sulfate
Summary
Arylsulfatase A (cerebroside sulfate sulfohydrolase) was purified 3500-fold at a 7% yield from human urine. A crude urinary protein concentrate was prepared by treating pooled urine with ammonium sulfate and subsequently drying the precipitate with acetone. The powder obtained was extracted with buffer and was subjected to chromatographic and electrophoretic procedures as follows: (a) ammonium sulfate reverse gradient solubilization chromatography; (b) DEAEcellulose chromatography; (c) Sephadex G-200 gel filtration; (d) preparative polyacrylamide gel electrophoresis; (e) SPSephadex chromatography; and (f) antialbumin-Sepharose chromatography. The enzyme was judged to be essentially homogeneous by: (a) a single band on polyacrylamide gel electrophoresis at two pH values; (b) formation of a single precipitin line on immunodiffusion against its antiserum; (c) complete freedom from albumin, the major contaminating protein; and (d) a single band on sodium dodecyl sulfate gel electrophoresis
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