Purification to homogeneity of the human skin chymotryptic proteinase "chymase".
The chymotrypic proteinase "chymase" has been purified to apparent homogeneity from human skin. Our procedure differs from previously published partial purifications in that it does not involve affinity chromatography, most of the steps are carried out in 2 M KCl which stabilizes the enzyme, detergent is used to protect the enzyme in low-ionic-strength media, and troublesome concentration steps are avoided by using very small columns of high-capacity exchangers. The high-salt skin extract is applied successively to columns of hydroxyapatite, copper chelate Sepharose, and Sephadex G-100 in 2 M KCl. After dialysis against a zwitterionic detergent, the enzyme is adsorbed onto a 0.4-ml column of CM-Sepharose. An alkaline wash removes the remaining contaminants from the highly cationic enzyme, which is then eluted with 1 M KCl in a final volume of 2 ml. Sodium dodecyl sulfate electrophoresis reveals a single diffuse band of Mr 30,000. Recoveries range from 20 to 40% with yields of 0.2 to 0.4 mg of enzyme from 200 g of skin. Specific activities vary from 600 to 1400 units/mg for the hydrolysis of acetyltyrosine ethyl ester.
D002845:Chromatography / Q000379:methods; D053818:Chymases; D003300:Copper; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D006801:Humans; D012685:Sepharose; D012697:Serine Endopeptidases; D012867:Skin / Q000201:enzymology*
D003911:Dextrans; D003300:Copper; D012685:Sepharose; C025614:sephadex; D010450:Endopeptidases; D012697:Serine Endopeptidases; D053818:Chymases