Purification of uricase
AssigneeEastman Kodak Company
InventorsHarry Wayne Harris, James Robert Schaeffer, Roy Eugene Snoke
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Abstract
Catalase is effectively removed from a uricase preparation by amphiphilic chromotography using as the column material cyanogen bromide-activated polysaccharide having a hydrophobic ligand attached thereto. Particularly useful hydrophobic ligands are the alkanediamines.
Drawings
Claims14
A process for separating uricase from catalase comprising:
- applying a uricase preparation having uricase and catalase activities to a chromatographic column comprising a cyanogen bromide-activated polysaccharide material having a hydrophobic ligand attached thereto, thereby leaving the uricase and the catalase on the column, and
- eluting the uricase from the column separately from the catalase to obtain a uricase preparation that is effectively free from catalase.
A process for effectively removing catalase from a uricase preparation obtained by growing Micrococcus leuteus NRRL B-8166, which process comprises chromatographing a buffered solution of the uricase on a column comprising a cyanogen-bromide activated polysaccharide having a hydrophobic ligand attached thereto and eluting the uricase from the column separately from the catalase.
A process for purifying and effectively removing catalase from a uricase preparation obtained by growing Micrococcus luteus NRRL B-8166, which process comprises:
- precipitating the uricase from solution with ammonium sulfate to obtain an ammonium sulfate pellet;
- dissolving the pellet in a buffer solution;
- applying the buffer solution containing the dissolved pellet to a chromatographic column comprising a cyanogen-bromide activated polysaccharide material having a hydrophobic ligand attached thereto, thereby retaining the uricase on the column;
- eluting the uricase from the column using a salt, surfactant, buffer solution to obtain an eluted uricase solution;
- applying the eluted uricase solution to a triethylaminoethyl-cellulose chromatographic column, thereby retaining the uricase on the column;
- eluting the uricase from the triethylaminoethyl-cellulose column using a buffered salt solution;
- and
- dialyzing the buffered salt solution containing the uricase to obtain a purified uricase solution.
A uricase preparation effectively free from catalase activity produced by purifying a uricase preparation obtained by growing Micrococcus luteus NRRL B-8166, the purification steps comprising:
- precipitating the uricase from a solution of the uricase preparation with ammonium sulfate to obtain an ammonium sulfate pellet;
- dissolving the pellet in a buffer solution and applying it to a chromatographic column comprising a cyanogen-bromide activated polysaccharide material having a hydrophobic ligand attached thereto, thereby leaving the uricase on the column;
- eluting the uricase from the column using a salt, surfactant, buffer solution to obtain an eluted uricase solution;
- applying the eluted uricase solution to a triethylaminoethyl-cellulose chromatographic column, thereby leaving the uricase on the column;
- eluting the uricase from the triethylaminoethyl-cellulose column using a salt, buffer solution to obtain a second eluted solution;
- and
- dialyzing the second eluted solution to obtain said uricase preparation.
A process for producing a catalase-free uricase preparation, said process comprising chromatographing a buffered solution of an enzyme preparation having catalase and uricase activity on a column comprising a cyanogen bromide-activated polysaccharide having a 1,6-hexanediamine ligand attached thereto, said polysaccharide having an elemental nitrogen content of at least 6.7 percent, and eluting the uricase from the column.
Dependencies are inferred from the claim text.