Preparation of protease-free and ribonuclease-free pancreatic deoxyribonuclease.
When pancreatic DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, freedom from contaminating ribonucleases or proteases is an important property of the enzyme preparation. A simple one-step procedure has been developed to effect complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose (a column (0.9 X 60 cm) operated in series with a regeneratable 1-ml bed). Commercial preparations of DNase (about 10 mg) give a quantitative yield of the enzyme that is protease-free as evidenced by full stability for more than 10 days at pH 8 and 37 degrees C even in the absence of the protecting action of Ca2+. Removal of the last traces of RNase has been accomplished by affinity chromatography on a column (0.4 X 72 cm) of 5'-(4-aminophenyl-phosphoryl)-uridine 2'(3')-phosphate-Sepharose; the product is a highly active DNase that gives no detectable hydrolysis of RNA by assay on radioactive substrates.
D002846:Chromatography, Affinity / Q000379:methods; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification
D003851:Deoxyribonucleases; D012260:Ribonucleases; D010447:Peptide Hydrolases