Affinity chromatography of porcine pancreas deoxyribonuclease I on DNA-binding sepharose under non-digestive conditions, using its substrate-binding site.
1. DNase I from porcine pancreas, if Mg2+ was present, hydrolyzed both sDNA and dDNA, whether free or bound to Sepharose. The hydrolysis rates were maximum at pH 7.5 with the bound DNAs and at pH 7.0 with the free DNAs negligible at pH 4.0 and pH 10.5 with the free and bound DNAs. The hydrolysis was completely inhibited by 50 mM sodium citrate. 2. With 50 mM citrate buffer (Ph 4.0), DNase I was effectively adsorbed on the DNA-Sepharoses in the absence of 5 mM Mg2+. The adsorbed enzyme was effectively eluated by the buffer containing 1 M KCl (eluate). The amounts of the eluated enzyme were approximately 1.5 X 10(5) units/mg DNA with sDNA-Sepharose and approximately 3.0 X 10(5) units/mg DNA with dDNA-Sepharose. This simple adsorption-elution of the pancreas extract resulted in approximately 300-fold purification of DNase I with a yield of 95%. In the elute, the ratios in activity of trypsin, chymotrypsin and RNase to DNase I were 1/(4.0 X 10(5)), 1/(5.3 X 10(3)), and 1/(4.1 X 10(2)) as low as in the extract, respectively. In addition, the eluate was not contaminated by kallikrein or carboxypeptidases A and B. 3. Upon repeating the adsorption-elution described above, the adsorbing capacities of DNA-Sepharoses gradually deteriorated with the whole pancreas extract, but not with the precipitate of the extract formed on 60% ammonium sulfate saturation, which contained 90% of the DNase I. With the precipitate, one dDNA-Sepharose solumn was repeatedly usable at least 20-times without deterioration. The DNase I preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis. 4. Conceivably, the above-mentioned adsorption of DNase I on DNA-Sepharoses was mainly due to the steric and electrostatic affinity of a relatively large moiety of the DNA molecule to the substrate-binding site, but not to the catalytic site, of the enzyme.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002951:Citrates; D004247:DNA; D004277:DNA, Single-Stranded; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D004720:Endonucleases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010179:Pancreas / Q000201:enzymology*; D012685:Sepharose; D013552:Swine
D002951:Citrates; D004277:DNA, Single-Stranded; D004247:DNA; D012685:Sepharose; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I