Purification and properties of deoxyribonuclease from human urine.
The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine*; D004720:Endonucleases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D011489:Protein Denaturation; D013379:Substrate Specificity
D003851:Deoxyribonucleases; D004720:Endonucleases