Deoxyribonuclease contamination

Note Lysis buffer (without proteinase K) and the TE buffer should be autoclaved to remove contaminating deoxyribonuclease activity. 

Two examples where metal ions confer stability or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme ), and Factor VIII. In the case of rhDNase, Ca2+ ions (up to 100 mM) increased the stability of the enzyme through a specific binding site (64). In fact, the removal of calcium ions from the solution with EGTA caused an increase in deamidation and aggregation. However, this effect was observed only with Ca+2 ions other divalent cations, Mg2+, Mn2+, and Zn2+, were observed to destabilize rhDNase. Similar effects were observed in Factor VIII. Ca2+ and Sr2+ ions stabilized the protein, whereas others such as Mg2+, Mn2+ and Zn2+, Cu2+, and Fe2+ destabilized the enzyme (65). In a separate study with Factor VIII, a significant increase in the aggregation rate was observed in the presence of Al3+ ions (66). The authors note that other excipients like buffer salts are often contaminated with Al3+ ions and illustrate the need to use excipients of appropriate quality in formulated products. 

Among the contaminants of DNA preparations obtained from mammalian tissue, protein and RNA are the most conspicuous. Proteins can be separated by denaturation with chloroform and amyl alcohol, a procedure that may be followed by repeated washings with sodium dodecyl sulfate. RNA can be eliminated by repeated digestion of the preparation with ribonu-clease. (Even crystallized RNase has deoxyribonuclease activity therefore, the RNase preparation must be boiled for 3 minutes before using it for treating the DNA preparation.)... 

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