Description

• Application: DNAse I is used to nick DNA as a first step to incorporate labeled bases into DNA. The enzyme from Sigma has been used in the processing of rat brain tissue. This study showed that axonal growth on astrocytes is not inhibited by oligodendrocytes.[1] In another study, thawed fixed samples of E. coli were digested with DNAse I from Sigma along with other enzymes. The digestion was done before permeabilization and staining of the nucleic acids.[2]
• Biochem/physiol Actions: DNase I is an endonuclease that acts on phosphodiester bonds adjacent to pyrimidines to produce polynucleotides with terminal 5′-phosphates. In the presence of Mg2+, DNAse I cleaves each strand of DNA independently and the cleavage sites are random. Both DNA strands are cleaved at approximately the same site in the presence of Mn2+.[3] The pH optimum is found to be between 7 and 8. Divalent cations such as Mn2+, Ca2+, Co2+, and Zn2+ are activators of the enzyme.[4] A concentration of 5 mM Ca2+ stabilizes the enzyme against proteolytic digestion. DNAse I from bovine pancreas consists of four chromatographically distinguishable components, A, B, C, and D, with their molar ratios being 4:1:1. Only minor amounts of D are found.[5] 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS)[6] and actin[7] are known to inhibit the enzyme activity.
• Unit Definition: One Kunitz unit will produce a ΔA260 of 0.001 per min per mL at pH 5.0 at 25 °C, using DNA, Type I or III as substrate.
• Physical form: Lyophilized powder containing calcium chloride
• Preparation Note: 10 mg/mL solution of DNAse I in 0.15 M NaCl may lose <10% of its activity when stored for a week in aliquots at −20 °C. The same solutions stored in aliquots at 2-8 °C can lose approximately 20% activity. It remains active for up to five hours at 60 °C between pH 5 and 7, and loses activity in <10 minutes at 68 °C. It loses activity at the rate of 6%/hour in acetate buffer (pH 5.0) and tris buffer ((pH 7.2) at 1 mg/mL concentration.
• Analysis Note: Protein determined by biuret.