Non disponible en dehors du Royaume-Uni et de l'Irlande

Analysis Note

Protein determined by biuret.

Application

DNAse I from Sigma has been compared with human urine-derived interleukin 1 inhibitor for the ability to hydrolyze [¹⁴C]DNA [¹⁴C]DNA. It has also been used to cleave a 139 base pair Hind III/Nci I restriction fragment to investigate the stability of the enzyme for use in footprinting experiments. DNase I is widely used as a footprinting agent for studying drug and protein binding to DNA.

Deoxyribonuclease I from bovine pancreas has been used in a study to investigate a new approach to obtaining high-activity RNase, DNase, cholesterolesterase, and trypsin from cattle pancreas. Deoxyribonuclease I from bovine pancreas has also been used in a study to investigate the isolation and further characterization of carp liver DNase.

Used for the removal of DNA from protein samples.

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⁺. The pH optimum lies between 7 and 8. Divalent cations such as Mn²⁺, Ca²⁺, Co²⁺, and Zn²⁺ are activators of the enzyme. 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 respectively. Only minor amounts of D are found. 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS) and actin are known to inhibit the enzyme activity.

Physical form

Lyophilized powder containing calcium chloride

Preparation Note

The enzyme powder may be reconstituted in water or any buffer at pH 4.0-9.0, except phosphate buffer. Calcium chelators should be avoided. 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.

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. (1 unit = 1 Kunitz unit)