脱氧核糖核酸酶 I(Sigma代理)DN25-10MG

Analysis Note

Protein determined by biuret.

Application

用于从蛋白质样品中除去 DNA。

DNAse I is used to nick DNA as a first step to incorporate labeled bases into DNA. The enzyme from Sigma has been used during the isolation of plasma membrane vesicles from Neurospora crassa cell culture.¹ It has also been used along with trypsin for the preparation of single cell suspension from rat testes.²

Deoxyribonuclease I from bovine pancreas has been used in a study to compare several procedures for reducing RNase contamination in preparations of DNase. Deoxyribonuclease I from bovine pancreas has also been used in a study to investigate the effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis.

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.

Derived from New Zealand-sourced pancreas

Unit Definition

在 pH 5.0 和 25°C 下以 I 型或 III 型 DNA 为底物，一个 Kunitz 单位每分钟产生的 ΔA₂₆₀ 为 0.001。[Mg²⁺] = 4.2 mM。

Physical form

Crude preparation, contains calcium chloride

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 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. The pH optimum is found to be between 7 and 8.⁴ 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.⁵ 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS)⁶ and actin⁷ are known to inhibit the enzyme activity.