Deoxyribonuclease

Chemical Properties

Off-white lyophilized powder, soluble in water, slightly soluble in 30% ethanol and acetone, optimal pH 7.8.The addition of 5mmol/L Ca2+ or diisopropylfluorophosphoric acid (DFP) to the aqueous solution can increase its stability. The inhibitors are EDTA and sodium dodecyl sulfate; the activators are divalent metal ions, and the best effect can be obtained by combining Mg2+ and Ca2+. The absorption coefficient of 1% aqueous solution at 280 nm is 11.1. The average chain length of the limiting digestion is tetranucleotide, and the enzyme reaction: deoxyribonucleic acid ═dinucleotide-5'-phosphate+oligonucleotide-5'-phosphate.

Uses

Deoxyribonuclease I is used for the removal of DNA from protein samples. 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. 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

Uses

Deoxyribonuclease I RNase-free solution from bovine pancreas has been used to digest DNA from various samples.

Uses

Amplification grade DNase I has been used for the digestion of DNA during isolation and purification of RNA. The purified RNA can be used for the synthesis of cDNA using RNA reverse transcriptase.

General Description

DNase I, or Deoxyribonuclease I, is an endonuclease isolated from bovine pancreas.

• Our DNase I Digests double str and single stranded DNA into oligo and mononucleotides.
• Bovine pancreatic DNase exists as four isozymes, having isoelectric points for A, B, C and D: 5.22, 4.96, 5.06 and 4.78.3. The predominant form is A, with smaller amounts of B and C, and only minor amount of D.
• DNase I structure resembles the structure of to exonuclease III. It includes two central sheets. Each β sheet is composed of six β-strands. This complex of β sheets is surrounded by extensive loop and α-helical regions. This enzyme shares structural similarity to exonuclease III.^[1]

Flammability and Explosibility

Not classified

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 is found to be 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. Only minor amounts of D are found. 2-Mercaptoethanol, chelators, sodium dodecyl sulfate (SDS) and actin are known to inhibit the enzyme activity.

Clinical Use

DNAse is a human endonuclease, normally present insaliva, urine, pancreatic secretions, and blood. The enzymecatalyzes the hydrolysis of extracellular DNA into oligonucleotides.Aerosolized recombinant human deoxyribonucleaseI (rhDNAse), dornase alfa, Pulmozyme, has beenformulated into an inhalation agent for the treatment of pulmonarydisease in patients with CF.Among the clinical manifestations of CF are obstructionof the airways by viscous, dehydrated mucus. Pulmonaryfunction is diminished, and microbes can become entrappedin the viscid matrix. A cycle of pulmonary obstruction andinfection leads to progressive lung destruction and eventualdeath before the age of 30 for most CF patients. The immunesystem responds by sending in neutrophils, and theseaccumulate and eventually degenerate, releasing largeamounts of DNA. The high levels of extracellular DNA releasedand the mucous glycoproteins are responsible for thedegenerating lung function. The DNA-rich secretions alsobind to aminoglycoside antibiotics typically used to treat theinfections. In vitro studies showed that the viscosity of thesecretions could be reduced by application of DNAse I.
Pulmozyme is approved for use in the treatment of CFpatients, in conjunction with standard therapies, to reducethe frequency of respiratory infections requiring parenteralantibiotics and to improve pulmonary function. The dose isdelivered at a level of 2.5 mg daily with a nebulizer.Pulmozyme is not a replacement for antibiotics, bronchodilators,and daily physical therapy.