Colorimetric Determination of DNase I Activity with a DNA-Methyl Green Substrate

Sinicropi, Dominick; Baker, Dana Lee; Prince, William S.; Shiffer, KA; Shak, Steven

doi:10.1006/abio.1994.1502

Cited by 73 publications

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“…DNA-Methyl Green Assay-The methyl green assay was used to measure DNA hydrolytic activity of DNase I in the presence of 2 mM Mg 2ϩ and 2 mM Ca 2ϩ as described previously (14). Salmon testes DNA (Sigma), up to ϳ25,000 bp 3 in size, was mixed with methyl green (Sigma) to create the DNA-methyl green substrate with a final DNA concentration of 770 g/ml, used for the assay.…”

Section: Methodsmentioning

confidence: 99%

Hyperactivity of Human DNase I Variants

Pan¹,

Lazarus²

1998

Journal of Biological Chemistry

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Human DNase I, an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight forms by introducing positively charged amino acids at positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA. A series of combination mutants having from one to six additional basic residues compared with the wild type has been constructed, expressed in human 293 cells, and characterized. The degree of hyperactivity for the mutants was highly dependent upon the conditions in various assays, including the concentration and length of the DNA substrate and the salt and divalent metal ion concentrations. The level of hyperactivity was inversely proportional to both DNA concentration and DNA length, consistent with the processive nicking mechanism for the hyperactive variants. Salt was inhibitory for wild type DNase I but actually enhanced the activity of the hyperactive variants. Under optimal conditions for wild type, variants with one additional positive charge possessed the highest activity, which was only severalfold greater than that for wild type. However, in the presence of low DNA concentrations and molecular weights, no Ca 2؉ , and 150 mM NaCl, the variant with six engineered basic residues was most active, having >10,000-fold higher activity than the wild type enzyme. Therefore, any potential increase in potency for the hyperactive variants in vivo will be determined by the concentration, length, and environment of the DNA.

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Section: Methodsmentioning

confidence: 99%

Hyperactivity of Human DNase I Variants

Pan¹,

Lazarus²

1998

Journal of Biological Chemistry

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“…Methods for the quantification of DNase I include colorimetry [4,5], precipitation, fluorometry and viscometry. There are drawbacks with these methods due to difficulties in spectroscopy as a result of turbidity of the solution and the need for large amounts of material.…”

Section: Introductionmentioning

confidence: 99%

Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay

Macanović

Lachmann

1997

Clinical and Experimental Immunology

103

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SUMMARYA new radial enzyme diffusion (RED) method for the measurement of DNase activity in serum and urine is described. The sensitivity of the assay is in the range of 15 . 6-500 ng/ml. The assay is based on the hydrolysis of double-stranded (ds) DNA (or nucleosomes) in agarose. The specificity of the reaction for DNase I was established by showing that either EDTA in the reaction buffer or G-actin abolished DNase activity. Being a functional assay, RED has advantages over radioimmunoassay (RIA) or ELISA, since antigenic assays may also measure complexes of DNase with actin. This method was used to measure DNase activity in the sera and urine of lupus-prone mice (NZB/NZW F 1 hybrids, aged 4-6 weeks). Serum DNase activity in these mice was significantly lower (mean 9 ng/ml) than in control, normal mice of the same age and sex (mean 37 ng/ml). Concentration of DNase in the urine of 4-6-week-old female NZB/NZW F 1 hybrids (24 ng/ml) was significantly lower then in control mice (521 ng/ml). The RED method was used to measure the concentration of actin as the DNase inhibitor in serum. G-actin in the presence of ATP binds DNase and inhibits its nucleolytic activity. Since ATP is necessary for the actin inhibition of DNase I, this shows that there is actin as well as DNase I in the serum. Actin is not only ATP-dependent, but also heat-labile. Heating the sera for 10 min at 50ЊC increases DNase activity. This is an alternative method for measuring the concentration of actin in the serum. An almost identical estimate of actin concentration in sera of normal mice was found from the difference of DNase activity in the presence or absence of ATP (mean actin concentration ¼ 21 ng/ml) or from the difference of DNase activity in heated and non-heated serum (mean actin concentration 18 ng/ml). We were not able to demonstrate DNase inhibitors in the urine of either control or NZB/W F 1 hybrid mice.

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“…Compared to bovine DNase I, the clinically more relevant human enzyme shares 78% identity (Shak et al, 1990) and is 2.4-fold less active (Sinicropi et al, 1994). In the present work, we have systematically analyzed the functional significance of each one of the above 22 residues and five additional positions in the DNA-binding interface by constructing site-directed human DNase I mutants, expressing them in human 293 cells, and characterizing the cell culture media in two activity-based assays.…”

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confidence: 99%

Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence

Cq¹,

Js²,

Herzka³

et al. 1998

Protein Science

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Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gtn 9, Arg 41, p r 76, Arg 1 1 1 , Asn 170, Tyr 175, and Tyr 21 1 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn*+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.Keywords: DNase I; protein-DNA interactions; site-directed mutagenesis; structure-function analysis Human deoxyribonuclease (DNase I), a pancreatic endonuclease that catalyzes the hydrolysis of double-stranded DNA, is currently used for the treatment of pulmonary complications in cystic fibrosis patients (Ramsey, 1996). The recombinant human enzyme is inhaled into the airways where it degrades DNA to lower molecular weight forms, thus reducing the viscoelasticity of CF sputum and improving lung function (Fuchs et al., 1994, Ramsey et al., 1993. In addition, the use of DNase I in a murine model of systemic lupus erythematosus (SLE) has been investigated with encouraging results (Macanovic et al., 1996). The pharmacological and clinical significance of DNase I has led us to recently engineer actin-resistant Reprint requests to: Robert A. Lazarus, Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080: e-mail: lazarus.bob@gene.com.Abbreviations: DNase I, deoxyribonuclease I; CF, cystic fibrosis; SLE, systemic lupus erythematosus: EDTA, ethylenediamine tetraacetic acid: TBE, tris-borate EDTA: Hepes, N-2-hydroxylethylpiperazine-N'-2-ethanesulfonic acid; SMase, sphingomyelinase. One or three letter codes are used to represent naturally occurring L-amino acids. When referring to mutants the wild-type amino acid is followed by the residue number and the new amino acid as in H134Q; DNase I variants with multiple mutation...

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Colorimetric Determination of DNase I Activity with a DNA-Methyl Green Substrate

Cited by 73 publications

References 0 publications

Hyperactivity of Human DNase I Variants

Hyperactivity of Human DNase I Variants

Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay

Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence

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