A label-free near-infrared fluorescent assay for the determination of deoxyribonuclease I activity based on malachite green/G-quadruplexes.

Owing to the biological and clinical significance of deoxyribonuclease I (DNase I), it is highly desirable to develop near-infrared (NIR) fluorescent assays for the determination of DNase I activity. Here we report a label-free NIR fluorescent assay for selective determination of DNase I activity based on malachite green (MG)/G-quadruplexes. In the presence of Na(+) or K(+), single stranded DNA (ssDNA) is able to form a G-quadruplex structure, thus to increase the rigidity of MG structure and result in a remarkable NIR fluorescence. As DNase I is capable of cleaving all types of DNA indiscriminately to release nucleotide products, the G-quadruplexes are cleaved into oligonucleotides in the presence of DNase I. As a result, the rigidity of MG structure is reduced, and the NIR fluorescence of the solution decreases with increase of DNase I activity, providing a useful platform for low-cost, label-free and convenient detection of DNase I activity. Under the optimum conditions, the proposed label-free NIR fluorescent assay gave a detection limit of 1 u mL(-1), and a relative standard deviation of 3.2% for eleven replicate detections of 50 u mL(-1) DNase I. The proposed assay was applied to the determination of DNase I activity in spiked human urine samples with recoveries from 99.1 to 109.0%.

Immunochemical assay for deoxyribonuclease activity in body fluids.

We have developed two microtiter plate assays to quantify the deoxyribonuclease activity in biological fluids. Both assays are based on hydrolysis of biotinylated and fluorescein-labeled DNA substrates, with subsequent immunochemical detection of non-digested DNA. The assay based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers is more sensitive (0.05 U/ml) and convenient for quantifying the DNase activity in biological fluids than the assay based on hydrolysis of double-labeled 20 bp oligonucleotide. The DNase activity in urine and blood plasma of healthy donors was measured using the PCR product-based assay. Urine samples revealed greater activity, 1.49+/-1.41 U/ml; blood plasma DNase I-like activity was 0.36+/-0.20 U/ml. DNase II-like activity was not detected in the plasma samples. The data obtained confirm that DNase I-like enzymes are responsible for the majority of deoxyribonuclease activity in blood plasma.

A label-free fluorescent assay for deoxyribonuclease I activity based on DNA-templated silver nanocluster/graphene oxide nanocomposite.

A novel label-free system for the sensitive fluorescent detection of deoxyribonuclease I (DNase I) activity has been developed by utilizing DNA-templated silver nanocluster/graphene oxide (DNA-AgNC/GO) nanocomposite. AgNC is first synthesized around C-rich template DNA and the resulting DNA-AgNC binds to GO through the interaction between the extension DNA and GO. The resulting DNA-AgNC/GO would show quite reduced fluorescence signal because the fluorescence from DNA-AgNCs is quenched by GO. In the presence of DNase I, however, it degrades the DNA strand within DNA/RNA hybrid duplex probe employed in this study, consequently releasing RNA which is complementary to the extension DNA. The released free RNA then extracts DNA-AgNC from GO by hybridizing with the extension DNA bound to GO. This process would restore the quenched fluorescence, emitting highly enhanced fluorescence signal. By employing this assay principle, DNase I activity was reliably identified with a detection limit of 0.10U/ml which is lower than those from previous fluorescence-based methods. Finally, the practical capability of this assay system was successfully demonstrated by its use to determine DNase I activity in bovine urine.

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies.

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.

Human genetically polymorphic deoxyribonuclease: purification, characterization, and multiplicity of urine deoxyribonuclease I.

A deoxyribonuclease I was purified from the urine of a 46-year-old male (a single individual) by using a series of column chromatographies to a homogeneous state as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to be a glycoprotein, containing 1 fucose, 7 galactose, 10 mannose, 6 glucosamine, and 2 sialic acid residues per molecule. The N-terminal amino acid sequence up to the 27th residue of the enzyme was similar to that of pancreatic deoxyribonuclease I from bovine and other species. The catalytic properties of the enzyme derived from a single individual closely resembled those of deoxyribonuclease I purified from human urine collected from several volunteers [Ito, K. et al. (1984) J. Biochem. 95, 1399-1406]. The purified enzyme was found to consist of multiple forms with different pI values. These findings are compatible with the existence of genetic polymorphism of deoxyribonuclease I in human urine previously reported [Kishi, K. et al. (1989) Hum. Genet. 81, 295-297]. This multiplicity of the urine enzyme might be due to variations in the primary structure and/or differences in the content of sialic acid.

Genetic control of urinary deoxyribonuclease I (DNase I) activity levels in mice.

Genetic factors controlling the deoxyribonuclease I (DNase I) activity level were examined in mice. A survey of inbred strains of mice revealed genetic variation in urinary and kidney DNase I activity levels, though a sex difference, males having significantly higher DNase I activity levels than females, was observed in all mouse strains tested. The sex difference in the urinary DNase I activity level was eliminated by testosterone administration to females or gonadectomy to males. The urinary DNase I level was closely correlated to that of the kidneys but no relationship between serum and urinary DNase I activity suggests that the production ratio of DNase I in the kidneys is responsible for strain variation in urinary DNase I levels. Inheritance of quantitative variation of urinary DNase I activity levels was studied by a test cross. The segregation ratio of backcross progenies fitted the model showing that urinary DNase I activity level was controlled by an autosomal single locus, Dna 1 (chi 2 = 0.1053, P > 0.90). The allele Dna 1 ra determines high DNase I inducibility in the kidneys and occurs in BALB/c, C3H/He and A/J strains. The allele Dna 1 rb determines low DNase I inducibility in the kidneys and occurs in DBA/2 and C57BL/6 strains.

Tissue distribution of deoxyribonuclease I (DNase I) activity level in mice and its sexual dimorphism.

The distribution of deoxyribonuclease I (DNase I) in mouse tissue and body fluids, the effects of sex on the enzyme activity, and the postnatal developmental regulation of the enzyme were examined in C3H/He mice. DNase I activity was high in the salivary gland and urine; and moderate in the liver, kidney, spleen, preputial gland and seminal vesicle. Weak activity was detected in the pancreas, heart, lung, brain, small intestine, thymus, coagulating gland and serum. Sexual dimorphism, of which males had significantly higher activity than females, was observed in the kidney, urine and liver. This sexual dimorphism in renal DNase I activity was eliminated by gonadectomy to males and testosterone treatment to females. These results reveal sex hormonal control of DNase I production in the kidney. DNase I activity in the liver of infant mice was almost equivalent to that of adult mice while the enzyme activity in the salivary gland, kidney and spleen remained at a low level; less than 5% at one week old and less than 20% at four weeks old. These results suggest tissue specific control of DNase I activity levels during postnatal development.

Usefulness of deoxyribonuclease I (DNase I) polymorphism for individualization from small aged urine stains.

We devised a procedure that combines a simple extraction method, isoelectric focusing and activity staining using the dried agarose film overlay method, for deoxyribonuclease I (DNase I) typing from aged urine stains. DNase I types were determined without difficulty from urine stains kept at room temperature for 3 months or more in all of the samples tested. The amounts of urine stains required for typing after 3 months of storage were estimated to be equivalent to 60-120 microl of liquid urine. Therefore, considering that useful PCR-based DNA typing has not yet been developed for urine stains, DNase I polymorphism could be considered the first biochemical marker found to be well suited for individualization from small aged urine stains.

A new individualization marker of sweat: deoxyribonuclease I (DNase I) polymorphism.

We confirmed for the first time, both biochemically and immunologically, the existence of deoxyribonuclease I (DNase I) in human liquid sweat. Isoelectric focusing of sweat samples on polyacrylamide gels (pH 3.5 to 5), followed by dried agarose film overlay detection, was used to determine the phenotypes of sweat DNase I. Because this detection method not only had high sensitivity, but also high band resolution, it was possible to determine DNase I types from sweat samples of 50 to 100 microL. Pretreatment of sweat samples with sialidase was essential for typing to enhance markedly the sensitivity accompanied by simplification of the isozyme pattern. The DNase I types in all sweat samples were consistently related to the types found in corresponding blood, urine, and semen samples. DNase I typing could, therefore, provide a novel discriminant characteristic in the forensic examination of sweat.

Genetic polymorphisms detectable in human urine: their application to forensic individualization.

This review describes several types of genetic polymorphism, which have recently been identified in human urine in our laboratory, and have also been found in other human body fluids such as blood, saliva and semen. These include uropepsinogen, ribonuclease, deoxyribonuclease I (DNase I), deoxyribonuclease II (DNase II), 43-kDa glycoprotein, alpha-L-fucosidase, glutamate pyruvate transaminase, alpha-2-HS-glycoprotein, transferrin and vitamin D-binding protein. Several substances can be detected more easily in urine than in plasma. The concentrations of uropepsinogen, DNase I and DNase II in blood plasma are too low for analysis, whereas those in urine are high enough for easy typing. In practice, DNase I-polymorphism is one of the most useful genetic markers for practical purposes, because of its higher content in various body fluids including urine, a well-balanced gene frequency, and its easy and accurate detectability. Furthermore, several genetic markers previously identified in blood and/or other forensic samples can be phenotyped reproducibly and easily from the corresponding urine samples. Thus, urine, in addition to the convenience and non-invasive nature of its collection, is by no means inferior to blood as a sample source for typing in the field of forensic science. Biochemical and serological typing of genetic polymorphisms present in human urine could offer useful information to practising forensic biologists for forensic individualization of urine samples.

Genetic polymorphism of human urine deoxyribonuclease I.

A genetic polymorphism of human urine deoxyribonuclease I (DNase I) has been detected by the technique of polyacrylamide gel isoelectric focusing (IEF-PAGE) followed by immunoblotting with anti-DNase I antibody. Family studies showed that the three common phenotypes - DNASE1 1, 1-2, and 2 - and the other four rare phenotypes - DNASE1 1-3, 2-3, 2-4, and 3-4 - represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1*1, *2, *3, and *4. The frequencies of the DNASE1*1, DNASE1*2, DNASE1*3, and DNASE1*4 alleles in a studied Japanese population were 0.5453, 0.4396, 0.0117, and 0.0034, respectively.

Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method.

In the single radial enzyme-diffusion (SRED) method for assay of deoxyribonuclease I, a precisely measured volume of the enzyme solution is dispensed into a circular well in an agarose gel layer in which DNA and ethidium bromide are uniformly distributed. A circular dark zone is formed as the enzyme diffuses from the well radially into the gel and digests substrate DNA. The diameter of the dark circle of hydrolyzed DNA increases in size with time and correlates linearly with the amount of enzyme applied to the well. Thus, the SRED can be used for quantitation of deoxyribonuclease I with a limit of detection of 2 x 10(-6) unit. This corresponds to 1 pg of purified urine deoxyribonuclease I. We measured the deoxyribonuclease I activity of 17 different human tissues and body fluids from healthy donors. Urine samples showed the greatest activity, 6.0 +/- 2.2 kilo-units/g protein (mean +/- SD). Serum deoxyribonuclease I activity was 4.4 +/- 1.8 units/L.

Genetic polymorphism of urine deoxyribonuclease I isomerases of subterranean mole rats, Spalax ehrenbergi superspecies, in Israel: ecogeographical patterns and correlates.

Genetic polymorphism of urine deoxyribonuclease I (DNase I) of mole rats was analyzed by isoelectric focusing in a thin-layer polyacrylamide gel (IEF-PAGE). One hundred and three subterranean mole rats, comprising 13 populations belonging to the four chromosomal species (2n = 52, 54, 58, 60) of the actively speciating Spalax ehrenbergi superspecies in Israel, were tested. The following results were indicated. (i) Spalax DNase I consisted of 6-12 major isozymes. (ii) Four phenotypes (numbers in parentheses) were 1 (92), 1-2 (5), 1-3 (4), and 2 (1). The decreasing order of genetic diversity, He, in the four species was 0.37, 0.13, 0.10, and 0.0 for 2n = 58, 52, 54, and 60, respectively. (iii) Spearman rank correlations and multiple regression analyses indicated associations of allele frequencies and genetic diversity with climatic and vegetation factors. We concluded that (a) climatic selection, either directly or indirectly through plant (i.e., food resources) diversity, plays an important role in DNase genetic differentiation and (b) no gene flow and introgression occur between the recent derivative of speciation (2n = 60) and its ancestor (2n = 58), suggesting the operation of reproductive isolation between both species despite natural hybridization.

Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing.

We have devised two independent detection methods for investigating possible molecular heterogeneity and genetic polymorphism in human DNase I, in terms of both its antigenicity and enzymatic activity. One was an immunoblotting method using an antibody specific to DNase I following polyacrylamide gel isoelectric focusing (IEF-PAGE). The DNase I-specific antibody was raised in a rabbit using purified enzyme from human urine as the immunogen. DNase I in urine was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 3.5-4.0. This method was able to detect as little as 0.1 micrograms of the purified DNase I and facilitated classification of desialylated urine samples from different individuals into several groups according to differences in DNase I isozyme patterns. About 0.5 ml of the original urine was sufficient for analysis of the isozyme patterns. The other method was the zymogram method, which had a high sensitivity and resolution almost identical to those of the immunoblotting method for analysis of DNase I patterns. It was easier to perform, more time-saving, and more useful since it did not require antibody specific to DNase I. These two methods should prove valuable for biochemical and genetic analysis of DNase I isozymes.

Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.

Population studies of human deoxyribonuclease I polymorphism.

We have improved the resolution of the conventional method for phenotyping deoxyribonuclease I (DNase I), which makes use of isoelectric focusing, by the addition of amphoteric separators. The distribution of DNase I phenotypes was extensively examined using this improved method in 1,212 unrelated individuals from a Japanese population. In order to investigate a possible difference in phenotype distribution between different populations, DNA samples from Germans and African Americans were genotyped using the polymerase chain reaction. The DNASE1*2 allele in the German population was found to be predominant among the four alleles of DNase I, in contrast to the Japanese population. These results are the first to demonstrate a wide distribution of DNase I polymorphism in the Japanese population as well as in two other populations.

DNA-hydrolyzing activity of Bence Jones proteins.

Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status.

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.

Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes.

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.