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Technical Briefs |
1 Department of Legal Medicine and Molecular Genetics, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan; Departments of2 Biology and 3 Forensic Medicine, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan
aauthor for correspondence: fax 81-27-220-8035, e-mail kkoichi{at}med.gunma-u.ac.jp
Deoxyribonuclease I (DNase I; EC 3.1.21.1) is genetically polymorphic (1), and has been postulated to be a candidate molecule for the endonucleolytic activity involved in apoptosis or programmed cell death (2) and involved in the initiation of systemic lupus erythematosus (3). We have reported the presence of high DNase I activity and its gene expression in the anterior lobe of the pituitary gland of both sexes and the abrupt increase in its gene expression at the onset of puberty (4). These findings suggest that DNase I has other, unknown biological roles as well as a digestive role. The single radial enzyme diffusion (SRED) method (5)(6) has been used to quantify DNase I activity and allows the measurement of very low DNase I activity in serum samples, although it requires a long incubation period (10–20 h). Recently we proposed that determination of serum DNase I activity is useful in the diagnosis of acute myocardial infarction (AMI) in the early phase after onset (7). However, the present SRED assay takes too long to be useful in the emergency room, and the development of a more rapid assay for DNase I is desirable for therapeutic decision-making. In this report, we describe a novel assay method that can be used to quantify DNase I activity in 1-µL serum samples within 30 min.
Human DNase I was purified from urine as described previously (8). Antibodies specific to human DNase I and II were produced in rabbits (8)(9)(10). Cellulose acetate membrane (CAM) was purchased from Sartorius, SYBR Green I (SG) was from Cambrex, and salmon testicular DNA (type III) was from Sigma.
An assay reagent set for DNase I activity was prepared in two steps: production of a gel plate for keeping the CAM wet, and preparation of CAM containing reaction buffer. In step 1, 20 mL of 5 g/L molten agarose GP-36 (Nacalai Tesque) in distilled water at 55 °C was poured into a horizontal plastic tray (7.5 x 14.0 cm; 1.0 cm deep) with a lid (EIKEN). After solidification at room temperature, the lid was placed on the gel dish, and the dish was stored at 4 °C. In step 2, we prepared two working solutions. For the first solution, we dissolved 10 g/L of salmon testicular DNA in distilled water, stirred it for 2–3 h, and stored the solution at 4 °C. For the second solution, we diluted the SG 250-fold with dimethyl sulfoxide. To prepare the reaction buffer, we mixed the SG (1.0 µL) and DNA (0.4 mL) solutions and buffer [9.6 mL, containing 0.1 mol/L MES (pH 6.5), 20 mmol/L MgCl2, 2 mmol/L CaCl2] well and poured the mixture into a plastic tray (7.5 x 14.0 cm; 1.0 cm deep). When stored at 4 °C under dark conditions, the reaction buffer can be used for more than 2 weeks. A row of sample spots with centers 15 mm apart was marked with a soft-leaded pencil on the CAM sheet, which must always be handled with a pair of clean tweezers. A 7.0 x 14.0 cm CAM accommodates 32 (4 x 8) samples. The CAM sheet was immersed in the reaction buffer for 30 s. The sheet was then removed from the buffer, and the excess solution was discarded by tapping on a dry filter paper. The CAM sheet was then carefully placed on top of the gel produced in step 1. We thus developed this reagent set consisting of the CAM sheet containing the reaction buffer and the gel in a tray. The reagent set, when covered and kept at 4 °C in the dark, is usable for at least 3 weeks.
Purified human DNase I of known activity (1 µL) was placed on the penciled spot on the CAM sheet with a Pipetman P2 micropipette (Gilson). The 1-µL sample solutions instantly spread and soaked into the sheet. Application of a sample volume of more than 3 µL is not suitable in this assay because the absorptive capacity of the CAM is low and the formation of a dark uniform circle and accurate measurement of its diameter are difficult. After sample application, the tray was covered with a lid, kept horizontal, and incubated at 37 °C. DNase I activity was monitored by periodic measurement of the fluorescence at 312 nm with a CSF-10BF ultraviolet transilluminator (COSMO BIO) or at 470 nm with a Bio-Image Analyzer LAS-1000 (Fujifilm). A circular dark zone formed as the DNase I diffused radially from the spot to which the sample was applied and digested the DNA substrate. Incubation continued until test samples showed well-defined dark circles with diffusion diameters (d) of 2.0–15.0 mm. Differences in length were measured in 0.1-mm increments. A calibration curve was constructed by plotting log10 DNase I activity against d.
The SRED/CAM method is based on the fact that SG shows fluorescence only with unhydrolyzed DNA and not with DNA digested by DNase I (5)(6). A dark circular zone is formed on the fluorescent background as DNase I diffuses from the sample spot position into the CAM sheet containing DNA, SG, divalent cations, and buffer and subsequently hydrolyzes DNA (Fig. 1
, inset). We found that the d was linearly proportional to the logarithm of DNase I activity within the range of 0.01 to 50 x 10-5 U, corresponding to a range of 0.05–250 pg calculated from the specific activity of purified human DNase I after a 15-min incubation. Longer incubation times of 30 and 45 min increased sensitivity 1.7- and 2.3-fold, respectively (Fig. 1
). The lower limit of detection was 0.5 x 10-8 U for both the 30- and 45-min incubations. The upper limit of linearity was 2 x 10-4 U for all three incubation times (15, 30, and 45 min).
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The within- and between-run imprecision studies were performed with the buffer described above and purified human DNase I at two selected concentrations (at the low and high ends of the assay). The within-run imprecision (CV) at DNase I concentrations of 3.4 x 10-1 and 3.4 x 102 U/L was 5.0% (n = 12) and 4.3% (n = 12), respectively. The between-run imprecision at the same two concentrations was 5.9% (n = 12) and 5.1% (n = 12), respectively. Furthermore, known amounts of human DNase I could be quantitatively recovered from the serum sample [mean (SD) recovery, 
97 (3.5)%; n = 12].
There are two predominant DNases in human serum, DNase I and II (5)(9)(10). The DNase activities in serum samples detected by the SRED/CAM method were completely inhibited by anti-DNase I antibody but not by anti-DNase II antibody, confirming that serum DNase activity was derived from DNase I.
The enzyme is able to diffuse more rapidly in the CAM than in agarose gel because the pore size in the CAM matrix may be larger than that in the agarose gel matrix on the SRED plate. Moreover, even a small amount of the enzyme can efficiently digest substrate DNA because the amount of DNA retained in the thin CAM is smaller than that contained in the gel. These properties of the SRED/CAM method produced better results, and the assay was more rapid and sensitive for determination of DNase I activity in this study than the conventional SRED method. When we assayed known amounts of human DNase I separately by the SRED/CAM and conventional SRED methods, we observed no significant difference between the amounts of enzyme measured by each method (n = 20; P >0.5, t-test).
We have developed ethidium bromide-SRED and SG-SRED methods for DNase activity (5)(6). The SG-SRED method is very sensitive and able to measure DNase I concentrations in the picogram range after incubation of 16 h at 37 °C (6). Because DNase I activity in serum samples derived from healthy individuals is low, a long incubation period is required for its determination by conventional SRED methods (5)(6). In our recent study, DNase I activities in serum were found to be transiently increased in patients with AMI within 2 h after the onset of chest pain, whereas only a few patients showed an increase in cardiac markers, such as creatine kinase MB and cardiac troponin T, during this period (7). As an example, in a typical case, serum DNase I activity in an 80-year-old female patient with AMI was measured periodically by this method and gave results of 30 U/L for a sample collected 2 h after onset, 9.0 U/L for a sample collected 12 h after onset, and 8.9 /L for a sample collected 24 h after onset; the 24-h value was similar to her outpatient values (reference interval, 7.1–14.3 U/L). Thus, an abrupt increase in serum DNase I activity accompanying the onset of AMI could serve as a novel biochemical diagnostic marker for AMI in the very early phase after onset. Early diagnosis of AMI allows more appropriate and earlier therapy, such as reperfusion and thrombolysis, to be administered to patients.
In conclusion, we have succeeded in developing a sensitive and rapid SRED/CAM DNase I assay method that is convenient and reliable for determining picograms to femtograms of DNase I in 1-µL serum samples within 30 min.
Acknowledgments
We gratefully acknowledge M. Itoi for technical assistance. This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (Grants 12307011 and 14657111 to K.K., and Grants 12357003 and 15209023 to T.Y.) and a grant from the Japan Foundation of Cardiovascular Research (to K.M.).
Footnotes
1 these authors contributed equally in this study 
References
The following articles in journals at HighWire Press have cited this article:
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  N. Morikawa, Y. Kawai, K. Arakawa, T. Kumamoto, I. Miyamori, H. Akao, M. Kitayama, K. Kajinami, J.-D. Lee, H. Takeshita, et al. Serum deoxyribonuclease I activity can be used as a novel marker of transient myocardial ischaemia: results in vasospastic angina pectoris induced by provocation test Eur. Heart J., December 2, 2007; 28(24): 2992 - 2997. [Abstract] [Full Text] [PDF]
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T. Kumamoto, Y. Kawai, K. Arakawa, N. Morikawa, J. Kuribara, H. Tada, K. Taniguchi, R. Tatami, I. Miyamori, Y. Kominato, et al. Association of Gln222Arg polymorphism in the deoxyribonuclease I (DNase I) gene with myocardial infarction in Japanese patients Eur. Heart J., September 1, 2006; 27(17): 2081 - 2087. [Abstract] [Full Text] [PDF]
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K. Arakawa, Y. Kawai, T. Kumamoto, N. Morikawa, M. Yoshida, H. Tada, R. Kawaguchi, K. Taniguchi, I. Miyamori, Y. Kominato, et al. Serum deoxyribonuclease I activity can be used as a sensitive marker for detection of transient myocardial ischaemia induced by percutaneous coronary intervention Eur. Heart J., November 2, 2005; 26(22): 2375 - 2380. [Abstract] [Full Text] [PDF]
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 Y. Kawai, M. Yoshida, K. Arakawa, T. Kumamoto, N. Morikawa, K. Masamura, H. Tada, S. Ito, H. Hoshizaki, S. Oshima, et al. Diagnostic Use of Serum Deoxyribonuclease I Activity as a Novel Early-Phase Marker in Acute Myocardial Infarction Circulation, May 25, 2004; 109(20): 2398 - 2400. [Abstract] [Full Text] [PDF]
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), 30 (•), and 45 min (
). (Inset), SRED/CAM, demonstrating the dark circles produced by diffused DNase I. Purified DNase I at 13, 8.0, 2.0, and 0.0 x 10-7 U in 1 µL was applied to the spots from left to right, respectively, and incubated at 37 °C for 30 min.