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

Abstract

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.

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

“…The test is similar in principle to the radial immunodiffusion method for measuring antigen concentration in agarose gels using a specific antibody [27]. A comparable method, but with different solutions and conditions, was used for measuring serum DNase I by Chitrabamrung et al [8] and Nadano et al [9]. Conditions to maximize detection of DNase-low salt concentration and a high ratio of DNA/DNasewere used.…”

“…DNase activity in murine serum has also been assayed by a synthetic end-labelled oligonucleotide substrate assay (D. Sinicropi, personal communication). Chitrabamrung et al [8] and Nadano et al [9] have reported the measurement of DNase I activity in human tissues and body fluids by single radial enzymediffusion methods. The method is based on the hydrolysis of DNA in a DNA-agar plate.…”

“…The method is based on the hydrolysis of DNA in a DNA-agar plate. The area of hydrolysed DNA was identified by either 0.1 amido black [8] or ethidium bromide [9] staining and was proportional to the amount of enzyme in the wells.…”

“…Units are given/ml of serum. and the actin is unable to repolymerize [9] even in the presence of high salt or other actin-binding proteins known to induce actin polymerization. Rat DNase I carries two mutations (E 13 to D and V 67 to I) responsible for the decrease in actin-binding of rat DNase I [3].…”

The candidate gene approach: have murine models informed the study of human SLE?

“…It is interesting to note that although there are three different isoforms of the enzyme, produced by six known polymorphisms, there are no significant differences in enzyme activity between these isoforms [26]. To fully elucidate the genetic contribution of DNASEI to the aetiology of SLE, identification of further SNPs across each isoform of the enzyme will be necessary, prior to detailed association studies in different racial groups.…”