Rapid Quantification of DNase I Activity in One-Microliter Serum Samples

Abstract

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 …