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

Abstract

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 p I 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 μg of the purified DNase I and facilitated classification of desialy-lated 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.