Determination of Pulmozyme (dornase alpha) stability using a kinetic colorimetric DNase I activity assay.

An enzymatic activity assay was developed for the determination of dornase alpha human recombinant desoxyribonuclease (DNase I) stability. The method was adapted from a colorimetric endpoint enzyme activity assay for DNase I based on the degradation of a DNA/methyl green complex. With the described modifications the kinetic measurement of enzyme activity is feasible on an automated analyzer system within a rather short time. The development of this assay was based on the need for reliable detection of a possible loss of enzyme activity after transferring the commercial therapeutic agent into sealed glass vials required for a placebo-controlled study. The measuring range of this stability test was from 0 to 3000 U/L corresponding to 0-120% of the original enzyme activity; CV values of control solutions inside the measuring range were between 3% and 5%. The enzyme activity decreased less than 15% during the observation period of 180 days. In conclusion the current kinetic assay is a reliable method for a simple time-saving determination of DNase I activity to test Pulmozyme stability as required for quality control. As dornase alpha is used for inhalation, this method also proved its reliability in testing DNase stability during aerosolization with new inhalation devices (e-flow).

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations.

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.