Effect of polyamines on DNase I activity

The biogenic polyamines putrescine, spermidine and spermine are the most prevalent polyamines in mammalian cells. They are involved in several biological processes one of which is to bind nucleic acid.It has been expected that this feature reduces accessibility to enzymes and in fact, in some experimental conditions, an inhibition of endonuclease activity is observed in DNA with the addition of polyamines [Pingoud, A et al. (1984) Biochemistry 23, 5697 - Kuosmanen, M and Poso, H (1985) FEBS Lett. 179, 17 - Baeza, I et al. (1987) Biochemistry 26, 6387 - D’Agostino, L et al. (2005) FEBS Journal 272, 3777]. With the aim to produce a polydeoxyribonucleotide(PDRN)-based hydrogel for dermatological purposes we tested if polyamines were able to protect PDRN from endonucleases’ digestion. Due to the molecular weight heterogeneity of our PDRN, extracted from trout, it was difficult to evaluate the enzyme activity by electrophoresis; thus we analyzed the activity of DNase I by the Kunitz assays: the hydrolysis of PDRN in the absence or presence of spermine and spermidine at different concentrations was monitored by measuring the absorbance increase at 260 nm. The incubation of 2.6 mg/ml PDRN with 0.12 mM spermine for 30’ at 37°C induced a significant decrease of the hydrolysis rate. This effect doesn’t result from the action of spermine on the DNase itself, because the addition of the polyamine just before the assay doesn’t inhibit the enzyme activity. The protective role against digestion disappears when the concentration of spermine was increased to 0.24 and 1.2 mM. Unlike spermine the pretreatment of PDRN with 0.12 mM spermidine produces a marked increase (200%) in the DNase I activity. As before the effect doesn’t result from an action of spermidine on the DNase itself. The results demonstrate that the effect of spermine on PDRN is concentration dependent as reported by other authors for its effect on DNA precipitation [Pelta, J et al. (1996) J. Biol. Chem. 271, 5656]. The contrary effects of activity between spermine and spermidine isn’t at the moment explainable supported by the controversial data from literature where it is reported that spermidine’s positive or negative effect is nuclease specific.