Purification and properties of a DNase inhibitor from Nicotiana tabacum cell cultures.

Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.