Activation of deoxyribonucleoprotein in human leucocytes stimulated by phytohemagglutinin: I. Kinetics of the binding of acridine orange to deoxyribonucleoprotein

Abstract

In accordance with previous findings a pronounced and rapid increase in the fluorescence intensity of fixed human leucocytes stained with acridine orange (AO) was observed only a few minutes after stimulation with phytohemagglutinin (PHA). This increase in fluorescence intensity was ascribed to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex. The measured AO fluorescence was not caused by staining artifacts due to altered autofluorescence of leucocytes, nonspecific AO binding or changes in cell membrane permeability. On the average, the number of AO binding sites increased by a factor of 2 after PHA stimulation. In activated cells, about half of all binding sites in DNP were accessible to AO. PHA-P and PHA-M induced the same final increase in AO binding sites in DNP although the rate of increase in AO binding sites was lower when PHA-M was used. Since the same decrease in rate could be obtained by lowering the PHA-P doses, it was concluded that the difference in the activity between the two PHA fractions might be due to different contents of the stimulating factor. Leucocytes sampled from infected or recently vaccinated donors exhibited a spontaneous increase in AO binding sites in DNP, which, in most cases, could not be further increased by PHA. The increase in AO binding sites in DNP, which seems to be one prerequisite for later cell growth, is apparently due to weakened interaction between DNA and concomitant proteins. This hypothesis was supported by the finding that acetylation of amino groups in the protein moiety of the DNP complex resulted in a marked increase in AO binding sites in nonstimulated cells while no such increase was found in PHA stimulated cells having a large number of accessible groups for AO binding even without acetylation.