6-IODOACETAMIDOFLUORESCEIN LABELING TO ASSESS THE STATE OF SULPHHYDRIL GROUPS AFTER THERMAL STABILIZATION OF ISOLATED-NUCLEI

Isolated nuclei and nuclear matrices, prepared from mouse erythroleuka emia cells, were reacted with the sulphhydryl-specific dye 6-iodoaceta midofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry an d confocal microscopy quantitative analysis did not reveal major diffe rences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanni ng laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. Ho wever, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix prepa ration. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between hea t-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.