Histone proteolysis in fish erythrocyte nuclei

The erythrocyte histones of trout, carp, white sucker, and chicken are subject to very different levels of autolytic activity. Carp erythrocyte histones extracted from typical nuclear preparations suffer extensively from degradation; histones 1, 5, and 3 (H1, H5, and H3 respectively) are preferentially cleaved and characteristic peptides designated P1, P2, and P3 appear during the course of proteolysis. Generally, erythrocytes from different fish species yield highly disparate proportions of H1 and H5, but this is not a consequence of the variable levels of proteolytic activity in these species. Phenylmethylsulfonyl fluoride (PMSF) (1.0 mM) was found to be superior to 50 mM sodium bisulfite as a protease inhibitor and was well suited for use in media employed for cell washes and the isolation of nuclei. Nonetheless, in carp erythrocytes residual protease activity (qualitatively the same as the uninhibited activity) persists even in the presence of PMSF. It is activated during cell lysis and remains associated with the nuclear fraction of the lysate during subsequent washes. The isolation of intact nuclei is important for the ultimate extraction of undegraded histone, especially from sources in which the risks of autolysis are high or unknown.