Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 1. Rotational diffusion indicates an extended structure at low ionic strength.
The fluorescence decay of ethidium intercalated into the DNA of nucleosome core particles increases in average lifetime from about 22 ns in H2O to about 39 ns in D2O. This increase, combined with the acquisition of large amounts of data (on the order of 10(8) counts per decay), allows measurement of anisotropy decays out to more than 350 ns. The overall slow rotational motions of the core particle may thereby be more clearly distinguished from the faster torsional motions of the DNA. In 10 mM NaCl at 20 degrees C, we recover a long correlation time of 198 ns in D2O (159 ns when corrected to a viscosity of 1.002 cP), in agreement with the value of 164 ns obtained in H2O. These values are consistent with hydrodynamic calculations based on the expected size and shape of the hydrated particle. To support our conclusion that this long correlation time derives from Brownian rotational diffusion, we show that the value is directly proportional to the viscosity and inversely proportional to the temperature. No significant changes in the rotational correlation time are observed between 1 and 500 mM ionic strength. Below 1 mM, the particle undergoes the "low-salt transition" as measured by steady-state tyrosine fluorescence anisotropy. However, we observe little change in shape until the ionic strength is decreased below approximately 0.2 mM, where the correlation time increases nearly 2-fold, indicating that the particle has opened up into an extended form. We have previously shown that the transition becomes nonreversible below 0.2 mM salt.
Brown
D W
DW
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Libertini
L J
LJ
Small
E W
EW
D000818:Animals; D002645:Chickens; D004058:Diffusion; D004996:Ethidium / Q000737:chemistry; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009707:Nucleosomes / Q000737:chemistry / Q000648:ultrastructure*; D009994:Osmolar Concentration; D013696:Temperature; D014783:Viscosity
D009707:Nucleosomes; D004996:Ethidium