Abstract
The characterization of a heteroassociating system is described in this article:
- The thermodissociation is studied using band sedimentation analysis;
- The electrostatic dissociation is studied using boundary sedimentation analysis.
The heterodimer stability of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) was examined recently by sedimentation equilibrium and velocity methods as a function of temperature and ionic strength. RT completely dissociates at 37°C in 50 and 100 mM Tris, pH 7.0, based on sedimentation equilibrium measurements. To obtain the temperature transition data for dissociation, we used band sedimentation velocity in D2O, which allows determination of the sedimentation coefficient, s20,w' using approximately 60 μg of RT. The dissociation of RT as a function of increasing temperature from 20°C to 37°C was monitored by measuring the decrease in sedimentation velocity. Different time periods of temperature incubation for RT generated different thermal dissociation curves that allow estimates of dissociation rate constants and respective equilibrium times for dissociation. To examine the effect of ionic strength on p66p51 association, we determined the changes in s20,w as a function of NaCl concentration. There is a sharp decrease in s20,w between 0.10 and 0.5 M NaCl, leading to apparent complete dissociation. The above results support a major role for electrostatic interactions in the stabilization of the RT heterodimer.