Perturbation of ribosome subunit interaction by glutaraldehyde fixation

Ultracentrifugal analysis of ribosomal purity is complicated by the rapid reequilibration of ribosomes with their subunits, and this is further enhanced by the effects of hydrostatic pressure. Fixation of the ribosome system prior to ultracentrifugal analysis supposedly freezes the reequilibration, and thus tends to obviate these difficulties. However, no redistribution of the ribosome-subunit population must be allowed to occur during fixation. Thus, it is necessary that fixation be extremely rapid compared to the ribosome-subunit reequilibration, in order to avoid errors in analysis. It was the purpose of this investigation to make a direct experimental comparison of the rates of these two processes, fixation and ribosome-subunit reequilibration, using the stopped-flow technique with a light-scattering detector, under a variety of buffer environment readjustments. The following findings resulted from this study: (1) Fixation in Tris buffers could not be followed by light scattering because of interaction of glutaraldehyde with Tris, leading to continuous production of high molecular weight contaminants. (2) Polymerization of glutaraldehyde leads to increased ultraviolet absorption, which must not be confused with scattering changes. (3) Undialyzed ribosome solutions prepared by dissolving stock suspensions stored at high levels of magnesium and univalent electrolyte into a known standard buffer produced solutions having free Mg2+ levels lower than those of original buffer, complicating kinetic observations and threatening ribosome stability. (4) Addition of malonic acid as a buffer for Mg2+ largely eliminated this problem. (5) The kinetics of glutaraldehyde were measured quantitatively by studying the perturbation of ribosome association and dissociation kinetics produced during shifts of Mg2+ levels. (6) At fixative levels not producing coagulation, fixation kinetics can be competitive with those of association-dissociation. (7) Mass action effects like those of dilution can be caused by fixation, and can give rise to excess subunits, which may be mistaken for loose couples in original ribosome preparations.