IRREVERSIBLE THERMAL DENATURATION OF ESCHERICHIA COLI RIBOSOMES

Irreversible thermal denaturation of high salt-washed Escherichia coli (B and Q13) ribosomes has been followed in terms of biological activity, sedimentation properties, and turbidity, as afunctionof solvent composition. In a buffer composed of 0.01 m Tris-0.01 m MgAc2 (pH 7.4) ribosomes lose 50% of their ability to form polyphenylalanine from polyuridylic acid in 5 min at 57° (Td,5). In this temperature range 70S ribosomes are destroyed, presumably by aggregation. The sharpness of this thermal transition is illustrated by the fact that in 0.01 m Tris-0.01 m MgAc2 several hours are required for 50% inactivation at 50°, while at 60° 50% inactivation occurs within 1 min. Reduction of the magnesium ionconcentration to 10-4 m reduces the Td,5 to 53.5° and under these conditions the normal 50S and 30S subunits are converted into slower sedimenting particles (~35-40 and 25S). Monovalent cations (K ⋝ NH4 > Na) in the presence of 0.01 m MgAc2 (but not 10-4 MgAc2) protect ribosomes from thermal inactivation presumably by preventing aggregation. In 0.01 m Tris- 0.01 m MgAc2-0.1 m KC1 (pH 7.4), ribosomes exhibit a 7d,5 of 60.5° and the loss in biological activity is closely paralleled by the destruction of the 30S subunit. The effect of heating on ribosome activity is characterized by an initial increase in activity (variable with the preparation) followed by a rapid but decreasing rate of inactivation. No explanation is provided for this activation, but it apparently results from an increase in the number of active ribosomes and is not due to a relaxation of the ribosomal structure which would also lead to translational ambiguity. Evidence is presented which suggests that the observed denaturation does not result from enzymatic degradation. It is also shown that ribosomes can be lyophilized from a variety of buffers with little or no alteration of physical or functional properties. © 1969, American Chemical Society. All rights reserved.