The possiblilty of using formaldehyde fixation for the quantitative studies of the reversibly interacting ribosomal system 50S-30S ~ 50S + 30S was investigated. Conditions have been found under which formaldehyde fixation of a reaction mixture of purified ribosomal particles proved to be possible without any change in the initial ratio of its components. This fixation technique was used in studying the dependence of the association level on Mg ยง + ion concentration. An estimation has been made of the number of Mg ยง ยง ions which bind during the association of 50S and 30S subparticles.
I. I N T R O D U C T I O N
It was shown [1] that the reaction mixture of 'purified' 50S and 30S ribosomal subparticles is a reversibly interacting system.
In studying equilibrium properties of such a system and, in particular, the kinetics of associationdissociation reactions, it is necessary that the system should be first fixed in its initial state. On the other hand, it is known that ribosomal subparticles treated with formaldehyde cannot associate even at high magnesium concentrations [2,3]. At the same time, formaldehyde-treated 70S ribosomes do not dissociate even when the magnesium ion concentration is decreased [2,4]. However, it is not known whether formaldehyde is able to fix the ribosomal system in its initial state, i.e. without changing the initial concentration ratios of the components.
In the present work, the conditions of effective formaldehyde fixation permitting quantitative studies of the reversibly interacting ribosomal system were determined. Using this technique, the dependence of ribosome association on the magnesium concentration was studied and the number of Mg + ยง ions involved in the reaction of association of ribosomal subparticles was estimated.
II. MATERIAL AND METHODS
Ribosomes prepared from E. coli MRE 600 were precipitated by adding 42 g of ( N H 4 ) 2 S O ~. per 100 ml of the initial solution [5]. The precipitate thus obtained was collected by centrifugation for 20 rain at 15000 rpm and resuspended in 10 mM Tris-HC1 buffer with 1 mM MgCI 2 and 500 mM NH4CI, pH 7.5 [6]. The ribosomal suspension was dialyzed against the same buffer for 12 hr at 4~ As a result, a mixture of 50S and 30S subparticles was obtained which were separated by