Contrast analysis of the composition of ribosomes extracted with differentpurification procedures

The composition and hydration of E. coli ribosomes isolated with different purification protocols has been analysed by combining two experimental tech niques: measurements of small-angle neutron scattering (SANS), for two diff erent isotopic solvent compositions, and refractive index (RI) increments. From the contrast between the solvent and solute scattering densities and t he molar polarizability, determined experimentally with SANS and RI measure ments, three independent equations are obtained and three unknown quantitie s are determined: (i) the volume of the solute hydrated skeleton Vs, (ii) t he material contained in it, namely the biological components, intrinsic (r RNA and proteins) and extrinsic, such as aminoacylsynthetase and elongation factors, (iii) the number of water molecules structurally bound to the rib osome and nonexchangeable with the solvent. From the form factor at infinit e contrast, a second definition of the solute volume is obtained, V-s(c), w hich represents the volume within the contour surface of the ribosome. This value is generally larger than Vs and can include a certain amount of wate r molecules, i.e. those inside the volume (V-s(c)-V-s). Considering the mol ar volume of this water to be equal to that of the bulk water, it is possib le to evaluate its amount. The particle density calculated from the ribosom e components in V-s(c), including proteins, RNA, bound and unbound water mo lecules, corresponds to the buoyant density measured for E. coli 70S partic les. The two ribosomal preparations display different performances in prote in synthesis; hence the results indicate that the optimal condition corresp onds to a wider skeleton and contour volume but containing a smaller amount of segregated water molecules. It is believed that the method provides a r eliable technique to determine the composition of ribosomes under various e xperimental conditions.