Experimental verification of the triple isotopic substitution method in small-angle neutron scattering

Abstract

The theoretical predictions of the triple isotopic substitution (TIS) method in small-angle neutron scattering have been confirmed experimentally. A unique advantage of the method, namely the possibility to make part of a complex particle 'invisible' in normal (H20) buffer, is demonstrated. Particle association, the addition of 'small' molecules (such as glycerol) or 'large' molecules (such as pyruvate kinase) in the solutions under study have been shown not to influence the difference scattering curve obtained. The possibility of extracting normalized interparticle interference functions from the TIS data is discussed. This enables the detection of the presence of aggregates (e.g. dimers) in the solution, even in cases where the molar mass of a particle and/or its concentration are unknown. A simple internal test is suggested which checks whether or not sufficient conditions for the applicability of the method are fulfilled.

Small‐Angle Scattering of X‐Rays and Neutrons

“…When the scattering from the third sample is subtracted from that of the mixture of samples 1 and 2, the difference curve is exclusively due to the nuclei in the component, whereas the rest of the complex vanishes (at any contrast). This was shown for a complex of the ribosomal elongation factor EF-TU -GDP with tRNA by Pavlov et al (1991). Mathematically, the difference curve is independent of the contrast of the rest of the complex with respect to the solvent.…”

Triple Isotopic Substitution Method in Small-Angle Neutron Scattering: Application to Some Problems of Structural Biology

“…The physical basis of this method has been reported in several publications (Serdyuk & Pavlov, 1988;Pavlov, Rublevskaya, Zacca'i, Leberman & Ostanevich, 1991 ;Serdyuk, Pavlov, Rublevskaya, Zaccai" & Leberman, 1994).…”