Intervent Dilution Chromatography: Concept for Separation of Strongly Interacting Macromolecules

Abstract

Intervent dilution chromatography separates interacting macromolecules by subjecting them to a dynamic environment in which the association constant is continuously varied. The dynamic environment is produced by using the sieving properties of a gel to repeatedly propel the molecular complex across an intervent boundary. Behind the boundary, at high inteirvent concentrations, the complex dissociates;'ahead of the boundary, the component molecules are separated by adsorption processes. By selective adjustment of the intervent composition of the sample and the conditions of column equilibration, the process is adapted to a particular need.This report describes the chromiatographic' concept and shows how parameters are adjusted to obtain the desired separation. In this study a particularly difficult separation, i.e., the separation of ribosomal proteins from ribosomal RNA, is chosen to illustrate the power of the procedure.Separation of macromolecules that interact strongly with each other is one of the most persistent problems of biochemistry. Although chromatography has been used to measure the amount of association between molecules (1), current chromatographic procedures have found only limited use in separation of molecules that significantly interact with each other. This limitation results from the fact that the basic chromatographic process demands that molecules behave independently. It is sometimes possible to alter the chromatographic environment to permit the molecules to behave independently while retaining sufficient interaction with the stationary phase to cause separation; however, such changes usually reduce the interactions necesary for separation to a point where resolution becomes impossible.Intervent dilution chromatography is a combination of molecular sieve (gel filtration) and adsorption chromatography that effectively circumvents the stated limitation in classical chromatography. Because molecular sieve processes may be combined with adsorption chromatography in several ways to optimize for various needs, we suggest'some systematic nomenclature. Chromatographic systems in general that use any active combination of molecular sieve and adsorption processes are called sievorptive chromatography. Intervent dilution chromatography, as well as the recently defined ion filtration chromatography (2), are specific modes of the general process of sievorptive chromatography. Although ion filtration and intervent dilution chromatography are conceptually distinct, in practice both processes may occur within the same column, as happens when ion filtration chromatography is used to "desalt" an enzyme sample.The chromatographic system that is introduced has broad utility and may be adjusted for a selective or complete dissociation of biological complexes. To illustrate the power of the new chromatographic approach, conditions were found to separate ribosomal proteins from ribosomal RNA, one of the strongest macromolecular complexes encountered in biochemistry. As a historical framework for this stu...

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