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Light-scattering from detergent-complexed biological macromolecules.

ABSTRACT: A theory is described for Rayleigh light-scattering from solutions of detergent-complexed macromolecules applicable to measurements carried out under conditions of Donnan equilibrium. The theory shows that when scattering measurements are made on detergent-solubilized macromolecules in the presence of detergent micelles the apparent Mr is dependent on the extent of detergent binding and effective charge on the detergent-macromolecule complex and the micellar charge and aggregation number. Equations are given for the apparent Mr of the macromolecule under limiting conditions of high salt and low salt concentration. Low-angle laser-light-scattering measurements were made on lysozyme complexed with sodium n-dodecyl sulphate both in the absence and in the presence of detergent micelles. These experimentally obtained data were used in conjunction with the detergent-binding isotherm to test the theory at high ionic strength. Light-scattering measurements were also made on detergent-saturated complexes as a function of ionic strength and pH. The results are in reasonable accord with both the qualitative and the quantitative predictions of the theory.

SUBMITTER: Jones MN

PROVIDER: S-EPMC1153556 | biostudies-other | 1984 May

REPOSITORIES: biostudies-other

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Project description:X-ray scattering of biological macromolecules in solution is an increasingly popular tool for structural biology and benefits greatly from modern high-brightness synchrotron sources. The upgraded MacCHESS BioSAXS station is now located at the 49-pole wiggler beamline G1. The 20-fold improved flux over the previous beamline F2 provides higher sample throughput and autonomous X-ray scattering data collection using a unique SAXS/WAXS dual detectors configuration. This setup achieves a combined q-range from 0.007 to 0.7 Å(-1), enabling better characterization of smaller molecules, while opening opportunities for emerging wide-angle scattering methods. In addition, a facility upgrade of the positron storage ring to continuous top-up mode has improved beam stability and eliminated beam drift over the course of typical BioSAXS experiments. Single exposure times have been reduced to 2 s for 3.560 mg ml(-1) lysozyme with an average quality factor I/σ of 20 in the Guinier region. A novel disposable plastic sample cell design that incorporates lower background X-ray window material provides users with a more pristine sample environment than previously available. Systematic comparisons of common X-ray window materials bonded to the cell have also been extended to the wide-angle regime, offering new insight into best choices for various q-space ranges. In addition, a quantitative assessment of signal-to-noise levels has been performed on the station to allow users to estimate necessary exposure times for obtaining usable signals in the Guinier regime. Users also have access to a new BioSAXS sample preparation laboratory which houses essential wet-chemistry equipment and biophysical instrumentation. User experiments at the upgraded BioSAXS station have been on-going since commissioning of the beamline in Summer 2013. A planned upgrade of the G1 insertion device to an undulator for the Winter 2014 cycle is expected to further improve flux by an order of magnitude.

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Light-scattering from detergent-complexed biological macromolecules.

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