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Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria
Abstract:
The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.
Insights
This study introduces a light scattering method to measure the size of beef heart mitochondria membrane fragments (ESMP). The technique provides a rapid and sensitive way to determine particle size and volume changes, revealing ESMP
Area of Science:
- Biophysics
- Biochemistry
- Materials Science
Background:
- Mitochondrial membrane fragments (ESMP) are complex structures with poorly defined size and hydration properties.
- Accurate characterization of ESMP size and volume changes is crucial for understanding their function and behavior.
- Existing methods like electron microscopy provide structural data, but dynamic changes require different approaches.
Purpose of the Study:
- To develop and validate a light scattering technique for determining the average particle diameter of ESMP.
- To measure the refractive index and osmotic behavior of ESMP using light scattering.
- To quantify water uptake in ESMP during energy-linked salt transport and compare it with other methods.
Main Methods:
- Light scattering measurements were performed on ESMP suspensions at various angles (45, 90, 135 degrees) using 546 nm light.
- The Rayleigh-Gans-Debye theory was applied to calculate average particle diameter (DLS) and refractive index.
- Scattering intensity was measured in varying sucrose concentrations to assess osmotic activity and water uptake.
Main Results:
- An average particle diameter (DLS) of 184-190 nm was determined, consistent with electron microscopy.
- The refractive index of ESMP was found to be 1.443, and particles were osmotically inactive towards sucrose.
- Light scattering quantified water uptake at 0.18 μL H2O/mg protein, significantly lower than dextran inaccessibility measurements (0.49 μL H2O/mg protein).
Conclusions:
- Light scattering offers a rapid, sensitive, and valid method for characterizing ESMP particle size (DLS) and volume changes.
- ESMP exhibit properties analogous to polyelectrolyte gels, suggesting applicability of Donnan equilibrium principles.
- The discrepancy in water uptake measurements highlights the sensitivity of light scattering to dynamic volume changes.