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Sedimentation analysis of SDS and albumin-SDS complexes

  • K. J. Tiefenbach
  • H. Durchschlag
  • R. Jaenicke
Biological Systems
Part of the Progress in Colloid & Polymer Science book series (PROGCOLLOID, volume 107)

Abstract

Knowledge of the structure of protein-SDS complexes and the constituent free proteins and SDS micelles is important for our understanding of protein-detergent interactions and for the application and improvement of physicochemical techniques connected with the purification and characterization of proteins. Several reasons are responsible for present controversies regarding the size and structure of macrosolutes, e.g. presence of a variety of low- and high-molecular solutes, the fact that macrosolutes show heterogeneity and may exhibit equilibria that are strongly influenced by the environmental conditions, and the point that techniques available for their structural investigation refer to different global or local properties. Sedimentation velocity and equilibrium experiments of the detergent-protein complexes and their constituents were performed under a variety of experimental conditions, such as rotor speed, scanning wavelength, choice of the baseline, the solvent or additives, and the concentrations of the components. Monitoring the sedimentation profiles of micellar SDS is facilitated by labeling the detergent micelles using a fluorescent dye, and discriminating the species under analysis by scanning at specific wavelengths. Interpretation of the results is facilitated by monitoring absorption spectra at discrete radial distances in the centrifuge cells, in addition to spectra recording of mixtures and components outside the centrifuge. In order to estimate the amount of bound detergent, sedimentation data (sedimentation coefficients, particle weights and mass distributions) were complemented by size-exclusion chromatographic studies. In the case of nonreduced proteins about 0.4 g SDS/g protein are bound at low detergent concentrations, and about 0.8 to 1.2 g/g at elevated concentrations.

Key words

Sodium dodecyl sulfate protein-detergent complexes micelles analytical ultracentrifugation absorption spectroscopy size-exclusion chromatography 

Abbreviations

AC

ammonium chloride

AF

amplification factor

AUC

analytical ultracentrifugation

BSA

bovine serum albumin

ChA

chymotrypsinogen A

CMC

critical micelle concentration

CMT

critical micelle temperature

DB

dextran blue

HSSE

high-speed sedimentation equilibrium

ßLg

ß-lactoglobulin

Lys

lysozyme

MyG

myoglobin

NaP

sodium phosphate buffer

NPN

N-phenyl-1-naphthylamine

OvA

ovalbumin

PAGE

polyacrylamide gel electrophoresis

SDS

sodium dodecyl sulfate

SEC

size-exclusion chromatography

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Copyright information

© Dr. Dietrich Steinkopff Verlag GmbH & Co. KG 1997

Authors and Affiliations

  • K. J. Tiefenbach
    • 1
  • H. Durchschlag
    • 1
  • R. Jaenicke
    • 1
  1. 1.Institute of Biophysics and Physical BiochemistryUniversity of RegensburgRegensburgGermany

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