ultracentrifugation can be found in several books on this subject
that have been published recently, underlining the growing importance of this method [1–4].
Since AUC experiments investigate molecules in solution, measurements are not disturbed by interactions with surfaces, as seen
with methods like surface plasmon resonance (SPR) or sizeexclusion chromatography (SEC). For example, immobilization
of one interaction partner is a prerequisite for SPR, which in turn
might result in the occlusion of binding sites. SPR analysis of
proteins involved in bacterial DNA replication yielded, for instance,
that in the absence of DNA only 2.2 χ subunits of DNA polymerase
III bind per immobilized single-stranded DNA-binding protein
(SSB) tetramer [5], whereas AUC experiments revealed that, as
expected, four χ molecules bind, one to each C-terminus of SSB
[6, 7]. An advantage of AUC over SEC is that even weak interactions can be detected, since the complex always sediments in the
presence of excess reactants and can therefore reform after dissociation. SEC, however, typically leads to a separation of complex and
reactants, precluding re-association of the complex after dissociation and thus the detection of weak interactions. Hence, the interaction of χ and SSB in the absence of DNA could be detected by
AUC and SPR [5, 6] but not by SEC [5].
In sedimentation velocity experiments, uniform solutions of
the macromolecules of interest are applied and concentration gradients formed by sedimentation that are partially balanced by diffusion are observed as a function of time. From the velocity of the
boundary movement, the sedimentation coefficient s can be
inferred; the broadening of the sedimentation boundary contains
information about the diffusion coefficient D. If s and D are known
for a given particle, its molar mass can be calculated. Several methods are available for the measurement of the concentration gradients that form during sedimentation. The absorbance optical
system allows the specific and sensitive detection of molecules like
proteins, nucleic acids, and extrinsic chromophores that absorb in
the UV/Vis range. This method cannot be used, however, when
material that significantly absorbs at the detection wavelength is
included in the reaction buffer. For example, investigations of
proteins or nucleic acids in the presence of nucleotides in the higher
micromolar or even millimolar concentration range are impaired by
the UV absorbance of these components. Alternatively, the Rayleigh interference optical system can be used that measures differences in the refractive index between sample and the buffer in
which the macromolecules are dissolved. The interference signal
depends generally on the mass concentration and is therefore sensitive to differences in buffer composition of sample and reference.
Therefore, extensive dialysis or size-exclusion chromatography of
the sample is required in order to assure that the buffer composition of sample and reference are as similar as possible since even
398
Andrea Bogutzki and Ute Curth
that have been published recently, underlining the growing importance of this method [1–4].
Since AUC experiments investigate molecules in solution, measurements are not disturbed by interactions with surfaces, as seen
with methods like surface plasmon resonance (SPR) or sizeexclusion chromatography (SEC). For example, immobilization
of one interaction partner is a prerequisite for SPR, which in turn
might result in the occlusion of binding sites. SPR analysis of
proteins involved in bacterial DNA replication yielded, for instance,
that in the absence of DNA only 2.2 χ subunits of DNA polymerase
III bind per immobilized single-stranded DNA-binding protein
(SSB) tetramer [5], whereas AUC experiments revealed that, as
expected, four χ molecules bind, one to each C-terminus of SSB
[6, 7]. An advantage of AUC over SEC is that even weak interactions can be detected, since the complex always sediments in the
presence of excess reactants and can therefore reform after dissociation. SEC, however, typically leads to a separation of complex and
reactants, precluding re-association of the complex after dissociation and thus the detection of weak interactions. Hence, the interaction of χ and SSB in the absence of DNA could be detected by
AUC and SPR [5, 6] but not by SEC [5].
In sedimentation velocity experiments, uniform solutions of
the macromolecules of interest are applied and concentration gradients formed by sedimentation that are partially balanced by diffusion are observed as a function of time. From the velocity of the
boundary movement, the sedimentation coefficient s can be
inferred; the broadening of the sedimentation boundary contains
information about the diffusion coefficient D. If s and D are known
for a given particle, its molar mass can be calculated. Several methods are available for the measurement of the concentration gradients that form during sedimentation. The absorbance optical
system allows the specific and sensitive detection of molecules like
proteins, nucleic acids, and extrinsic chromophores that absorb in
the UV/Vis range. This method cannot be used, however, when
material that significantly absorbs at the detection wavelength is
included in the reaction buffer. For example, investigations of
proteins or nucleic acids in the presence of nucleotides in the higher
micromolar or even millimolar concentration range are impaired by
the UV absorbance of these components. Alternatively, the Rayleigh interference optical system can be used that measures differences in the refractive index between sample and the buffer in
which the macromolecules are dissolved. The interference signal
depends generally on the mass concentration and is therefore sensitive to differences in buffer composition of sample and reference.
Therefore, extensive dialysis or size-exclusion chromatography of
the sample is required in order to assure that the buffer composition of sample and reference are as similar as possible since even
398
Andrea Bogutzki and Ute Curth