Review articles have been published on the theory and application of static light scattering combined with SEC (SEC/MALS) for
determination of the molecular weight of proteins in solution [3–7]
and for analysis of protein complexes [5, 8], including protein
interactions with DNA [2, 9–14] and RNA [1, 12, 15, 16].
The SEC/MALS analysis utilizes three detectors: an absorbance (UV) detector, a static light scattering (LS) detector and a
refractive index (RI) detector, which are placed in series with a SEC
column. The UV detector monitors absorbance at a selected wavelength, the RI detector monitors changes in the refractive index,
and the LS detector records the excess of scattered light; SEC serves
solely as a fractionation step. Since static light scattering provides
only the weight-average molar mass of the species in solution, the
SEC separation plays an integral role in the overall analysis,
although the elution from SEC does not need to correlate with
the molecular weight of the species being studied. The SEC/MALS system is validated in a buffer of choice by analyzing protein
and nucleic acid standards. The computation of molecular weight is
based on the theory that the excess of scattered light is proportional
to the product of the molecular weight and concentration
[3, 6]. Scattered light is measured by the light scattering detector;
concentration is measured by the RI and UV detectors. The
responses from the three detectors are processed by the software
to calculate the molecular weight of the eluting macromolecule
[6]. The accuracy of molecular weight measurement by SEC/MALS is ~ Æ3% [6] and in most cases allows determination of the
oligomeric state of the protein and stoichiometry of protein–
nucleic acid complexes. In addition, the responses collected by
the refractometer and the absorbance detector allow an “online”
measurement of extinction coefficient of the eluting material
[2, 17], which aids in computation of protein–nucleic acid
stoichiometry.
The examples used in this chapter result from the SEC/MALS
analyses of two well-characterized protein–nucleic acid complexes
with known crystal structures: reverse transcriptase (RT) domain
from group II intron–encoded protein from Eubacterium rectale
(E.r. RT domain) [1] and its complex with intron RNA (D4A) and
of FUSE binding protein-interacting repressor (FIR) that binds to
FUSE DNA [2]. These results presented in Figs. 1, 2 and 3 and
summarized in Tables 1, 2 and 3 illustrate two different scenarios
observed when studying protein complexes via SEC/MALS: slow
dissociating complex that retains molecular weight despite changes
in concentration (E.r. RT domain; Fig. 1b, c), and fast dissociating
complexes that dissociate due to dilution during the fractionation
step (FIR; Fig. 2b, c) [18]. Additionally, these examples illustrate
clearly that SEC elution volume alone is not a reliable method to
determine the mass and oligomeric state of these complexes
because their elution from SEC does not correlate with their size
SEC Coupled with Light Scattering
383
determination of the molecular weight of proteins in solution [3–7]
and for analysis of protein complexes [5, 8], including protein
interactions with DNA [2, 9–14] and RNA [1, 12, 15, 16].
The SEC/MALS analysis utilizes three detectors: an absorbance (UV) detector, a static light scattering (LS) detector and a
refractive index (RI) detector, which are placed in series with a SEC
column. The UV detector monitors absorbance at a selected wavelength, the RI detector monitors changes in the refractive index,
and the LS detector records the excess of scattered light; SEC serves
solely as a fractionation step. Since static light scattering provides
only the weight-average molar mass of the species in solution, the
SEC separation plays an integral role in the overall analysis,
although the elution from SEC does not need to correlate with
the molecular weight of the species being studied. The SEC/MALS system is validated in a buffer of choice by analyzing protein
and nucleic acid standards. The computation of molecular weight is
based on the theory that the excess of scattered light is proportional
to the product of the molecular weight and concentration
[3, 6]. Scattered light is measured by the light scattering detector;
concentration is measured by the RI and UV detectors. The
responses from the three detectors are processed by the software
to calculate the molecular weight of the eluting macromolecule
[6]. The accuracy of molecular weight measurement by SEC/MALS is ~ Æ3% [6] and in most cases allows determination of the
oligomeric state of the protein and stoichiometry of protein–
nucleic acid complexes. In addition, the responses collected by
the refractometer and the absorbance detector allow an “online”
measurement of extinction coefficient of the eluting material
[2, 17], which aids in computation of protein–nucleic acid
stoichiometry.
The examples used in this chapter result from the SEC/MALS
analyses of two well-characterized protein–nucleic acid complexes
with known crystal structures: reverse transcriptase (RT) domain
from group II intron–encoded protein from Eubacterium rectale
(E.r. RT domain) [1] and its complex with intron RNA (D4A) and
of FUSE binding protein-interacting repressor (FIR) that binds to
FUSE DNA [2]. These results presented in Figs. 1, 2 and 3 and
summarized in Tables 1, 2 and 3 illustrate two different scenarios
observed when studying protein complexes via SEC/MALS: slow
dissociating complex that retains molecular weight despite changes
in concentration (E.r. RT domain; Fig. 1b, c), and fast dissociating
complexes that dissociate due to dilution during the fractionation
step (FIR; Fig. 2b, c) [18]. Additionally, these examples illustrate
clearly that SEC elution volume alone is not a reliable method to
determine the mass and oligomeric state of these complexes
because their elution from SEC does not correlate with their size
SEC Coupled with Light Scattering
383