5
thermal unfolding. Researchers add an extrinsic
fluorescence dye (usually SYPRO orange) to the
sample and measure the fluorescence intensity
of a polarity sensitive probe at gradually
increasing temperatures to obtain transition
temperatures of exposure of the hydrophobic
regions of proteins (Th). The change in
fluorescence intensity correlates directly with
protein unfolding levels and temperatures
3
.
Circular dichroism (CD) measures thermal
or chemical unfolding. It is a spectroscopic
technique where the difference between the
absorption of le -handed be εL and right
be εR, circularly polarized light is measured
during temperature or chemical increase. It
can determine the van't Hoff enthalpy (ΔH)
and entropy (ΔS) of unfolding, the midpoint
of the unfolding transition (T
m
or C
1/2
), and the
free energy (ΔG) of unfolding. Sample volume
is high, and resolving the spectra of complex
proteins can take several hours
4
.
nanoDSF is a modified form of DSF that does
away with the need to add an extrinsic dye.
It measures the intrinsic fluorescence from
tryptophan or tyrosine residues to get a readout
to monitor either thermal or chemical protein
unfolding. The fluorescence intensity ratios at
350 nm and 330 nm detect any changes in protein
structure from thermal or chemical denaturation.
With nanoDSF, scientists can get precise unfolding
temperatures (T
m
and T
onset
), critical denaturant
concentrations (Cm), and free folding energy (ΔG
and ΔΔG), while using very little sample, which
can accelerate the biotherapeutics development
process.
