Modular Spectroscopy Tools
for Measuring Intrinsic
The availability of powerful LED UV light sources, when combined with spectroscopy
methods, is opening up a new world of fluorescence measurements.
Intrinsic fluorescence is a powerful indica- tor of protein structure and function. The amount of fluorescence can often give the researcher insight into the protein’s conformational states or activity under different
biological conditions including changes in temperature, pH and ion concentration.
For fluorescence measurements, a reliable
excitation source is required. Light-emitting
diodes (LEDs) are a cost-effective choice for
these measurements, with high-power UV LEDs
available today that can yield valuable information about proteins and amino acids.
To demonstrate, Ocean Optics, Dunedin,
Fla., used a 280-nm UV LED and a back-
thinned CCD array spectrometer to measure
intrinsic fluorescence from samples of lyso-
zyme and bovine serum albumin (BSA) in
different conformational states. Lysozyme is
a naturally occurring enzyme often used as a
bacterial agent; BSA is a protein important to
biochemical functions in the body. The results
illustrated the power of the modular spectros-
copy approach to monitoring fluorescence from
proteins, where flexibility among components
allows users to configure setups for biomol-
ecules and even non-biological samples that
require UV excitation to fluoresce.
Most proteins contain aromatic amino acids
that fluoresce when excited with UV light. The
fluorescence spectrum of the sample depends
on the amino acid composition and conforma-
tional state of the protein. As the protein goes
from a native (folded) to a denatured (unfolded)
state, the local environment surrounding the
aromatic amino acids changes, which affects
the fluorescence properties of the amino acids.
These changes in intrinsic protein fluorescence
can be used to monitor protein unfolding. This
is valuable information for applications such as
medical diagnostics, where researchers are inves-
tigating neurodegenerative and
other diseases associated with
improper protein unfolding.
The native state of a protein
can be altered using elevated
temperature, chaotropic or other
chemical agents such as urea or
guanidine hydrochloride, and
adjustments in pH. As the protein unfolds, amino acids that
were previously buried in the
hydrophobic core of the protein are exposed to the solvent.
Solvent exposure and the resulting susceptibility to quenching
agents reduce the fluorescence of
tryptophan (Trp), tyrosine (Tyr)
and phenylalanine (Phe).
A 280-nm UV LED was used
with a high-sensitivity spectrometer to measure fluorescence from samples of lysozyme
and BSA. To show the impact
of protein conformation, fluorescence spectra were measured
for the proteins diluted in phos-phate-buffered saline (1X PBS
Figure 1: Fluorescence spectra of bovine serum albumin, a major constituent of blood plasma, reveal changes in protein structure with exposure to a low-pH buffer. Source: Ocean Optics