Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 67
Figure 3.7. Fluorescence intensity of NADPH titrated into buffer (!)
or a solution of 17β-HSD ("). Revised from [29].
used to study binding of NADPH to proteins (Figure 3.7).
In the absence of protein the emission increases linearly
with NADPH concentration. In the presence of protein the
intensity initially increases more rapidly, and then increases
as in the absence of protein. The initial increase in the
intensity of NADPH is due to binding of NADPH to 17$-
HSD, which occurs with a fourfold increase in the quantum
yield of NADPH. Once the binding sites on 17$-HSD are
saturated, the intensity increases in proportion to the concentration
of unbound NADPH. In contrast to NADH,
emission from FAD and flavins is usually quenched upon
binding to proteins.
3.2. EXTRINSIC FLUOROPHORES
Frequently the molecules of interest are nonfluorescent, or
the intrinsic fluorescence is not adequate for the desired
experiment. For instance, DNA and lipids are essentially
devoid of intrinsic fluorescence (Figure 1.18). In these
cases useful fluorescence is obtained by labeling the molecule
with extrinsic probes. For proteins it is frequently
desirable to label them with chromophores with longer
excitation and emission wavelengths than the aromatic
amino acids. Then the labeled protein can be studied in the
presence of other unlabeled proteins. The number of fluorophores
has increased dramatically during the past decade.
Useful information on a wide range of fluorophores can be
found in the Molecular Probes catalogue. 30
3.2.1. Protein-Labeling Reagents
Figure 3.8. Reactive probes for conjugation with macromolecules.
Numerous fluorophores are available for covalent and noncovalent
labeling of proteins. The covalent probes can have
a variety of reactive groups, for coupling with amines and
sulfhydryl or histidine side chains in proteins. Some of the
more widely used probes are shown in Figure 3.8. Dansyl
chloride (DNS-Cl) was originally described by Weber, 31