is thus a valuable tool for assessment of the degree of non-polarity. It can also be used
in competition assays to monitor binding of ligands and prosthetic groups.
Reagents such as fluorescamine,o-phthalaldehyde or 6-aminoquinolyl-N-hydro-
xysuccinimidyl carbamate have been very popular conjugating agents used to deri-
vatise amino acids for analysis (see Section 8.4.2).o-Phthalaldehyde, for example, is a
non-fluorescent compound that reacts with primary amines andb-mercaptoethanol to
yield a highly sensitive fluorophore.
Metal-chelating compounds with fluorescent properties are useful tools for a variety
of assays, including monitoring of metal homeostasis in cells. Widely used probes for
calcium are the chelators Fura-2, Indo-1 and Quin-1. Since the chemistry of such
compounds is based on metal chelation, cross-reactivity of the probes with other metal
ions is possible.
The intrinsic fluorescence of nucleic acids is very weak and the required excitation
wavelengths are too far in the UV region to be useful for practical applications. Numerous
extrinsic fluorescent probes spontaneously bind to DNA and display enhanced emission.
While in earlier days ethidium bromide was one of the most widely used dyes for this
application,it has nowadays been replaced by SYBRGreen, as the latter probe posesfewer
hazards for health and environment and has no teratogenic properties like ethidium
bromide. These probes bind DNA by intercalation of the planar aromatic ring systems
ANS Fluorescein o-Phthalaldehyde SYBR Green
Fura-2 Dansyl N-(Lissamine rhodamine B sulfonyl)
NH SO 3 H
HO O O
COOH
CHO
CHO
N
N
N
+N
S
COO–
O
O O
N O
COO–
COO–
N COO–
CH 3
5 Na+
5–
N
O S O
~
NO
O S O
~
S
N+
O
O
O–
Fig. 12.11Structures of some extrinsic fluorophores. Fura-2 is a fluorescent chelator for divalent and higher
valent metal ions (Ca^2 þ,Ba^2 þ,Sr^2 þ,Pb^2 þ,La^3 þ,Mn^2 þ,Ni^2 þ,Cd^2 þ).
499 12.3 Fluorescence spectroscopy
