Biology Reference
In-Depth Information
Table 9.2 Macromolecular backbone-fluorophore conjugated probes and their
activation with a model protease in vitro
Fluorescence
Intensity
increase after
trypsin
b
activation
times at
37
C, 30 min
FL change,
before
and after
trypsin
activation
(ns)
Molar
extinction
coefficient
«
Excitation
maximum
(nm PBS)
Emission
maximum
(nm PBS)
Number
dyes/
mol
a
NIR dye
(m.w.)
(l/mol cm)
Cy5.5 mono
NHS ester
(1128)
674
688
250,000
35
53
6
0.21/1.1
IRDye
680RD
(1003.5)
672
694
150,000
30
52
7
0.14/0.92
IRDye 35
(1151)
682
696
200,000
30
33
7 D
IRDye
800CWNHS
ester (1166)
774
789
240,000
40
108
4
0.27/0.50
a
Molecular mass of PGC was calculated as 350 kD.
b
0.01 mg/ml trypsin (130 U/mg).
IRDye 35 is a research dye provided by LI-COR Biosciences and is not commercially available.
and FL is longer than for NIR dyes such as Cy7 and IRDye 800CW. How-
ever,
in vivo
imaging experiments suggest that heptamethine dyes (such as
IRDye 800CW
Ò
) have advantages that are not limited to the spectral range
(i.e., red-shifted excitation and emission) of these fluorophores. It has been
suggested that MPEG protective chains could theoretically interfere with en-
zymatic lysis of the bonds within PGC sensors. The assumption in this case is
that the sensitivity of the probe would suffer. However, such interference is
unlikely, given the relatively small hydrodynamic diameters of lysosomal hy-
drolases. The interference is of greater importance for larger proteins that are
secreted and activated in the extracellular space, for example, in the extracel-
lular matrix. In any case, the extent of PGC sensor fragmentation will depend
on whether the protease and the sensor are allowed to react in the same com-
partment for a sufficient length of time. While model serine proteases have
high catalytic rates, real
in vivo
target's catalytic constants may be much lower,
which undoubtedly will influence the sensitivity of any sensor, including mac-
romolecular dye-conjugated PGC.
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