Principles of Fluorescence Spectroscopy

542 PROTEIN FLUORESCENCE
mined from the intensity observed with a given excitation
wavelength, divided by the absorbance at the excitation
wavelength. If energy transfer is 100% efficient, then the
tryptophan emits irrespective of whether tyrosine or tryptophan
is excited. In this case the relative quantum yield Q(λ)
is independent of excitation wavelength. If there is no tyrto-trp
energy transfer then the relative tryptophan quantum
yield decreases at shorter wavelengths. This decrease
occurs because light absorbed by the tyrosine does not
result in emission from the tryptophan. This can be understood
by recognizing that the absorbance is below 290 nm
due to both tyrosine and tryptophan. In the absence of energy
transfer only absorption by tryptophan results in tryptophan
emission.
The fractional absorbance of each type of amino acid
can be calculated from the absorbance due to each amino
acid. The fractional absorbance due to tryptophan is given
as follows:
atrp(λ) ftrp(λ)
atrp(λ) atyr(λ) (16.1)
where ε i (λ) is the absorbance of the individual residues at
wavelength λ. For simplicity we deleted from this expression
the minor term due to absorption by phenylalanine.
Figure 16.19 shows that only tryptophan absorbs at wavelengths
longer than 295 nm. The relative quantum yield at
295-nm excitation is taken as a reference point at which all
the light is absorbed by tryptophan. The fluorescence is
monitored at 350 nm or longer to avoid tyrosine emission.
The relative quantum yield at any excitation wavelength is
given by
Q(λ) a trp(λ) Ea tyr(λ)
a trp(λ) a tyr(λ)
(16.2)
If E is 100% then the relative quantum yield of tryptophan
fluorescence is independent of excitation wavelength. If E
is zero then the relative quantum yield is given by the fractional
absorption due to tryptophan.
Equation 16.1 is written in terms of absorbance due to
each type of residues. If the number of tyrosine and tryptophan
residues is known the fractional absorbance of tryptophan
can be approximated from
nεtrp(λ) ftrp(λ)
nεtrp(λ) mεtyr(λ) (16.3)
Figure 16.20. Excitation wavelength dependence of the relative quantum
yield of the dipeptide tryptophanyltyrosine and an equimolar mixture
of tyrosine and tryptophan. The lines correspond to transfer efficiencies
of 0, 50, and 100%. Reprinted with permission from [85].
Copyright © 1969, American Chemical Society.
where ε i (λ) are the extinction coefficients, n is the number
of tryptophan residues per protein, and m is the number of
tyrosine residues per protein. This expression can be slightly
in error due to shifts in the absorption of the residues in
different environments.
The use of the relative quantum yield to determine the
energy transfer efficiency is illustrated by data for a dipeptide—tyr–trp—and
for an equimolar mixture of tyrosine
and tryptophan (Figure 16.20). In the dipeptide the donor
and acceptor are well within the Förster distance and the
transfer efficiency is expected to be near 100%. This prediction
is confirmed by the independence of the tryptophan
quantum yield from the excitation wavelength. All the energy
absorbed by tyrosine or tryptophan appears as tryptophan
fluorescence. For the mixture of unlinked tyrosine and
tryptophan the relative quantum yield closely follows the
fractional absorbance due to tryptophan (E = 0), indicating
the absence of significant energy transfer.
Such data can be used to estimate the efficiency of
tyrosine–tryptophan energy transfer in proteins. The excitation
wavelength-dependent tryptophan quantum yields are
shown for interferon-γ in Figure 16.21. These values are
measured relative to the quantum yield at 295 nm, where
only tryptophan absorbs. The relative quantum yield
decreases with shorter excitation wavelengths in both the
monomeric and dimeric states. The quenching efficiency
(E) is estimated by comparison with curves calculated for
various transfer efficiencies. Using this approach the trans