As expected, for high quencher conc

As expected, for high quencher concentration the probability of
finding a quencher molecule within the critical sphere increases and
the Stern-Volmer plot departs from the linear behavior [25,26] (see
Fig. 3B). Using Eq. (1), values for KSV=6.0±0.4 M−1 and V=748±
133 Å3 were obtained (full line in Fig. 3B). Assuming a spherical shape
for V, a radius r=5.6±0.3 Å was obtained, in good agreement with the
sum of the molecular radii for tryptophan and acrylamide rcalc=6.3 Å
(calculated using the algorithm implemented in http://www.molinspiration.
com/cgi-bin/properties).
Using the second line in Eq. (1) and assuming a life timeτ0=3 ns for
the tryptophan excited state, a value of kq=(2.0±0.1)×109 M−1 s−1
was obtained for the apparent bimolecular quenching constant.
Regarding the value obtained for kq and the measure λmax, the
wavelength for the maximum of the fluorescence spectrum, Eftink
[28] described a correlation between the position of the emission
maximum (λmax) and the value for kq for acrylamide quenching of
tryptophan emission for single tryptophan proteins. Although a high
molecular weight protein like PV2 is not likely to have a single
tryptophan, our quenching experiments suggest the existence of a
single class of tryptophan residues. Following Eftnik's empirical
correlation it is possible to conclude that those tryptophan residues
are placed in occluded, yet not totally buried, positions inside the
tertiary structure of PV2.