Principles of Fluorescence Spectroscopy

548 PROTEIN FLUORESCENCE
Figure 16.31. Fluorescence emission spectra of apoazurin Ade from
Alcaligenes denitrificans in the absence (top curve) and presence (bottom
curve) of 0.45 M Kl. The middle line is the difference spectrum
(DS) and is due to the easily quenched emission (presumably from
Trp-118). The buffer was 0.05 M sodium acetate, pH 5.0. Revised
from [101].
varies from almost completely inaccessible for azurin and
asparaginase to completely accessible for glucagon.
Glucagon is a relatively small peptide with 29 amino acids
that opposes the action of insulin. Glucagon has a random
structure in solution. 103–105 Hence, there is little opportunity
for the protein to shield the single trp-25 residue from acrylamide
quenching.
The extent of collisional quenching can also be
dependent on protein conformation and/or the extent of
subunit association. This effect has been observed for melit-
Figure 16.33. Dependence of the apparent acrylamide bimolecular
quenching constant (k q ) on the emission maximum for single-tryptophan
proteins: 1, glucagon; 2, adrenocorticotropin; 3, melittin
monomer; 4, melittin tetramer; 5, gonadotropin; 6, phospholipase A 2 ;
7, human luteinizing hormone; 8,9, monellin; 10, gonadotropin; 11,
human serum albumin, N form; 12, human serum albumin, F form;
13, myelin basic protein; 14, elongation factor Tu-GDP; 15, nuclease;
16, fd phage; 17, ribonuclease T 1 ; 18, parvalbumin; 19, calciumdepleted
parvalbumin; 20, asparaginase; 21, apoazurin Pae; 22,
mastopartan X. Revised from [101].
tin, which is a small peptide of 26 amino acids containing a
single-tryptophan residue at position 19. Depending on
ionic strength, melittin can exist as a monomer or tetramer.
In the tetramer, the four tryptophan residues, one per monomer,
are located in a hydrophobic pocket between the helices.
106 The tryptophan residues in melittin are more easily
quenched when melittin is in the monomer state (Figure
16.33).
Acrylamide and iodide are polar molecules, and do not
readily penetrate the nonpolar regions of proteins. However,
small nonpolar oxygen molecules readily penetrate all
Figure 16.32. Acrylamide quenching of representative single-tryptophan proteins and NATA. Left: intensity versus acrylamide concentration. Right:
Stern-Volmer plots. Data from [80], [86], and [99].