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protein-like fluorescence signals was demonstrated by Maie et al. (
2007
) for estuarine and
Florida coastal waters. In this study, weak correlations were observed between the T-peak
(associated commonly with tryptophan) and the high molecular weight fraction of ultra-
filtered DOM. A large portion of the T-peak was found to be associated with nonproteina-
ceous compounds by using size-exclusion chromatography combined with a fluorometer
tuned to measure T-peak fluorescence. Maie et al. (
2007
) proposed that phenols associ-
ated with tannins were the likely fluorophores contributing to T-peak fluorescence in their
samples.
Because the fluorescence properties of tryptophan within a protein molecule are sensi-
tive to local conditions, tryptophan fluorescence is important in many biochemical studies
related to the properties of proteins (Lackowicz, 2006). Of particular significance in the
study of DOM are the effects of reactions of proteins with other organic compound classes
on protein fluorescence. It is well known, for instance, that tannins bind proteins (Robinson,
1991
; Kraus et al.,
2003
). Labieniec and Gabryelak (
2006
) demonstrated that interaction
of the protein bovine serum albumin with tannic, ellagic, and gallic acids (all polyphenols)
resulted in a marked decrease in protein fluorescence. A representative, but not inclusive,
list of DOM relevant compounds known to influence protein fluorescence includes caffeine
(Kriško et al.,
2005
); cinnamic acid and its hydroxyl derivatives, coumaric acid and caffeic
acid (Min et al.,
2004
; Bian et al.,
2007
); chlorogenic and ferulic acids (Kang et al.,
2004
);
and flavanols, such as quercitin (Mishra et al.,
2005
). These studies commonly involve the
measurement of the quenching of protein fluorescence resulting from binding of the com-
pound of interest to the proteins. The results of these measurements provide information
about the nature (e.g., number of binding sites, binding constants, binding distances, and
conformational changes) of the interactions. Min et al. (
2004
) demonstrated that interac-
tions of coumaric and caffeic acids with human serum albumin resulted in both quenching
and a red-shift of the tryptophan fluorescence signal. In the case of the flavanol quercitin,
the absorption spectrum of quercitin itself was red-shifted and its fluorescence intensity
increased, while the fluorescence intensity of albumin was quenched. These results indicate
an excitation energy transfer between tryptophan and the bound quercitin. Reactions such
as these are possible in soils and natural waters and may strongly influence fluorescence of
the protein-like fluorophores, as well as contributing to other portions of the fluorescence
spectra of natural samples. Despite the sensitivity of different fluorophores to reactions
such as those discussed, these reactions have received little attention in the study and inter-
pretation of DOM fluorescence.
2.4.2 Simple Phenols
Simple phenols are important compound groups produced by higher plants (Robinson,
1991
) and algae (Geiselman and McConnell,
1981
; Connan et al.,
2004
) that comprise
DOM. In addition, compounds such as cresol (methylphenol;
Figure 2.4c
) and sal-
icylic acid (2-hydroxybenzoic acid) are often present in wastewater effluents. They are
found as individual molecules (Thurman,
1985
), as the fundamental structures in tannins
