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the enzyme’s active site. More importantly, the catechin-bearing
probe 1a can discriminate interactions with different proteins,
as shown herein using LDOX and BSA, and can still capture
LDOX in low abundance in a complex protein mixture.
This proof-of-concept study thus constitutes a solid ground-
work for future utilization of such catechin-bearing probes (or
flavonoid analogues thereof) in chemical proteomic work aimed
either at identifying proteins involved in flavonoid biosynthetic
metabolism or at profiling ranges of flavonoid target proteins
pulled-down from plant cell extracts or human cell lysates.
The authors thank the Conseil Interprofessionnel du Vin de
Bordeaux (CIVB) for their generous financial support, including
Fig. 3 Coomassie blue-stained SDS-PAGE analysis of competitive bind-
ing assays of probe 1a and catechin with the LDOX and BSA proteins; 1100
fold-excess; see the ESI† for details.
´ `
´
Helene Carrie’s doctoral research assistantship, the government
of Vietnam for Dong Tien Tran’s doctoral research assistantship
serum albumin protein (BSA), which is known to form a 1 : 1
complex with catechin.15 Following the same experimental protocol,
probe 1a was thus co-incubated with LDOX, BSA or a 1:1 mixture of
LDOX and BSA, and then oxidatively activated using NaIO4. Analysis
by SDS-PAGE (Fig. 3) shows that BSA is captured by probe 1a in the
absence of LDOX (lanes 2 and 6). However, when a 1 : 1 mixture of
LDOX and BSA (lane 3) is treated with probe 1a (lane 7), LDOX is
quasi exclusively captured at a level seemingly identical to that
observed in the control experiment run in the absence of competing
BSA (see lane 7 as compared to lane 4).
´
and Thierry Dhakli (IECB, UMS 3033/US 001, Universite de
Bordeaux) for his help in LDOX expression and purification.
Notes and references
´
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The higher affinity of the catechin-bearing probe 1a for LDOX
as compared to that for BSA is quite remarkable if one considers
the relatively low Km value of 175 mM of LDOX with its catechin
ligand.13a
The specificity of binding of probe 1a to the LDOX enzyme was
also unambiguously confirmed by using a 100-fold molar excess of
catechin, in which case 1a was prevented from binding to the
LDOX’s active site thereby occupied by catechin. This is evidenced
by the disappearance of the corresponding probe-labeled LDOX
band (Fig. 3, see lane 5 as compared to lane 4). These results
confirmed that the flavanol-bearing probe 1a (and 1b, data not
provided) can be used to capture the LDOX enzyme. Thus, we next
evaluated the capacity of probe 1a to capture the LDOX enzyme
present as a minor component in a complex protein mixture.
To this aim, we used a bacterial lysate of E. coli supplemented with
0.5%/w of LDOX in the presence of only ca. 2 equiv. of 1a relative to
the added LDOX. Under these conditions of low abundance of the
LDOX target, we could not visualize the result of its oxidative
¨
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In summary, we have successfully designed and prepared simple
affinity-based probes to investigate flavanoid-protein specific
interactions. We have demonstrated that the (epi)catechin-
bearing probes 1a and 1b can be used to efficiently capture the
LDOX enzyme under oxidative activation by simply exploiting the
inherent chemical reactivity of the polyphenolic entity, and
provided evidence that these probes form a covalent adduct with
the LDOX enzyme, most likely involving nucleophilic residues at
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Chem. Commun., 2014, 50, 9387--9389 | 9389