ChemComm
For the second set of experiments, the cross-linked PLL-S-TP/HA
Communication
films were brought into contact with b-Gal-mal enzymes in the
presence of TCEP (tris(2-carboxyethyl) phosphine hydrochloride) to
deprotect the thiopyril moieties (S-TP) of PLL which then react with
the maleimide groups of b-Gal. b-Gal-mal become thus covalently
linked to the film. The deprotection reaction was monitored by
measuring the supernatant absorbance at 343 nm (Fig. S5 in ESI†).
The increase in absorbance corresponds to the release of the
thiopyridone molecules in solution. The reaction takes place for
approximately 30 minutes. Beyond this time, no more coupling
reaction occurs. The buildup process is presented in Fig. 1b.
By using a calibration curve, the enzyme concentration in
À1
the film was estimated to be of the order of 850 mg mL (see
details in §3 in ESI,† and Fig. S4). These cross-linked PLL/HA
films containing covalently attached b-Gal-mal enzymes were
then stretched in a stepwise manner up to 80–100%. Fig. 2b
shows a typical evolution of the fluorescence intensity moni-
tored when a FDG solution comes into contact with the film
during stretching. First in the non-stretched state, the films are
enzymatically active indicating that the covalent immobiliza-
Fig. 3 Evolution of the mean enzymatic activity monitored via production
of fluorescence for different strains. (a) Enzymes not covalently linked to
the film and (b) enzymes covalently linked to the film. The activities have
been normalized to the rates measured in the initial, non-stretched state.
tion of the enzymes within the cross-linked polyelectrolyte The rate values correspond to the mean value of 2 experiments in (a) and
network does not affect their activity. Upon stretching, the up to 7 experiments in (b) and error bars correspond to standard deviations.
The Kruskal–Wallis test reveals non-significant influence of the strain in (a)
fluorescence production rate monitored in the solution decreased
(
p = 0.166) whereas significant influence is suggested in (b) (p = 0.021) if one
and this diminution was amplified when the strain was increased.
This evolution of the enzymatic activity was different from that
observed with non-cross-linked enzymes where the fluorescence
refers to the risk level of 0.05.
production remained almost constant or even increased slightly highlight the enzymatic mechano-responsive properties of our
upon stretching. This experiment was repeated several times and designed films.
the results are summarized in Fig. 3a and b where the fluores-
This original approach constitutes a very general strategy to
cence production rates, averaged over different experiments, are construct enzymatically mechano-responsive systems. Unlike
plotted as a function of the strain. The stretching of immobilized many other chemo-mechano-responsive systems reported so
enzymes in the PEM film affected their enzymatic activity by about far, it is based on very low energy demanding processes, namely
30% compared to the non-stretched state. It is expected that the conformational changes, instead of covalent bond breaking.
decrease of the enzymatic activity is due to a stretching-induced This is one of the routes chosen by nature to induce mechano-
change in the enzyme conformation. This hypothesis is in agree- transduction processes. The next step in this field is now to
ment with our previously reported observations on the stretching extend and generalize this approach by using artificial enzymatic
of GFP molecules covalently coupled onto an elastomeric substrate: systems.
one changes by up to 40% the fluorescence intensity by stretching
the substrate by 100%, an effect directly related to changes of the ‘‘Biostretch’’ ANR-10-BLAN-0818), IRTG, icFRC (Labex CSC),
conformation of GFP. A 30–40% catalytic decrease by stretching is IUF and USIAS.
This research was supported by grants from ANR (project
11
also in accordance with reductions found by the early experiments
6,7
of Berezin et al.
References
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