Enzyme Activity in a Supramolecular Hydrogel
FULL PAPER
conditions used to obtain the photoimage shown in Figure 6a are as fol-
lows: [1]=0.25 wt%, [b-Glc]=0.7nm in an acetate buffer (pH 5), [b-
Gal]=0.3mm in a Tris/HCl buffer (pH 8), [AP]=0.5nm in a Tris/HCl
buffer (pH 9), [Throm]=0.3mm in a Tris/HCl buffer (pH 8) containing
100mm NaCl and 10mm CaCl2, [Tryp]=1mm, in a Tris/HCl buffer (pH 8)
containing 100mm CaCl2, [LEP]=1mm, in a Tris/HCl buffer (pH 8),
[Chym]=1mm in a Tris/HCl buffer (pH 8) containing 100mm CaCl2,
[V8]=5mm in a Tris/HCl buffer (pH 8), [BSA]=10mm in a Tris/HCl
buffer (pH 8), [MUGlc, MUGal, MUP, VPR-AMC]=20mm, [pep-1, pep-
2, pep-4, pep-5]=100mm, [ASP]=200mm. The aqueous solution used to
prepare hydrogels containing ASP contained 1 vol% of MeOH.
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Lysate analysis: Lysates were prepared from the corresponding bacteria
or cell by homogenizer-disruption of cells following standard proce-
dures.[21] Each lysate (2 mL) was dropped onto hydrogel spots containing
a substrate and the resultant fluorescence change was recorded with a
digital camera.
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Enzyme/inhibitor assay using the soaking method: In this method,
0.5 wt% of hydrogel was employed. After quickly dipping a semi-wet
substrate array into an enzyme/inhibitor solution, the array was incubat-
ed in a sealed box with high humidity for at least 15 min. Photoimages of
the resultant arrays were collected. The conditions used to obtain the
photoimages shown in Figure 6c are as follows: [1]=0.5 wt%,
[MUGlc]=20mm in an acetate buffer (pH 5), [MUGal]=20mm in a Tris/
HCl buffer (pH 8), [MUP]=20mm in a Tris/HCl buffer (pH 9), [VPR-
AMC]=20mm in a Tris/HCl buffer (pH 8) containing 100mm NaCl and
10mm CaCl2, [pep-1]=100mm in a Tris/HCl buffer (pH 8) containing
100mm CaCl2, [pep-3]=100mm in a Tris/HCl buffer (pH 8) containing
100mm CaCl2, [pep-2]=100mm in a Tris/HCl buffer (pH 8), [b-Glc]=
0.1nm, [b-Gal]=3mm, [AP]=0.05nm, [Throm]=0.06mm, [Tryp]=2mm,
[Chym]=2mm, [V8]=5mm, [SBTI]=6mm.
Trypsin inhibitor assay using the hydrogel chip: A trypsin inhibitor solu-
tion (1 mL) was dropped onto a hydrogel chip containing an enzyme and
the hydrogel was incubated in a sealed box with high humidity at room
temperature for 30 min. The corresponding substrate solution (1 mL) was
then dropped onto the hydrogel chip and the time course of the fluores-
cence spectral change and the photoimages of the resulting arrays in the
enzyme inhibitor assay were collected following the procedure described
above. The conditions used to obtain the photoimages shown in Figure 6g
are as follows: [1]=0.25 wt%, [b-Glc]=0.7nm in an acetate buffer
(pH 5), [b-Gal]=0.3mm in a Tris/HCl buffer (pH 8), [AP]=0.5nm in a
Tris/HCl buffer (pH 9), [Throm]=0.3mm in a Tris/HCl buffer (pH 8) con-
taining 100mm NaCl and 10mm CaCl2, [Tryp]=1mm in a Tris/HCl buffer
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(pH 8) containing 100mm CaCl2, [LEP]=1mm in
a Tris/HCl buffer
(pH 8), [Chym]=1mm in a Tris/HCl buffer (pH 8) containing 100mm
CaCl2, [SBTI]=6mm, [BA]=10mm, [MUGlc, MUGal, MUP, VPR-
AMC]=20mm, [pep-1, pep-4]=100mm.
Atomic force microscopy: A suspension of gelator 1 (1 mg) in water
(400 mL) was heated to form a homogeneous solution. This hot solution
(3 mL) was placed on a mica substrate and a thin film was formed follow-
ing the spin-coating procedure (3000 rpm, 20 s). The resultant film was
observed by atomic force microscopy (AFM) using the noncontact mode
at room temperature under ambient air conditions.
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Confocal laser scanning microscopy: A hydrogel spot was prepared on a
dish for CLSM observation following the procedure described above. An
enzyme solution (1 mL) was dropped onto the hydrogel spot and then the
progress of the enzymic reaction progress was monitored by CLSM.
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mass spectrometry.
Acknowledgements
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We thank Prof. N. Kato for fruitful discussions. S.-i.T. and S.K. thank
JSPS for their postdoctoral fellowships.
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