Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2001
Electronic supplementary information (ESI)
Post-photoaffinity Labeling Modification Using Aldehyde Chemistry
to Produce a Fluorescent Lectin toward Saccharide-Biosensors
Tsuyoshi NAGASE, Seiji SHINKAI and Itaru HAMACHI*+
Department of Chemistry & Biochemistry, Graduate School of Engineering,
Kyushu University, Fukuoka, 812-8581, JAPAN
+
PRESTO, JST
Identification of the photoaffinity labeling reagent 1
IR (KBr) 0-H 3300, N3 2100 / cm-1, 1H-NMR (250 MHz, CD3OD, 25 ˚C, TMS) /ppm;
3.58 (m, 1H, H5), 3.72 (m, 3H, H4 and H6), 3.88 (dd, J= 4.0, 12.0 Hz, 1H, H3), 3.99 (dd,
J= 4.2, 2.8, 1H, H2), 5.42 (d, J= 2.8 Hz, 1H, H1), 6.98 (d, J= 8.8 Hz, 2H, aromatic), 7.15
(d, J= 8.8 Hz, 2H, aromatic)
Purification of 1-labeled ConA by affinity column chromatography.
The 1-labeled ConA was purified by the affinity column chromatography (Sephadex
G100).
In this purification, the three peaks were observed in the elution curve
(ESI-Figure 1). The 1st, 2nd and 3rd peaks were identified by MALDI TOF-MS to be
the homo-dimer of the 1-labeled ConA, the hetero-dimer of the 1-labeled ConA and
non-labeled ConA, and the homo-dimer of non-labeled ConA, respectively.
It is
reported that ConA exists as dimer in the purification condition (pH 5.0). The 1st
fraction was concentrated by ultrafiltration (YM10 filters, Amicon) and dialyzed against
the buffer A (50 mM HEPES (pH 7.0), 1 mM MnCl2, CaCl2, 0.1 M NaCl) at 4 ˚C. The
2nd fraction was concentrated by ultrafiltration (YM10 filters, Amicon) and purified by
size exclusion chromatography (Biogel P-30, 10 mM acetate buffer (pH 5.0), 2 cm  20
cm) and dialysis against the buffer A at 4 ˚C. The resultant solutions were utilized for
saccharide titration experiments.
ESI-1
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2001
3rd
1st
40 mM
]/ mM
[D-Glucose
Abs.
2nd
Fraction number
ESI-Fig. 1 Fraction curves in the affinity purification of the 1-labeled ConA.
Lysyl-endopeptidase digestion
Lysyl-endopeptidase digestion of 1-labeled ConA (100 µM, 1st fraction in the
affinity purification) was performed under the conditions of a 50:1 weight ratio of
substrate to enzyme in 200 µL of Tris buffer (pH 9.0) in the presence of 3 M urea at 37
˚C for 15 h. The hydrolysis was stopped by the addition of 0.1 % TFA, and the solution
was analyzed by HPLC (ODS column, gradient; H2O:CH3CN = 95:5 to 45:55, 100 min).
As a control experiment, native ConA was also digested by lysyl-endopeptidase in the
same condition as 1-labeled ConA.
In the native ConA digestion, 8 peaks, which are shown in ESI-Fig. 2 as the peptide
fragments (A1,4-10), were observed in the HPLC peptide mapping (ESI-Fig. 3). All
peaks in the HPLC peptide mapping were identified as shown in Fig. 3 by MALDI-TOF
MS analyses.
ESI-2
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2001
A1
A2
A3 A4
A5
A6
A7
A8
A9
A10
ESI-Fig. 2 Amino acid sequence of Con A. The ideal 10 peptide fragments digested
by lysyl-endopeptidase were shown as A1-10. The amino acids surrounded by squares
were the one closely involved with saccharide binding site.
A10
A1
Native conA
A7
A6
A4
A9
A5
A8
ESI-Fig. 3 Peptide mapping of native ConA
ESI-3
Supplementary Material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2001
In the digestion of the 1-labeled ConA, two peaks, which were not in the native ConA
peptide mapping, were newly observed at the retention time of 51 and 68 min, which
were identified by MALDI-TOF MS to be the 1-labeled A1 fragment (Calcd. for
[1-labeled A1 +Na]+, 3,534; Found, 3,540 ± 10) and the 1-labeled A6 fragment (Calcd.
for [1-labeled A6 +H]+, 4,796; Found, 4,790 ± 10), respectively (ESI-Fig. 4).
A10
1-labeled A6
A7
1-labeled A1
A1
A9
A4
A5
A8
ESI-Fig. 4 Peptide mapping of 1-labeled ConA.
ESI-4