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Post-photoaffinity

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Post-photoaffinity

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

    -11IR (KBr) 3300, 2100 / cm, H-NMR (250 MHz, CDOD, 25 ?C, TMS) ;/ppm; 0-HN33

    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 MnCl, CaCl, 0.1 M NaCl) at 4 22

    ?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

    2nd

    Abs.

    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 [D-Glucoseaffinity 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

    ] / mM?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; HO:CHCN = 95:5 to 45:55, 23

    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

    A1A2A3A5A4

    A6

    A7A8A9

    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

    A7A6A4

    A9A5

    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 A1A1

    A9

    A5A4

    A8

     ESI-Fig. 4 Peptide mapping of 1-labeled ConA.

     ESI-4

Supplementary Material (ESI) for Chemical Communications

This journal is ? The Royal Society of Chemistry 2001

ESI-5

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