Supporting Information s85

Supporting Information

A water-soluble selenoxide reagent as a useful probe for the reactivity and folding of polythiol peptides

Kenta Araia, Masato Noguchia, Beena G. Singhb, K. Indira Priyadarsinib, Katsuhiko Fujioa, Yurika Kuboc, Kyoko Takayamac, Setsuko Andoc, and Michio Iwaokaa*

aDepartment of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan.

bRadiation and Photochemistry Division Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India.

cDepartment of Chemistry, School of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.

Contents:

Figure S1–S2. Typical HPLC chromatogram obtained by short-term oxidation experiment of Ins-A and Rlx-A using 1 equivalent of DHSox for 1 min at 25 °C.

Table S1–S3. Observed and expected molecular masses of the folding intermediates fractionated from HPLC of Ins-A, Rlx-A and Ins-B as determined by ESI(+)-TOF-MS spectrometry.

Figure S3-S35. Relative populations of SS intermediates of CX-397, Ins-A, Rlx-A, and Ins-B as a function of the reaction time under various conditions.

Figure S36. UV absorbance changes at 310 nm and the plots of the second-order rate constant against the pH obtained by the short-term oxidation of DTTred with DHSox using a stopped-flow instrument.

Figure S37. HPLC chromatograms of the peptide fragments obtained from 3Sº and 3S by digestion with thermolysin.

Figure S1. Typical HPLC chromatogram obtained by short-term oxidation experiment of Ins.-A using 1 equivalent of DHSox for 1 min at 25 °C. The fraction number is corresponding to that in the table S2. The symbols of x is a unknown impurities.

Table S1. Observed and expected molecular masses of the folding intermediates and the native state of Ins-A as determined by ESI(+)-TOF-MS spectrometry.

Figure S2. Typical HPLC chromatogram obtained by short-term oxidation experiment of Rlx-A using 1 equivalent of DHSox for 1 min at 25 °C. The fraction number is corresponding to that in the table S2. The symbols of x is a unknown impurities.

Table S2. Observed and expected molecular masses of the folding intermediates and the native state of Rlx-A as determined by ESI(+)-TOF-MS spectrometry.

Table S3. Observed and expected molecular masses of the folding intermediates and the native state of Ins.-B as determined by ESI(+)-TOF-MS spectrometry.

Figure S36. UV absorbance changes at 310 nm (panel A) and the plots of the second-order rate constant against the pH obtained at 25 °C by the short-term oxidation of DTTred with DHSox using a stopped-flow instrument. The reaction conditions of panel A were (a) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 200 mM acetate buffer at pH 4.0, (b) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 100 mM Tris-HCl buffer at pH 7.0, (c) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 100 mM Tris-HCl buffer at pH 8.0, and (d) [DTTred]0 = 2 mM, [DHSox]0 = 0.1 mM in 25 mM NaHCO3 buffer at pH 10.0. See the text for details of determination of the second-order rate constants.

Figure S37. HPLC chromatogramss of the peptide fragments of (A) 3S° and (B) 3S digested by thermolysin. For detailed digestion and HPLC conditions, see experimental section in the text.