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

Kenta Arai; Masato Noguchi; Beena G Singh; K Indira Priyadarsini; Katsuhiko Fujio; Yurika Kubo; Kyoko Takayama; Setsuko Ando; Michio Iwaoka

doi:10.1016/j.fob.2012.12.004

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

FEBS Open Bio. 2012 Dec 29:3:55-64. doi: 10.1016/j.fob.2012.12.004. Print 2013.

Authors

Kenta Arai¹, Masato Noguchi, Beena G Singh, K Indira Priyadarsini, Katsuhiko Fujio, Yurika Kubo, Kyoko Takayama, Setsuko Ando, Michio Iwaoka

Affiliation

¹ Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292, Japan.

Abstract

A water-soluble selenoxide (DHS(ox)) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS(ox) would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS(ox) was extensively studied at pH 4-10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS(ox) (375 mV at 25 °C) compared to l-cystine supported the high ability of DHS(ox) for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pK a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT(red)) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.

Keywords: 1S, 2S, 3S, and 4S, ensembles of SS intermediates with one, two, three, and four SS bonds, respectively; 1S°, 2S°, and 3S°, ensembles of SS intermediates of CX-397 with one, two, and three kinetically formed SS bonds, respectively; 4-Dihydroxyselenolane oxide; AEMTS, 2-aminoethyl methanethiosulfonate; CD, circular dichroism; CX-397, recombinant hirudin variant CX-397; DHSox, trans-3,4-dihydroxyselenolane oxide; DHSred, reduced DHSox; DTTox, oxidized dithiothreitol; DTTred, dl-dithiothreitol; Disulfide; ESI, electron spray ionization; GSSG, oxidized glutathione; Gdn-HCl, guanidine hydrochloride; HPLC, high-performance liquid chromatography; HV-1, recombinant hirudin variant-1; HV-3, recombinant hirudin variant-3; Ins-A, insulin A-chain; Ins-B, insulin B-chain; N, native protein; NHE, normal hydrogen electrode; Oxidative folding; R, reduced polypeptide; RNase A, ribonuclease A; Redox potential; Rlx-A, relaxin A-chain; R°, reduced CX-397 at acidic conditions; SH, thiol; SS, disulfide; SeSe, diselenide; S−, thiolate; TFA, trifluoroacetic acid; Tris, tris(hydroxymethyl)aminomethane.; pI, isoelectric point; trans-3.