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

Abstract

A water-soluble selenoxide (DHSox) 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 DHSox 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 DHSox 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 DHSox (375 mV at 25 °C) compared to l-cystine supported the high ability of DHSox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pKa 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 (DTTred) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.

Reinvestigation of the Oxidative Folding Pathways of Hen Egg White Lysozyme: Switching of the Major Pathways by Temperature Control

“…In this study, oxidative folding of HEL was carried out by using DHS ox as a strong oxidant, which enables rapid and stoichiometric SS formation in polythiol substrates [10]. As a result, the three des intermediates, i.e.…”

“…, the temporary separation of the SS rearrangement process from the SS formation process by using trans -3,4-dihydroxyselenolane oxide (DHS ox ) as a strong oxidant, for the study of the oxidative folding pathways of SS-containing proteins [9]. DHS ox enables rapid, irreversible, and stoichiometric SS formation in various polythiol peptides in an aqueous medium [10]. For example, when this analytical method was applied to the oxidative folding of bovine pancreatic RNase A having four SS linkages in the native state, the SS formation and SS rearrangement phases were clearly separated from each other, and the oxidative folding pathways could be easily characterized [11,12].…”

Synthesis, characterization, and structure of trans-3,4-dihydroxy-1-selenolane {DHS(OH)2} substituted derivatives

“…Similarly some water-soluble diorganyl tellurides exhibit peroxide decomposing and chain-breaking antioxidative capacity. 20 In view of potential biological applications of XV, it was considered worthwhile to synthesize its derivatives. 16 In earlier papers XV has been denoted as DHS red , but to indicate substitution here it is referred as DHS(OH) 2 .…”

Oxidative folding pathways of bovine milk β‐lactoglobulin with odd cysteine residues

“…1B) [24], was employed. To enable clear observation of these intermediates, a strong and specific oxidant, trans-3,4-dihydroxyselenolane oxide (DHS ox ) (Fig.…”

“…To enable clear observation of these intermediates, a strong and specific oxidant, trans-3,4-dihydroxyselenolane oxide (DHS ox ) (Fig. When applied to oxidative protein folding, DHS ox allows rapid (< 1 min) and quantitative SS formation over a wide pH range (at least from pH 3 to 10) [24]. DHS ox is a unique water-soluble selenoxide reagent developed in our laboratory and has been utilized for oxidative folding of various SScontaining proteins, such as bovine pancreatic ribonuclease A [25][26][27], recombinant hirudin CX-397 [28], hen egg white lysozyme [29], and bovine milk a-lactalbumin [30], as well as native chain assembly of bovine and human insulin [31] and human relaxin-2 [31], but has never been applied to the oxidative folding of a protein with odd Cys residues.…”