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.

The inhalation of asbestos is a risk factor for the development of malignant mesothelioma and lung cancer. Based on the broad surface area of asbestos fibers and their ability to enter the cytoplasm and nuclei of cells, it was hypothesized that proteins that adsorb onto the fiber surface play a role in the cytotoxicity and carcinogenesis of asbestos fibers. However, little is known about which proteins adsorb onto asbestos. Previously, we systematically identified asbestos-interacting proteins and classified them into eight sub-categories: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. Here, we report an adsorption profile of proteins for the three commercially used asbestos compounds: chrysotile, crocidolite and amosite. We quantified the amounts of adsorbed proteins by analyzing the silver-stained gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis with ImageJ software, using the bands for amosite as a standard. We found that histones were most adsorptive to crocidolite and that chromatin-binding proteins were most adsorptive to chrysotile. The results suggest that chrysotile and crocidolite directly interact with chromatin structure through different mechanisms. Furthermore, RNA-binding proteins preferably interacted with chrysotile, suggesting that chrysotile may interfere with transcription and translation. Our results provide novel evidence demonstrating that the specific molecular interactions leading to carcinogenesis are different between chrysotile and crocidolite.