Selenoglutathione:  Efficient Oxidative Protein Folding by a Diselenide

Abstract

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG). Thus, the diselenide bond of GSeSeG is 7 kcal/mol more stable than the disulfide bond of GSSG. Nonetheless, we found that GSeSeG can be used to oxidize cysteine residues in unfolded proteins, a process that is driven by the gain in protein conformational stability upon folding. Indeed, the folding of both ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) proceeded efficiently using GSeSeG as an oxidant, in the former case with a 2-fold rate increase relative to GSSG and in the latter case accelerating conversion of a stable folding intermediate to the native state. In addition, GSeSeG can also oxidize the common biological cofactor NADPH and is a good substrate for the NADPH-dependent enzyme glutathione reductase (kcat = 69 +/- 2 s-1, Km = 54 +/- 7 microM), suggesting that diselenides can efficiently interact with the cellular redox machinery. Surprisingly, the greater thermodynamic stability of diselenide bonds relative to disulfide bonds is not matched by a corresponding decrease in reactivity.

Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier

“…The likely reason for this is that diselenide bonds are more stable than disulfide bonds, with a lower redox potential. 19 This property is beneficial for transfection in vivo because disulfide bonds may be cleaved by glutathione at the concentrations present in blood, while diselenide bonds remain stable. In the gel retardation assay, the ability of OEI 800 -SeSe x and OEI 800 -SS x to complex with the DNA decreased after treatment with DTT, which implies a reduction-sensitive ability for diselenide bonds and that the reductive reaction between DTT and OEI 800 -SeSe x was already efficient enough for release of DNA (Figure 4).…”

“…As reported elsewhere, selenoglutathione can oxidize the common biological cofactor NADPH, and reacts strongly with glutathione reductase, which is an NADPH-dependent enzyme. 19 Therefore, diselenide bonds can be efficiently cleaved in a cell reduction environment and have the same reduction-sensitive ability as disulfide bonds.…”

“…Compared with OEI 800 , OEI 800 -SeSe x with its high molecular weight possessed a better ability to condense plasmid DNA, and could form tight nanoparticles and endocytose easily. It was hypothesized that the reductionsensitive disulfide and diselenide bonds could be cleaved in the intracellular environment, triggered by glutathione or NADPH, 19 and the OEI 800 -SeSe x and OEI 800 -SS x degraded to pieces of OEI 800 , which led to release of plasmid DNA to get high transfection ability and lower cytotoxicity. These results are in agreement with those of other studies of OEI 800 cross-linked by degradable bonds.…”

“…18 Furthermore, other intracellular reductive reagents, such as nicotinamide adenosine dinucleotide phosphate (NADPH), can change oxidative glutathione to reduced glutathione. 19 These factors can be used as triggers for disassociation of polycation and gene payload. Various chemical linkers responsive to different triggers have been designed, including acetals, [20][21][22] hydrazone bonds, [23][24][25][26] esters, 6 disulfides, [27][28][29][30][31][32][33] and matrix metalloproteinase substrate peptide-containing linkers.…”

“…As reported recently, diselenide bonds possess reductive biodegradable ability similar to that of disulfide bonds. 19,40 Micelles formed by polymers containing diselenide bonds were quite stable under physiological conditions, but were sensitive to reductive stimuli. 36 In addition, disulfide bonds were too fragile for the reductive environment, but can be cleaved by a reductive reagent in low concentration.…”