Peskin, A. V. | Search bioRxiv

bioRxiv2024

Pharmacological concentrations of vitamin C inhibit hypochlorous acid generation by myeloperoxidase, and oxidized vitamin C inhibits critical thiol proteins and growth of Jurkat cells

High-dose vitamin C therapy for cancer, originally advocated by Linus Pauling (Proc Natl Acad Sci, 1976, 73, 3685-3689), remains a subject of ongoing debate. In this study, we investigate why only pharmacological doses are effective and explore the reasons behind inconsistent therapeutic outcomes. Our data suggest that the bona fide cause of toxicity was oxidized vitamin C rather than hydrogen peroxide. We found that vitamin C at millimolar concentrations, directly inhibits hypochlorous acid generation by myeloperoxidase, through competition with chloride rather than by scavenging the hypochlorous acid that is formed. Products of vitamin C oxidation reacted with the thiols of peroxiredoxin 2 and GAPDH, but failed to react with the cysteine of p16INK4a. The growth and viability of Jurkat cells were affected by oxidized vitamin C. These experiments were conducted in the presence of catalase, demonstrating that the biological effects were due to the products of vitamin C oxidation and not hydrogen peroxide. These findings may have practical implications for the treatment of cancer and diseases in which the deleterious effects of neutrophil activation are observed. For intravenous administration of pharmacological vitamin C to have a beneficial effect, its concentration in the blood must be maintained at millimolar levels and this can only be achieved via maintenance infusion. As a proof-of-concept, our data suggest that to enhance anticancer therapy interventions, it is crucial to implement treatments that facilitate the oxidation of vitamin C in the bloodstream.O_QDProgress in the art of healing has always been induced by progress in basic knowledge. C_QD O_QDAlbert Szent-Gyorgyi (Woods Hole, Mass., USA, 1973) C_QD Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/627077v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1fef8c6org.highwire.dtl.DTLVardef@7ebe59org.highwire.dtl.DTLVardef@102e623org.highwire.dtl.DTLVardef@153252d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 {+/-} 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 {+/-} 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 {+/-} 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

biochemistry↗