Browsing by Subject "Titration"
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Item Disulfide dithiol redox titrations of proteins(2005-05) Mason, Jeremy Todd; Knaff, David B.; Pare, PaulRedox-active disulfide/dithiol couples in proteins play important regulatory roles within cells. Disulfide/dithiol redox reactions of regulatory proteins found in the purple photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus are used to regulate the expression of genes encoding photosystem components in response to the presence oxygen and light. Disulfide/dithiol redox reactions of regulatory proteins found in the yeast Saccharomyces cerevisiae regulate expression of genes encoding the production of peroxide-scavenging proteins in response to intracellular H2O2 tension. AppA and PpsR are two proteins present in R. sphaeroides that have been shown to regulate gene expression in response to oxygen through disulfide/dithiol redox chemistry. AppA, which contains FAD, also functions as a blue light receptor. It has been proposed that AppA reduces PpsR, causing PpsR to lose its abililty to bind DNA and repress transcription of photosynthesis genes. Redox titrations of the disulfide/dithiol couples in PpsR and AppA were carried out at pH 7.0 and the two proteins were shown to be isopotential, with both having Em values of -320 mV at pH 7.0. Em vs. pH profiles for PpsR and RegB, a protein involved in regulation of gene expression in R. capsulatus, were generated in an attempt to detect pKa values for groups involved in proton uptake/release that is coupled to the disulfide/dithiol redox chemistry. However, neither protein showed a pKa for redox-linked residues at physiological pH values. Yap1 is a key regulator of gene expression in S. cerevisiae in response to peroxides. Gpx3 and Trx2 are two additional components of this S. cerevisiae regulatory cascade. Em values for the two disulfide bonds in Yap1 have been determined (Em1 = -330 mV and Em2 = -155 mV), as has an Em value of -315 mV for Gpx3, a component thought to serve as the physiological oxidant for Yap1. Trx2 has an Em of -275 mV, which is capable of reducing the disulfide in Yap1 that corresponds to Em2, but not Em1.Item Disulfide/Dithiol redox titrations of proteins(Texas Tech University, 2005-05) Mason, Jeremy Todd; Knaff, David B.; Pare, PaulRedox-active disulfide/dithiol couples in proteins play important regulatory roles within cells. Disulfide/dithiol redox reactions of regulatory proteins found in the purple photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus are used to regulate the expression of genes encoding photosystem components in response to the presence oxygen and light. Disulfide/dithiol redox reactions of regulatory proteins found in the yeast Saccharomyces cerevisiae regulate expression of genes encoding the production of peroxide-scavenging proteins in response to intracellular H2O2 tension. AppA and PpsR are two proteins present in R. sphaeroides that have been shown to regulate gene expression in response to oxygen through disulfide/dithiol redox chemistry. AppA, which contains FAD, also functions as a blue light receptor. It has been proposed that AppA reduces PpsR, causing PpsR to lose its abililty to bind DNA and repress transcription of photosynthesis genes. Redox titrations of the disulfide/dithiol couples in PpsR and AppA were carried out at pH 7.0 and the two proteins were shown to be isopotential, with both having Em values of -320 mV at pH 7.0. Em vs. pH profiles for PpsR and RegB, a protein involved in regulation of gene expression in R. capsulatus, were generated in an attempt to detect pKa values for groups involved in proton uptake/release that is coupled to the disulfide/dithiol redox chemistry. However, neither protein showed a pKa for redox-linked residues at physiological pH values. Yap1 is a key regulator of gene expression in S. cerevisiae in response to peroxides. Gpx3 and Trx2 are two additional components of this S. cerevisiae regulatory cascade. Em values for the two disulfide bonds in Yap1 have been determined (Em1 = -330 mV and Em2 = -155 mV), as has an Em value of -315 mV for Gpx3, a component thought to serve as the physiological oxidant for Yap1. Trx2 has an Em of -275 mV, which is capable of reducing the disulfide in Yap1 that corresponds to Em2, but not Em1.Item Synthetic studies towards the total synthesis of cortistatin A synthesis of the pentacyclic core of citreamicin µ and GA-ring model studies(2016-08) Blumberg, Shawn Thomas; Martin, Stephen F.; Krische, Michael J; Anslyn, Eric V; Kerwin, Sean M; Willson, Carlton GThe route to the key bicyclic intermediate was streamlined to eight steps in 16% yield, using a TMSI promoted coupling of a furan and an enone. Additionally, methodology for the selective ozonolysis of the bicyclic intermediate was developed via ozone titration. Work on the dihydroxylation step led to the discovery and development of a new pH-neutral Sharpless-style asymmetric dihydroxylation. The synthesis of pentacyclic core of citreamicin µ was accomplished in 12 steps. New methodologies were developed, including: an ortho- selective bromination of a vanillin derivative and the use of 4-(Phenylazo)diphenylamine (PDA) as an internal indicator for the acetylide coupling. The usefulness of PDA led to its development as a general all-purpose indicator for the titration of strong bases, Lewis acids and reducing agents. The discovery of (n-Bu)4NOAc as a privileged additive led to the development of new methods for the synthesis of isocoumarins and new methodology for the condensation of amino acids using LiMe4 was developed.Item The use of scanning electrochemical microscopy for the detection and quantification of adsorbed intermediates at electrodes(2010-08) Rodriguez Lopez, Joaquin, 1983-; Bard, Allen J.; Crooks, Richard M.; Stevenson, Keith J.; Mullins, C. B.; Henkelman, GraemeScanning electrochemical microscopy (SECM) was used for the study and characterization of catalytic and electrocatalytic processes occurring at electrodes. The Surface Interrogation mode (SI-SECM) was introduced for the detection and quantification of adsorbed intermediates and products of catalyzed chemical and electrochemical reactions at noble metals (Pt, Au). In SI-SECM two micro electrodes (i.e. an SECM tip and a substrate of the desired material) are aligned concentrically at a micrometric distance where SECM feedback effects operate. A contrast mechanism based on feedback effects allows for the detection of reactive adsorbed intermediates at the substrate: the SECM tip generates a reactive homogeneous species that “micro-titrates” the substrate adsorbates to yield an electrochemical signal that contains information about the amount of intermediate and about its kinetics of reaction with the redox mediator. The technique was used for the study of the reactivity of three model small adsorbates: 1) the reactivity of adsorbed oxygen on Au and Pt with a reducing mediator was explored and suggested the detection of “incipient oxides” at these surfaces; kinetic parameters of the reactivity of Pt oxides with mediators were obtained, fit to theory and used to explain observations about the electrocatalytic behavior of Pt under anodizing conditions; 2) the reactivity of oxidizing mediators with adsorbed hydrogen on Pt was studied and showed the cation of N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) to be a successful interrogation agent, the detection of hydrogen generated by the decomposition of formic acid on Pt at open circuit was investigated; 3) electrogenerated bromine was used to catalytically interrogate carbon monoxide at Pt, this reaction was previously unreported. The mentioned applications of SECM were validated through the use of digital simulations of diffusion in the complex SECM geometry through flexible commercial finite element method software.