The reactions between sodium nitrite and the polyamines spermidine and spermine were investigated. When reacted at 80°C for 1 hr at pH 3.5 with a 1:3 molar ratio of amine groups to sodium nitrite, five volatile nitrosamines were identified as products in the nitrosation of spermidine. 3HC1, and two volatile nitrosamines were identified from spermine 4HC1. The principal volatile nitrosation product both from spermidine and spermine was γ-butenyl(β-propenyl)nitrosamine (BPN). The identification of this compound was based on the spectral characteristics of the compound using mass, infrared, and nuclear magnetic resonance spectrometry. To confirm the identity, BPN was synthesized from γ-butenyl(β-propenyl)amine and sodium nitrite, and the spectra obtained for this substance and the unknown compound were compared. The amine precursor was synthesized from allylamine and l-Bromo-4-butene. Two hydroxylated, dialkyl nitrosamines were identified as products from spermidine 3HC1: γ-butenyl(γ-propanol)nitrosamine and δ-butanol(β-propenyl)nitrosamine. The compounds were characterized by mass and infrared spectrometry, and by the Griess test for nitrosamines. The structures were confirmed by making the trifluoroacetate derivatives of the compounds, and obtaining their mass spectra. In the presence of chloride ions, chlorinated dialkyl nitrosamines were tentatively identified as nitrosation products from spermidine. The identification was based on mass spectrometry, particularly the isotope effects by chlorine, and by the Griess test. It appeared that δ-butylchloride(β-propenyl)nitrosamine probably was a major isomer, but other structural isomers may also be formed. The nitrosamines described above have previously not been synthesized or characterized. Their individual carcinogenic potency therefore is not known. Nitrosopyrrolidine, which was a nitrosation product both from spermidine and spermine, is a potent carcinogen. The yields of the individual nitrosamines from spermidine 3HC1 and spermine 4HC1 were, respectively: BPN, 1.7%, 1. 4%; γ-butenyl(γ- propanol)nitrosamine, 0, 29%, 0%; δ-butanol(β-propenyl)nitrosamine, 0.18%, 0%; δ-butylchloride(β-propenyl)nitrosamine (and its isomers), 0, l2%, 0%; nitrosopyrrolidine3 0.60% and trace amounts. The yields were estimated on the basis of the amount of polyamine precursor. Maximum accumulation of all nitrosamines from spermidine was observed between pH 3.0 and 4.5, when reacting for 1 hr at 50°C, Increasing the reaction time to 25 hrs at 50°C, the yield of BPN at pH 5.0 exceeded the yield at pH 3.5. From the temperature effect on the nitrosation rate, activation energy for the formation of BPN from spermidine and nitrite was estimated to be 19 kcal/mol. In the presence of 0.1-1 M sodium chloride at pH 4.0, no significant effects on the yields of BPN, γ-butenyl(γ-propanol)nitrosamine and δ-butanol (β-propenyl)nitrosamine were observed, while the yield of δ-butylchloride (β-propenyl)nitrosamine (and its isomers) was strongly enhanced with increased sodium chloride concentration. The yields of all nitrosamines were drastically reduced in the presence of 1.5 M or 2.0 M sodium chloride in the system. In the nitrosation of proline, sodium chloride in concentrations up to 1 M strongly activated the reaction at pH 0.5. Small inhibiting effects were observed at pH 2.5, however, and moderate inhibition by sodium chloride was seen at pH's 4.0 and 5.5. Multiple regression analysis showed the best fitted model was of the form, log (initial rate of nitrosation) = a + b[ NaCl] + c[ NaCl]² at all pH levels tested. / Graduation date: 1975

A study of the reactions of S-nitrosothiols in solution was undertaken. S-Nitrosothiols were known to produce NO, a physiologically important regulatory chemical, in solution but the mechanism of the reaction was unknown. Kinetic measurement of the S-nitrosothiol decomposition spectroscopically was erratic and irreproducible. The reaction was found to be mediated by adventitious Cu(^2+) in the water supplies used. The reaction of Cu(^2+) with S-nitrosothiols was studied. The rate equation was established and the reaction was found to be first order in S-nitrosothiol and the added Cu(^2+). Second order rate constants for the reaction were determined so that the effect of structure could be studied. S-Nitirosothiols containing no other functional groups did not react with Cu(^2+). S-Nitrosothiols containing a P-amino group were found to be the most reactive. The S-niti-osothiols were thought to be bidentately bound about the Cu(^2+). which leads to rate limiting S-N bond breaking producing the thiyl radical and nitiic oxide. A similar reaction was found to occur between S-nitrosothiols and Cu+ and Fe2+ ions, but no other transition metals appeared to catalyse nitiic oxide formation from S-nitrosothiols. The reaction of-S-nitrosothiols with other thiols was also Studied. The ti-ansnitrosation reaction was rapid and the variation of the observed rate constant with pH over the range 6-13 produced a sigmoidal curve which was indicative of a reaction involving attack by tiuolate anion. It is believed that this reaction could be important in vivo as a mechanism for storing NO in the form of a stable S-niti-osothiol and then transferring the NO group to a more reactive S-niti-osothiol for rapid NO release. The effect of the thiol group alone was also investigated and depending on the concentration of thiol used the reaction was either catalysed or inhibited. Other conditions such as changing pH, adding metal chelators and the oxygen dependency of the reaction were studied. Methods of measuring NO levels in solution were tested, the best being the use of a NO specific electi-ode. The reactions of NO with thiols were also studied to see if a mechanism for in vivo formation of S-nitrosothiols could be determined.

No description available.

The kinetics of nitrosation of cysteine, cysteine methyl ester, N-acetylcysteine, penicillamine, N-acetylpenicillamine, glutathione and thioglycolic acid was undertaken. These thiols exhibited identical rate laws which are interpreted as nitrosation at sulphur by H(_2)NO(_2)(^+)/NO(^+). The rate constants determined show the high reactivity of thiols towards the nitrosating agent. The nucleophile catalyzed reactions were also investigated and the order of reactivity NOCl > NOBr > NOSCN was observed. Normally in these nucleophile catalyzed reactions there is a first order dependence on [thiol]. However, for N-acetylcysteine and thioglycolic' acid at high [thiol] the rate of formation of NOX tends to become the rate determining stage. The difference in rate constants between cysteine and penicillamine and their N-acetyl derivatives is explained in terms of internal stabilization. The decomposition of S-nitrosocysteine (S-NOCys) at pH 5.5, 7 and 9.8 in the presence and absence of C1(^-), Br(^-) and SCN(^-), and also alanine and sodium bicarbonate at pH 7, and S-nitrosoglutathione (GS-NO) at pH 7 in the presence and absence of alanine, C1(^-), and sodium bicarbonate was studied. The decomposition profiles were complex, but showed that S-NOCys was least stable at pH 7, and that GS-NO was more stable than S-NOCys. The addition of the aforementioned species did not significantly affect the rate of decomposition of the thionitrites. Finally the potential of S-NOCys, GS-NO and S-nitroso-N-acetyl- penicillamine as nitrosating agents towards amines was investigated at pH 7 and pH 8. These thionitrites nitrosated morpholine to give approximately the same yield of N-nitrosomorpholine (ca -17%) at pH 7, and less at pH 8 for S-NOCys and GS-NO. The addition of sodium acetate, sodium chloride, sodium bicarbonate, alanine and glucose, compounds liable to be present in vivo, did not significantly affect the yield of N-nitrosomorpholine. The transnitrosation reaction was complete before total decomposition of the thionitrite and a direct reaction between the thionitrite and morpholine is proposed.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed Oct. 16, 2007). Vita. Includes bibliographical references.

S-Nitrosothiols have been found to undergo nucleophilic attack by the hydroperoxide anion to effect electrophilic nitrosation of the nucleophile. Peroxynitrite anion is formed in almost quantitative yields and the kinetics of the reaction examined, confirming attack through the deprotonated form of hydrogen peroxide. Conversely, under slightly acidic conditions peroxynitrous acid, the neutral form of peroxynitrite, has been shown to nitrosate an excess of thiol in an indirect pathway. Initially two moles of thiol are oxidised to the corresponding disulfide with the concomitant production of nitrite. Under mildly acidic conditions nitrous acid is formed which can then nitrosate excess thiol present. The reaction of a 2;1 excess of thiol over peroxynitrous acid has been shown to generate nitrous acid, which remains relatively stable, as there is no thiol remaining due to its oxidation to the disulfide. At higher acidities an additional source of nitrosation is uncovered which is explained in the terms of the formation of a protonated from of peroxynitrous acid with analogies toward the nitrous acidium ion. A comparison of the antioxidant potential of S-nitrosothiols versus thiols has also been examined using the powerful oxidant potassium bromate. Complex kinetic traces were generated but evidence was obtained which showed that S-nitrosothiols have enhanced antioxidant potential over thiols due to the nitroso moiety. The alkaline hydrolysis of S-nitrosothiols was also investigated with attack of the hydroxide ion postulated to proceed via nucleophilic attack on the sulfur atom of S-nitrosothiols. Also the reaction between S-nitrosothiols and phenolic compounds was found to proceed through different mechanisms depending on the ring substituents on the phenol.

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of the proteins using UV-visible spectroscopy indicates that E75 has a b type heme with a low spin six-coordinate ferric iron. In the E75 heme binding domain, the heme exhibits an unstable ferrous state and only binds NO and CO at high non-physiological levels. These data place into doubt the suggested roles for E75 as a gas regulated transcription regulator.

by Chan Chi Wai, Michael. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 238-272). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Three biologically-active aminothiols cysteamine (CA), DL-cysteine (CYSH) and DL-homocysteine, were studied in this thesis. These aminothiols react with nitrous acid (HNO2), prepared in situ, to produce S-nitrosothiols (RSNOs): S-nitrosocyteamine (CANO), S-nitrosocysteine (CYSNO) and S-nitrosohomocysteine (HCYSNO). They also react with S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) through a transnitrosation reaction to produce their corresponding RSNOs. A detailed kinetics and mechanistic study on the formation of these RSNOs and their subsequent decomposition to release nitric oxide (NO) were studied. For all three aminothiols the stoichiometry of their reaction with nitrous acid is strictly 1:1 with the formation of one mole of RSNO from one mole of HNO2. In all cases, the nitrosation reaction is first order in nitrous acid, thus implicating it as a nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating another nitrosating agent, the nitrosonium cation, NO+ ( which is produced from the protonation of nitrous acid) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Nitric oxide itself was not detected as a nitrosant. Bimolecular rate constants for the nitrosation of CA, CYSH and HCYSH were deduced to be 17.9, 6.4, 0.09 M-1 s-1 for the nitrosation by nitrous acid and 8.25 x 1010, 2.89 x 1010 and 6.57 x 1010 M-1 s-1 for the nitrosation by nitrosonium cation respectively. A linear correlation was obtained between the rate constants and the pKa of the sulfur center of the aminothiols for nitrosation by NO+. The stabilities of the three RSNOs were found to be affected by metal ions. They were unstable in the presence of metal ions, with half-lives of few seconds. However, in the presence of metal ion chelators, they were found to be relatively stable with half-lives of 10, 30 and 198 hours for CYSNO, CANO and HCYSNO respectively. The relative stability of HCYSNO may be an advantage in the prevention of its metabolic conversion to homocysteine thiolactone, the major culprit in HCYSH pathogenesis. This dissertation has thus revealed new potential therapeutic way for the modulation of HCYSH related cardiovascular diseases.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 164-173). Also available on the Internet.