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PubMed1987

13C and 31P NMR study of gluconeogenesis: utilization of 13C-labeled substrates by perfused liver from streptozotocin-diabetic and untreated rats.

The metabolism of 13C-labeled substrates was followed by 13C and 31P NMR in perfused liver from the streptozotocin-treated rat model of insulin-dependent diabetes. Comparison was made with perfused liver from untreated littermates, fasted either 24 or 12 h. The major routes of pyruvate metabolism were followed by a 13C NMR approach that provided for the determination of the metabolic fate of several substances simultaneously. The rate of gluconeogenesis was 2-4-fold greater and beta-hydroxybutyrate production was 50% greater in liver from the chronically diabetic rats as compared with the control groups. Large differences in the distribution of 13C label in hepatic alanine were measured between diabetic and control groups. The biosyntheses of 13C-labeled glutathione and N-carbamoylaspartate were monitored in time-resolved 13C NMR spectra of perfused liver. Assignments for the resonances of glutathione and N-carbamoylaspartate were made with the aid of 13C NMR studies of perchloric acid extracts of the freeze-clamped livers. 13C NMR spectroscopy of the perfusates provided a convenient, rapid assay of the rate of oxidation of [2-13C]ethanol, the hepatic output of [2-13C]acetaldehyde, and the accumulation of [2-13C]acetate in the perfusate. By 31P NMR spectroscopy, carbamoyl phosphate was measured in all diabetic livers and an unusual P,P'-diesterified pyrophosphate was observed in one-fourth of the diabetic livers examined. Neither of these phosphorylated metabolites was detected in control liver. Both 13C and 31P NMR were useful in defining changes in hepatic metabolism in experimental diabetes.

Alanine↗
PubMed1982

Major high mobility group like proteins of Drosophila melanogaster embryonic nuclei.

Nuclei from Drosophila melanogaster embryos contain three major proteins which are extracted by 0.35 M NaCl and by 2% perchloric acid. One of these is histone H1, and we refer to the other two as A63 and A13 in accordance with their molecular weights determined by electrophoresis on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (63,000 and 13,000, respectively). The molecular weight of A13, based on its amino acid composition, is approximately 10,000. The amino acid analyses of A63 and A13 show that both of these proteins have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group proteins isolated from vertebrate tissues. While A13 comigrates with histone H2A on NaDodSO4-polyacrylamide gels and with H2B on acid/urea gels, it can be readily resolved from the histones by Triton/acid/urea-Na DodSO4 two-dimensional electrophoresis.

Amino Acids↗
PubMed1980

Evidence for a quantitative tissue-specific distribution of high mobility group chromosomal proteins.

The quantitative tissue specificity of the high mobility group (HMG) chromosomal proteins was investigated. Perchloric acid (PCA) extracts of four different chicken tissues and erythrocytes contained three proteins which comigrated on NaDodSO4-polyacrylamide gels with the HMG's 1,2, and E from erythrocyte nuclei. These three HMG's from embryonic skeletal muscle and erythrocytes also comigrated on two-dimensional gels, employing an acid-urea system in the first dimension and an NaDodSO4 system in the second. Interpretation of the two-dimensional gels suggested that the two low molecular weight proteins of this triplet arose from the HMG 2 band of the acid-urea gels. These have been designated HMG 2A and HMG 2B. Three proteins of similar molecular weights were also found in the PCA extracts of calf thymus. They were arranged in a similar but not identical pattern on two-dimensional gels. Thus, these three HMG's appear to be neither tissue nor species specific. In addition, the 2.0% PCA extracts of all chicken tissues examined contain a 38 000-dalton (38K) nuclear protein which coisolates with the HMG's. These four proteins are found in different relative amounts in each of the four chicken tissues and erythrocytes. They are found in the same relative amounts, however, in embryonic skeletal muscles from different chicken strains with widely different highly repetitive sequence content, suggesting that none of these individual proteins is selectively localized to constitutive heterochromatin. The quantitative tissue specificity of the HMG's and the 38K protein, however, suggests that they may participate in regulating cell-specific gene expression.

Animals↗
PubMed1976

Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.

Binding Sites↗
PubMed1976

Phosphorus-31 nuclear magnetic resonance detection of unexpected phosphodiesters in muscle.

In the examination of intact muscles by 31P nuclear magnetic resonance spectroscopy, a number of signals have been detected in the phosphodiester region (-0.5 to 0.5 ppm) of the spectrum which could not be correlated with the known common phosphates of muscle tissue. These signals arise from perchloric acid extractable compounds with several common chemical properties, one of which is a ready solubility in nearly anhydrous ethanol solutions. A component contributing to the major resonance has been identified as glycerol-3-phosphorylcholine. This characterization is based on both 31P nuclear magnetic resonance and chromatographic data.

Animals↗
PubMed1975

The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo.

The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.

Binding Sites↗
PubMed2001

Tetranuclear mixed-metal M(II)2Cu(II)2 complexes derived from a phenol-based macrocyclic ligand having two N(amine)2O2 and two N(imine)2O2 metal-binding sites.

The reaction of N,N'-dimethyl-N,N'-ethylenebis(5-bromo-3-formyl-2-hydroxybenzylaminato)copper(II) with ethylenediamine in aqueous DMF with excess perchloric acid resulted in the [2:2] cyclic condensation of the constituents, providing the dinuclear Cu(II) complex [Cu2(H2R)](ClO4)2. It crystallizes in the monoclinic space group P2(1)/c, with a = 19.603(3) A, b = 13.370(2) A, c = 21.072(3) A, beta = 98.87(1) degrees, V = 5456(1) A3, and Z = 4. The ligand R4- has two N(amine)2O2 and two N(imine)2O2 metal-binding sites sharing two phenolic oxygens, and [Cu2(H2R)](ClO4)2 has the two Cu(II) ions in the N(imine)2O2 sites and two protons in the N(amine)2O2 sites. [Cu2(H2R)](ClO4)2 was converted by neutralization into [Cu2(R)], from which mixed-metal Cu(II)2M(II)2 complexes [Cu2M2(R)Cl4] (M = Co(II), Ni(II), Zn(II)) were derived. [Cu2Co2(R)Cl4]*2CHCl3*H2O crystallizes in the monoclinic space group C2/c, with a = 32.514(3) A, b = 12.246(3) A, c = 19.827(2) A, beta = 126.082(1) degrees, V = 6380(1) A3, and Z = 4. [Cu2Zn2(R)Cl4]*2CHCl3*H2O crystallizes in the monoclinic space group C2/c, with a = 32.53(1) A, b = 12.242(2) A, c = 19.729(9) A, beta = 126.03(3) degrees, V = 6354(4) A3, and Z = 4. The two complexes are isotructural and have a dimer-of-dimers structure with two separated Cu(II)M(II) units. In each dinuclear unit, the Cu(II) is bound to the N(imine)2O2 site and the M(II) is bonded to a phenolic oxygen and two nitrogens of the N(amine)2O2 site. The Cu(II) and M(II) ions are bridged by a phenolic oxygen and an exogenous chloride ion. The Cu(II)2Ni(II)2 complex has a defect double-cubane structure. Cryomagnetic studies for the Cu(II)2Co(II)2 complex indicate an antiferromagnetic spin-exchange interaction within each dinuclear Cu(II)Co(II) unit (J = -9.5 cm(-1) based on H = -2JS(Cu)S(Co)). The Cu(II)2Ni(II)2 complex shows a weak antiferromagnetic interaction between the adjacent Cu(II) and Ni(II) ions (-3.5 cm(-1)) and a weak ferromagnetic interaction between the two Ni(II) ions (+2.0 cm(-1)).

Journal Article↗
PubMed2002

Mechanistic insight from a volume profile for electron transfer between promazine and hexaaquairon(III).

The kinetics of the outer-sphere electron-transfer reaction between promazine (dimethyl-(3-phenothiazin-10-yl-propyl)-amine) and hexaaquairon(III) was studied using a high-pressure stopped-flow technique. The effect of pressure (over the range 0.1-130 MPa at 25 degrees C and ionic strength 1.0 M) on the reaction rate in aqueous perchloric acid solution resulted in volumes of activation of -6.2 +/- 0.4 and -12.5 +/- 0.5 cm(3) mol(-)(1) for the forward and reverse processes, respectively. The effect of pressure on the overall equilibrium constant revealed a reaction volume of +5.0 +/- 0.2 cm(3) mol(-)(1). The reported volume profile reveals mechanistic information on the electron-transfer process in terms of volume changes along the reaction coordinate. The volume of activation for the promazine/promazine(+*) self-exchange reaction was calculated on the basis of the Marcus cross relationship.

Journal Article↗
PubMed2004

Kinetics and mechanism of bromate-bromide reaction catalyzed by acetate.

The initial rate of the bromate-bromide reaction, BrO3- + 5Br- + 6H+ --> 3Br2 + 3H2O, has been measured at constant ionic strength, I = 3.0 mol L(-1), and at several initial concentrations of acetate, bromate, bromide, and perchloric acid. The reaction was followed at the Br2/Br3- isosbestic point (lambda = 446 nm) by the stopped-flow technique. A very complex behavior was found such that the results could be fitted only by a six term rate law, nu = k1[BrO3-][Br-][H+]2 + k2[BrO3-][Br-]2[H+]2 + k3[BrO3-][H+]2[acetate]2 + k4[BrO3-][Br-]2[H+]2[acetate] + k5[BrO3-][Br-][H+]3[acetate]2 + k6[BrO3-][Br-][H+]2[acetate], where k1 = 4.12 L3 mol(-3) s(-1), k2 = 0.810 L4 mol(-4) s(-1), k3 = 2.80 x 10(3) L4 mol(-4) s(-1), k4 = 278 L5 mol(-5) s(-1), k5 = 5.45 x 10(7) L6 mol(-6) s(-1), and k6 = 850 L4 mol(-4) s(-1). A mechanism, based on elementary steps, is proposed to explain each term of the rate law. This mechanism considers that when acetate binds to bromate it facilitates its second protonation.

Journal Article↗
PubMed1997

Multielectron Reduction of alpha-[S(2)Mo(18)O(62)](4-) in Aprotic and Protic Media: Voltammetric Studies.

Detailed voltammetric studies are reported of the reduction of alpha-[S(2)Mo(18)O(62)](4)(-) at glassy carbon and platinum macro- and microdisk electrodes (stationary and rotated) in acetonitrile and in 95/5 acetonitrile/water mixtures containing 0.02 M perchloric acid. Experiments at -30 degrees C and at short time scales in acetonitrile detect eight one-electron processes in the potential range 0.1 to -2.7 V versus Fc(+)/Fc (Fc = ferrocene). The eight processes fall into four pairs. The differences in potential within each pair are remarkably constant (0.26 +/- 0.02 V), as are the separations between adjacent pairs (0.60 +/- 0.06 V). This periodicity is consistent with spin-paired [Mo(V)O](2)(&mgr;-O) fragments providing a significant contribution to the overall superexchange stabilization. The influence of the following chemical reactions (primarily protonation by trace water) increases with the number of electrons added, although the five-electron reduced species [S(2)Mo(18)O(62)](9)(-) has a significant lifetime on the voltammetric time scale. In the presence of aqueous acid and at short time scales, eight chemically reversible processes are observed in the range 0 to -1.5 V in which two, two, two, two, four, four, two, and eight electrons, respectively, are transferred. The four pairs of one-electron processes found in acetonitrile occur as the first four overall two-electron reduction steps, separated by about 0.1, 0.2, and 0.3 V, respectively. Addition of a further 10 electrons occurs in a range of 0.3 V only and results in an 18-electron reduced species, stable on the voltammetric time scale. This corresponds to formal reduction of all Mo(VI) centers in alpha-[S(2)Mo(18)O(62)](4)(-) to Mo(V). The species is able to accept a further eight electrons in a series of unresolved processes.

Journal Article↗
PubMed1999

[Co(4)O(4)](4+) Cubane Core as a Brønsted Base: Preparation and Properties of [Co(4)O(3)(OH)(O(2)CR)(2)(bpy)(2)](3+) and [Co(4)O(2)(OH)(2)(O(2)CR)(2)(bpy)(2)](4+) Salts.

The preparation and properties are described of complexes resulting from the mono- and diprotonation of the [Co(4)O(4)](4+) unit to give products containing [Co(4)O(3)(OH)](5+) and [Co(4)O(2)(OH)(2)](6+), respectively. Treatment of [Co(4)O(4)(O(2)CC(6)H(4)-p-OMe)(2)(bpy)(4)](2+) (bpy = 2,2'-bipyridine) in MeCN with 70% perchloric acid leads to isolation of [Co(4)O(2)(OH)(2)(O(2)CC(6)H(4)-p-OMe)(2)(bpy)(4)](ClO(4))(4) (5) in 61% yield. Treatment of [Co(4)O(4)(O(2)CC(6)H(4)-p-Me)(2)(bpy)(4)](2+) in MeCN with an acidic solution of (NH(4))(3)[Ce(NO(3))(6)] leads to isolation of [Co(4)O(3)(OH)(O(2)CC(6)H(4)-p-Me)(2)(bpy)(4)][Ce(NO(3))(6)] (6) in 96% yield. Complex 5.3MeCN.H(2)O crystallizes in monoclinic space group C2/c with (at -155 degrees C) a = 33.838(8) Å, b = 13.826(3) Å, c = 29.944(7) Å, beta = 98.84(1) degrees, and Z = 8. Complex 6.PhCN.H(2)O crystallizes in orthorhombic space group Pbca with (at -154 degrees C) a = 22.603(4) Å, b = 34.759(6) Å, c = 18.167(3) Å, and Z = 8. The complexes contain [Co(4)O(2)(OH)(2)](6+) (5) and [Co(4)O(3)(OH)](5+) (6) distorted-cubane cores, the sites of protonation being apparent by (i) the approach of ClO(4)(-) or [Ce(NO(3))(6)](3)(-) anions to within hydrogen-bonding distances (O.O approximately 2.7 Å) and (ii) the longer Co-OH(-) bond lengths compared with Co-O(2)(-) bond lengths. Peripheral ligation is provided by chelating bpy and syn,syn-bridging RCO(2)(-) groups. The Co(III) atoms are six-coordinate and approximately octahedral. Solution studies show that the diprotonated species is a strong diprotic acid analogous to H(2)SO(4); the first deprotonation goes to completion, but the monoprotonated species is a weak acid and is in equilibirium with the nonprotonated species. pH studies in aqueous solution yield a pK(a2) value of 3.15 for the second deprotonation. Electronic and (1)H NMR spectra of the diprotonated complexes in various solvents are consistent with the monoprotonated complex being the major species in solution. The combined results demonstrate that the [Co(4)O(4)](4+) core is capable of acting as a Brønsted base, undergoing either one or two protonation reactions.

Journal Article↗
PubMed2001

Electron self-exchange in the solid-state: cocrystals of hydroquinone and bipyridyl triazole.

Solid-state voltammetry, spectroscopy, and microscopy studies have been used to probe the proton and electron conductivity within a self-assembled cocrystal, HQBpt. This crystallographically defined material contains 3,5-bis(pyridin-2-yl)-1,2,4-triazole, HBpt, dimers that are pi-stacked and hydrogen bonded to 1,4-hydroquinone, H(2)Q, in a herringbone arrangement. When deposited onto platinum microelectrodes, the cocrystal exhibits a well-defined voltammetric response corresponding to oxidation of H(2)Q to the quinone, Q, across a wide range of voltammetric time scales, electrolyte compositions, and pH values. Scanning electron microscopy reveals that redox cycling in aqueous perchlorate solutions in which the pH is systematically varied from 1 to 7 triggers electrocrystallization and the extensive formation of rodlike crystals. Fast scan rate voltammetry reveals that the homogeneous charge transport diffusion coefficient, D(app), is independent of the perchlorate concentration for 0.1 < [ClO(4)(-)] < 1.0 M (pH 6.6) at 3.14 +/- 0.11 x 10(-)(9) cm(2) s(-)(1). Moreover, D(app) is independent of the perchloric acid concentration for concentrations greater than approximately 2.0 M, maintaining a value of 4.81 +/- 0.07 x 10(-)(8) cm(2) s(-)(1). The observation that D(app) is independent of the supporting electrolyte suggests that the rate-determining step for homogeneous charge transport is not the availability of charge-compensating counterions or protons, but the dynamics of electron self-exchange between H(2)Q and Q. We have used the Dahms-Ruff formalism to determine electron self-exchange rate constants which are 2.84 +/- 0.22 x 10(9) and 9.69 +/- 0.73 x 10(10) M(-)(1) s(-)(1) for pH values greater than approximately 2.0 and less than -0.3, respectively. Significantly, these values are more than 2 orders of magnitude larger that those found for benzoquinone self-exchange reactions in aqueous solution. These results indicate that hydrogen bonds play an important role in supporting rapid electron transfer. The increase in D(app) between pH 1.0 and -0.3 is associated with protonation of the HBpt moieties, which triggers a reversible change in the material's structure.

Journal Article↗
PubMed2005

The chlorate-iodine clock reaction.

A clock reaction produced by mixing chlorate and iodine solutions in perchloric acid media is reported. This is the first example of a clock reaction using chlorate as a reagent. Increasing chlorate and acid concentration reduces the induction period. Changing the initial iodine concentration does not affect the length of the induction period. The discovery of this clock reaction opens the possibility that a new family of oscillation reactions can be built using chlorate as reagent.

Journal Article↗
PubMed2001

Fluoride content of foods made with mechanically separated chicken.

The goal of the present study was to determine the extent to which foods made with mechanically separated chicken can contribute to total fluoride intake. Fluoride content of each blended sample was determined with a fluoride combination electrode following perchloric-acid-facilitated diffusion of hydrogen fluoride. Infant foods had the highest fluoride content followed by chicken sticks, luncheon meats, and canned meats. A single serving of chicken sticks alone would provide about half of a child's upper limit of safety for fluoride. Fluoride content of foods made with mechanically separated chicken was significantly correlated with calcium content, which is consistent with the possibility that the mechanical separation process was the source of the extra fluoride. Foods made with mechanically separated turkey were not a major source of fluoride.

Animals↗
PubMed2005

Bioactive compounds in cod (Gadus morhua) products and suitability of 1H NMR metabolite profiling for classification of the products using multivariate data analyses.

This work investigates the suitability of (1)H NMR spectroscopy to identify the fate of some bioactive compounds in seafood submitted to several processing conditions and examines the possibility of using (1)H NMR spectroscopy profiling to classify such products. Perchloric acid extracts of cod white muscle from newly killed and (i) unprocessed, (ii) boiled, and (iii) fried fillets and from (iv) frozen fillets, (v) the frozen fillets after thawing, and (vi) their drip loss and from (vii) rehydrated cod klippfish (n = 5) were analyzed by 500 MHz (1)H NMR spectroscopy. It was possible to identify taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO) in all extracts examined, and frozen fish was recognizable by the presence of dimethylamine (DMA). None of the heating procedures seemed to induce the loss of bioactive compounds from the fillet, but freezing and thawing did: the compounds were lost in what is known as drip loss. About 80% of the samples were correctly classified using a probabilistic neural network procedure having as inputs the scores of the first 20 principal components of the principal component analysis of a selected region of the NMR spectra.

Animals↗
PubMed1999

Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes.

The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 degrees C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the reaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270-280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 degrees C and increasing the reaction time from 2 to 4 h allows complete lignin solubilization but minimizes degradation of the xylans.

Acetates↗
PubMed1999

Reversed phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products. 1. Moist snuff.

The official methods for the determination of nicotine in commercial tobacco products (AOAC, CORESTA) are based on approaches that are not selective for nicotine (colorimetric measurement, steam distillation, perchloric acid titration), and the availability of published methods based on state-of-the-art chromatographic methods is limited. Reversed phase ion-pair liquid chromatography has been established as a viable alternative for the analysis of basic analytes. A reversed phase ion-pair liquid chromatographic method for the determination of nicotine in commercial tobacco products was developed and optimized in separate experiments (Ciolino, L. A.; Turner, J. A.; McCauley, H. A.; Smallwood, A. W.; Yi, T. Y. J. Chromatogr. 1999a, 852 (2), 451-463). An extensive within-laboratory performance study of the optimized method was subsequently conducted, and results are presented here for the determination of nicotine in commercial moist snuff. Results for the determination of nicotine in commercial cigarettes are presented in a subsequent paper.

Chromatography, Liquid↗
PubMed2000

Phosphorylation of the 12S globulin from rapeseed (Brassica napus L.) by phosphorous oxychloride: chemical and conformational aspects.

The effect of progressive phosphorylation by phosphorous oxychloride upon the conformation of the 300 kDa storage protein (cruciferin) from rapeseed has been studied using chemical analysis, SDS-PAGE, HPLC, analytical ultracentrifugation, viscometry, fluorescence spectroscopy, and hydrophobicity measurement. The amount of phosphorous in the protein increased with the excess of phosphorous oxychloride and the pH of reaction. The bulk of phosphorus was only loosely bound to the protein and was removed by washing with cold perchloric acid. The more stably bound phosphorus groups after reaction at pH 8 were found to be nearly equally attached to amino and hydroxyl groups, whereas phosphorylation at pH 10-11 led to predominant O-phosphorylation as detected by studying the acid- and alkali-lability of the protein-phosphorous bonds. A 50 kDa component appeared as a product of covalent cross-linking of the constituent alpha- and beta-polypeptide chains. A 2.5S fraction appeared as the main product of dissociation, which takes place after a critical step of modification. The higher the extent of phosphorylation, the larger was the percentage of higher molecular weight products, the percentage of which was most significant after modification under strongly alkaline conditions. They may be attributed both to products of chemical cross-linking and to noncovalently linked aggregates formed by interactions of partially unfolded derivatives exhibiting an increased surface hydrophobicity.

Brassica↗