Superoxide dismutase targets NO from GSNO to Cysbeta93 of oxyhemoglobin in concentrated but not dilute solutions of the protein

The role of hemoglobin (Hb) in transmitting the vasodilatory property of NO throughout the vascular system is of much current interest. NO exchange between Hb and low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO) has been speculated and reported in vitro. Previously, we reported that NO delivery from GSNO to Cysbeta93 of human oxyHb is prevented in the presence of the Cu chelators, neocuproine, and DTPA.(1) In the present work, 5 mM solutions of commercial human Hb were found by ICP-MS to contain approximately 20 microM Cu and Zn, suggesting the presence of Cu,Zn-superoxide dismutase (CuZnSOD), which was confirmed by Western blotting. SOD activity measurements were consistent with the presence of approximately 20 microM CuZnSOD monomer in 5 mM Hb solutions, which is the physiological concentrations of these proteins in the red blood cell. Incubation of 3.75 mM oxyHb (15 mM heme; 7.5 mM Cysbeta93) with 3.75 or 7.5 mM GSNO gave rise to 50% or 100% S-nitrosation, respectively, of Cysbeta93 as monitored by FTIR nu(SH) absorption, whereas excess GSNO over Cysbeta93 converted oxyHb to metHb due to the reaction, oxyHb + NO<==>metHb + NO(3)(-). Removal of CuZnSOD by anion-exchange chromatography yielded an oxyHb sample that was unreactive toward GSNO, and replacement with bovine CuZnSOD restored reactivity. Addition of 1 microM GSNO (Cysbeta93/GSNO = 1) to solutions diluted 10(4)-fold from physiological concentrations of oxyHb and CuZnSOD resulted largely in metHb formation. Thus, this work reports the following key findings: CuZnSOD is an efficient catalyst of NO transfer between GSNO and Cysbeta93 of oxyHb; metHb is not detected in oxyHb/GSNO incubates containing close to the physiological concentration (5 mM) of Hb and CuZnSOD when the Cysbeta93/GSNO molar ratio is 0.5 to 1.0, but metHb is detected when the total Hb concentration is low micromolar. These results suggest that erythrocyte CuZnSOD may play a critical role in preserving the biological activity of NO by targeting it from GSNO to Cysbeta93 of oxyHb rather than to its oxyheme.     

Constituents of microbial iron chelators. The synthesis of optically active derivatives of δ-N-hydroxy-L-ornithine.

δ-N-Hydroxy-L-ornithine derivatives were synthesized from L-glutamic acid by reduction of the γ-acid chloride to the aldehyde, formation of the substituted oxime, and reductive acylation.     

Activity of artificial mutant variants of human growth hormone deficient in a disulfide bond between Cys53 and Cys165

In order to understand the role of Cys53 and Cys165 of human growth hormone (hGH) in receptor-binding and biological activity, artificial mutant variants of hGH were prepared in Escherichia coli by in vitro mutagenesis. Variants of hGH were constructed by replacement of Cys165 with Ala ([Ala165]hGH) or Ser ([Ser165]hGH), by replacement of Cys53 with Ala ([Ala53]hGH), by replacement of Cys53 and Cys165 with Ala ([Ala53, Ala165]hGH), or by replacement of Cys53 with Ala and Cys165 with Ser ([Ala53,Ser165]hGH). All of the variants constructed as well as reduced hGH exhibited less biological activity than that of intact hGH, and the decreases in biological activity were almost equal, as measured by a sensitive biological assay for growth hormone: adipose conversion assay using 3T3-F442A cells. These variants also showed less receptor-binding activity than that of intact hGH. These results suggest that it is possible neither the residue Cys53 nor Cys165 is directly involved in the receptor binding, and that the disulfide bridge between Cys53 and Cys165 in hGH may not always be crucial for the biological activity, though necessary to express full hGH activity.     

Recent advances in therapeutical applications of the versatile hydroxypyridinone chelators

Journal of Inclusion Phenomena and Macrocyclic Chemistry - Hydroxypyridinones (HOPOs) are excellent class of chelators that have been gaining attention in the field of pharmaceutical drugs by their...     

猴金属硫蛋白MT1 α-结构域中Cys33和Cys34的突变对蛋白质稳定性的影响

重组猴金属硫蛋白MT1及其C33M,C34S和C33M/C34S突变体蛋白的蛋白质快速液相色谱(FPLC)分析表明,突变体蛋白C34S和C33M/C34S存在着两个稳定的流分f1和f2.CD光谱和重金属离子分析表明,突变体蛋白多流分不仅与半胱氨酸和Cd2+离子结合方式和构型有关,还与金属结合量有关.突变体蛋白多流分的结果表明,在把猴金属硫蛋白α-结构域中的Cys33和Cys34突变为非半胱氨酸残基以建立β-β结构域的金属硫蛋白时,可导致蛋白形成多种结构形式.这表明α-结构域在稳定金属硫蛋白的结构中起着重要的作用,半胱氨酸残基在肽链上的分布对金属硫蛋白的三级结构和金属硫簇的形成有重大的影响.     

Research in the imidazole series. 93. Synthesis of derivatives of benzimidazolyl-1-acetic acid

A simple method has been developed for obtaining esters of benzimidazolyl-1-acetic acid and its 2-alkyl (or aralkyl) derivatives, by reaction of benzimidazoles with esters of chloroacetic or bromoacetic acid in DMF in the presence of anhydrous potassium carbonate. The method of synthesis of amides of these acids has been simplified.For Communication 92, see [1].Translated from Khimiya Geterotsiklicheskikh Soedinenii, Vol. 29, No. 5, pp. 656–658, May, 1993.     

Reactions of Metal Salts and Alkali Metal Nitrates. Role of the Metal Precursors and Alkali Metal Ions in the Resulting Phase of Zirconia

Zirconia powders are prepared by reaction of a zirconium precursor with an alkali metal nitrate. The major part of the reactions takes place before the melting points and then the reactions go slowly to completion at 450°C in the molten salts. The roles of the precursor and the alkali metal ion are discussed considering the reaction between two precursors, octahydrated zirconium oxychloride and zirconium tetrachloride, and two nitrates, LiNO₃ and NaNO₃, and some resulting physico-chemical differences. The obtained zirconia powders contain very small amounts of alkali metal ions which act as stabilizing agent. Their effect on the balance tetragonal-monoclinic ZrO₂ depends upon the homogeneity of their distribution which is related to their ability to diffuse inside the bulk of particles and their polarizing power when located mainly on the surface.     

Reversed-phase liquid chromatography of biologically active lipophilic chelators. II. Improvement of chromatographic performance and selected applications in biochemical analysis

Lipophillic derivatives of strong calcium chelator BAPTA--DP-b99 and DP-109--are potential drug substances for cerebrovascular and neurodegenerative diseases. The previously published reversed phase HPLC methods for these compounds suffered from integration problems due to gradient dip, insufficient repeatability and peak shape. A C4 column rapidly aged. The addition of acetic acid to the organic part and of ammonium acetate to the aqueous part results in more symmetric peaks, improves method precision and solves integration problems. Washing the column in both directions with a combination of methanol, tetrahydrofuran and water extends its use. The improved methods are sensitive, selective, reproducible, and stability indicating. Impurities and degradation products were identified by LC-MS. Versatile detection techniques can be used with these HPLC methods, allowing performance of bioanalysis with mass spectrometric or flow scintillation detectors. The bioanalytical application of these methods is illustrated by examples of pharmacokinetic and metabolic studies with the labeled compounds.     

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.     

Metal cation dependence of interactions with amino acids: bond energies of Cs+ to Gly, Pro, Ser, Thr, and Cys

The interactions of cesium cations with five amino acids (AA) including glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), and cysteine (Cys) are examined in detail. Experimentally, the bond dissociation energies (BDEs) are determined using threshold collision-induced dissociation of the Cs(+)(AA) complexes with xenon in a guided ion beam tandem mass spectrometer. Analyses of the energy-dependent cross sections include consideration of unimolecular decay rates, internal energy of the reactant ions, and multiple ion-neutral collisions. Bond dissociation energies (0 K) of 93.3 ± 2.5, 107.9 ± 4.6, 102.3 ± 4.1, 105.4 ± 4.3, and 96.8 ± 4.2 kJ/mol are determined for complexes of Cs(+) with Gly, Pro, Ser, Thr, and Cys, respectively. Quantum chemical calculations are conducted at the B3LYP, B3P86, MP2(full), and M06 levels of theory with geometries and zero-point energies calculated at the B3LYP level using both HW*/6-311+G(2d,2p) and def2-TZVPPD basis sets. Results obtained using the former basis sets are systematically low compared to the experimental bond energies, whereas the latter basis sets show good agreement. For Cs(+)(Gly), theory predicts the ground-state conformer has the cesium cation binding to the carbonyl group of the carboxylic acid. For Cs(+)(Pro), the secondary nitrogen accepts the carboxylic acid hydrogen to form the zwitterionic structure, and the metal cation binds to both oxygens. Cs(+)(Ser), Cs(+)(Thr), and Cs(+)(Cys) are found to have tridentate binding at the MP2(full) level, whereas the density functional approaches slightly prefer bidentate binding of Cs(+) at the carboxylic acid moiety. Comparison of these results to those for the smaller alkali cations provides insight into the trends in binding affinities and structures associated with metal cation variations.     

Role of organic acid chelators in manganese regulation of lignin degradation byPhanerochaete chrysosporium

Applied Biochemistry and Biotechnology - Nitrogen, carbon, and manganese are potent regulators of lignin degradation, but although nitrogen and carbon elicit a generalizated response when cells are...