Determination of the primary structures of heparin- and heparan sulfate-derived oligosaccharides using band-selective homonuclear-decoupled two-dimensional 1H NMR experiments.

Band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments are applied to the assignment of 1H NMR spectra of oligosaccharides, using as an example a heparin-derived hexasaccharide. The anomeric (H1) region of the 1H NMR spectrum is band-selected in the F1 dimension. With the increased resolution that results from less truncation of interferograms in the t1 dimension, finer digital resolution in the F1 dimension, and collapse of multiplets to singlets in the F1 dimension, cross-peaks to the anomeric protons of the two iduronic acid residues, which overlap in normal two-dimensional total correlation spectroscopy (TOCSY) and rotating frame Overhauser enhancement spectroscopy (ROESY) spectra of the hexasaccharide, are resolved in BASHD-TOCSY and BASHD-ROESY spectra, leading to an unequivocal assignment of the 1H NMR spectrum of the hexasaccharide. Incorporation of the water attenuation by transverse relaxation method for the complete and selective elimination of the water resonance into two-dimensional BASHD experiments makes it possible to observe oligosaccharide resonances at the frequency of the water resonance, as demonstrated with the observation of cross-peaks to resonances at the frequency of the water resonance in BASHD-TOCSY spectra of a second heparin-derived hexasaccharide.

An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides.

The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin.

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.

A cleavage cocktail for methionine-containing peptides.

A new cocktail has been developed for cleavage and deprotection of methionine-containing peptides synthesized by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis methodology. The cocktail (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1,2-ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide 1.5% w/w) was designed to minimize methionine side-chain oxidation. Application of the new cocktail (Reagent H) is demonstrated with the synthesis of a model pentadecapeptide from the active site of DsbC, a periplasmic protein involved in protein disulphide bond formation. The model peptide, which contains one methionine and two cysteine residues, was cleaved with several cleavage cocktails, including Reagent H. The crude peptides obtained with the widely used cocktails K, R and B were found to be 15% to 55% in the methionine sulphoxide form, whereas no methionine sulphoxide was detected in the crude peptide obtained by cleavage and deprotection with Reagent H. Also, no methionine sulphoxide was detected when 1.5% w/w NH4I was added to cocktails K, R and B; however, the yield of the desired peptide was less than with Reagent H. A second 28 amino acid model peptide of the active site of DsbC was also cleaved and deprotected with Reagent H. The reduced dithiol form of the peptide was found to be the major component (51% yield) of the crude peptide obtained by cleavage for 3 h. When the cleavage time was extended to 10 h, the peptide was converted to the intramolecular disulphide form (35% yield). A proposed mechanism for the in situ oxidation of cysteine with Reagent H is presented.

Stability and structure-forming properties of the two disulfide bonds of alpha-conotoxin GI.

alpha-Conotoxin GI is a 13 residue snail toxin peptide cross-linked by Cys2-Cys7 and Cys3-Cys13 disulfide bridges. The formation of the two disulfide bonds by thiol/disulfide exchange with oxidized glutathione (GSSG) has been characterized. To characterize formation of the first disulfide bond in each of the two pathways by which the two disulfide bonds can form, two model peptides were synthesized in which Cys3 and Cys13 (Cono-1) or Cys2 and Cys7 (Cono-2) were replaced by alanines. Equilibrium constants were determined for formation of the single disulfide bonds of Cono-1 and Cono-2, and an overall equilibrium constant was measured for formation of the two disulfide bonds of alpha-conotoxin GI in pH 7.00 buffer and in pH 7. 00 buffer plus 8 M urea using concentrations obtained by HPLC analysis of equilibrium thiol/disulfide exchange reaction mixtures. The results indicate a modest amount of cooperativity in the formation of the second disulfide bond in both of the two-step pathways by which alpha-conotoxin GI folds into its native structure at pH 7.00. However, when considered in terms of the reactive thiolate species, the results indicate substantial cooperativity in formation of the second disulfide bond. The solution conformational and structural properties of Cono-1, Cono-2, and alpha-conotoxin GI were studied by 1H NMR to identify structural features which might facilitate formation of the disulfide bonds or are induced by formation of the disulfide bonds. The NMR data indicate that both Cono-1 and Cono-2 have some secondary structure in solution, including some of the same secondary structure as alpha-conotoxin GI, which facilitates formation of the second disulfide bond by thiol/disulfide exchange. However, both Cono-1 and Cono-2 are considerably less structured than alpha-conotoxin GI, which indicates that formation of the second disulfide bond to give the Cys2-Cys7, Cys3-Cys13 pairing induces considerable structure into the backbone of the peptide.

Quantitative characterization of the binding of histamine by heparin.

Tissue histamine is stored in mast cell granules, presumably as a histamine-heparin complex. Heparin is a polyelectrolyte, with a fraction of its anionic charge neutralized by condensed counterions. The interaction of heparin with histamine in aqueous solution was quantitatively characterized by 1H nuclear magnetic resonance (NMR) spectroscopy. Binding constants were determined from chemical shift-pH titration data for the C2H proton of the imidazolium ring for a wide range of histamine, heparin, and Na+ concentrations. The results indicate a binding stoichiometry of 1 histamine per heparin disaccharide repeat unit. The binding is electrostatic, as indicated by the strong dependence of the binding constant on Na+ concentration. From an analysis of the binding constants using the counterion condensation theory of polyelectrolytes, it was determined that the binding of H2A2+ results in displacement of 1.72 Na+ ions from the counterion condensation volume of heparin and that H2A2+ makes 2 ionic interactions with heparin. The displacement of Na+ from the counterion condensation volume of heparin by H2A2+ was also studied by 23Na NMR. From 23Na spin-lattice relaxation time data, it was determined directly that 1.78 Na+ ions are displaced per H2A2+ bound by heparin. The results are discussed in terms of the ion exchange process which takes place when histamine is released by mast cells.

Characterization of the thiol/disulfide chemistry of peptides corresponding to the 603-609 disulfide loop of the human immunodeficiency virus (HIV) envelope glycoprotein gp41.

The redox chemistry of two synthetic model peptides for the 603-609 disulfide loop found in envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) are reported. The two peptides: N-Ac-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys-Thr-Thr-NH2 (I) and N-Ac-Trp-Gly-Cys-Ser-Gly-Arg-His-Ile-Cys-Thr-Thr-NH2 (II) were synthesized by the solid phase method. Peptide I corresponds to amino acids 601-611 of gp41 of the North American/European strain of HIV-1. Peptide II incorporates amino acid replacements frequent in African HIV-1 isolates. The redox chemistry of the disulfide bonds in the two peptides was characterized in aqueous and aqueous/urea solution by studying their thiol-disulfide exchange reactions with the tripeptide glutathione (GSH). GSH reacts with the disulfide bonds to form mixed disulfides, which in turn react with another molecule of GSH to give the dithiol form of the peptide and GSSG. Equilibrium constants were determined for each step and for the overall reduction reactions. Redox potentials of -0.246V and -0.241V were calculated from the equilibrium constants for the disulfide bonds in peptides I and II in aqueous solution at 25 degrees C and pH 7.0. The overall equilibrium constants are less in 8 M urea solution, which indicates a stabilization of the reduced, dithiol form of both peptides by secondary structure which can be denatured by urea. This conclusion is supported by nuclear Overhauser enhancement data obtained from 2D-ROESY NMR spectra which provide evidence of elements of secondary structure for the reduced forms of both peptides. The results are discussed in terms of a protein disulfide isomerase catalyzed reduction of the disulfide bond in gp41.

Determination of acid dissociation constants of peptide side-chain functional groups by two-dimensional NMR.

An NMR method is described for determining residue-specific acid dissociation constants for peptides which contain more than one residue of the same acidic or basic amino acid. The method is based on using the differences in C alpha H proton chemical shifts which result from peptide sequence nearest-neighbor and possibly secondary structure effects to resolve resonances for carbon-bonded reporter protons adjacent to each side-chain acidic group in two-dimensional total correlation spectroscopy (TOC-SY) spectra. Acid dissociation constants were determined for each of the four lysine side-chain ammonium groups of the peptide Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2. Resonances for the C epsilon H2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the C alpha H-C epsilon H2 cross peaks in the TOCSY spectrum. Chemical shift-pH titration data were obtained for each lysine side-chain ammonium group from one-dimensional subspectra taken from two-dimensional TOCSY spectra measured as a function of pH. The pKAs of the Lys1, Lys5, Lys13, and Lys14 side-chain ammonium groups were determined to be 11.14 +/- 0.01, 10.95 +/- 0.01, 10.96 +/- 0.02, and 11.09 +/- 0.02, respectively. The chemical shift-pH titration data were also analyzed by a pH-independent procedure to obtain relative acid dissociation constants: KA(Lys1)/KA(Lys5) = 0.663 +/- 0.009, KA(Lys1)/KA(Lys13) = 0.703 +/- 0.014, and KA(Lys1)/KA(Lys14) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys1, Lys5, Lys13, and Lys14 of 1:1.508:1.422:1.099. To further demonstrate the utility of this method, acid dissociation constants were determined for the six acidic groups of the peptide Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2. Chemical shift-pH titration curves were obtained for the C beta H2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH. The pKAs of the CyS3 and Cys13 thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively. The relative acid dissociation constants (KA(Cys3)/ (KA(Cys13)) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.

Speciation and quantitation of underivatized and Ellman's derivatized biological thiols and disulfides by capillary electrophoresis.

Capillary electrophoresis (CE) methods have been developed for the speciation and quantitation of thiols and disulfides of biological interest, including the endogenous compounds glutathione, glutathione disulfide, cysteine, cystine, homocysteine, and homocystine and the therapeutic agents penicillamine, penicillamine disulfide, N-acetylcysteine, and captopril. Good speciation and quantitation were achieved for the underivatized thiols and disulfides using a detection wavelength of 200 nm; detection limits were in the range 20-90 microM (1-4 pmol) using a 50-microns-diameter capillary. To achieve lower detection limits, thiols were derivatized with the thiol-specific probe molecule, 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent). Good speciation and quantitation were achieved for the Ellman's derivatized thiols using a detection wavelength of 357 nm; detection limits were in the range 5-50 microM (0.03-0.3 pmol) using a 25-microns-diameter capillary. Both the underivatized and derivatized methods were applied to the determination of glutathione in human erythrocytes. Glutathione concentrations of 2-3 mM were obtained for the erythrocyte samples analyzed, with good agreement between results obtained by the two methods.

Multinuclear magnetic resonance studies of the interaction of inorganic cations with heparin.

The interaction of Na+, Ca2+, Mg2+, Zn2+ and La3+ with heparin, a highly negatively charged glycosaminoglycan, was studied by 1H and 23Na nuclear magnetic resonance spectroscopy. 1H chemical shift and nuclear Overhauser effect (NOE) data indicate that the counter ions Na+, Ca2+ and Mg2+ interact with the low pH, carboxylic acid form of heparin by delocalized, long-range electrostatic interactions. At higher pH, 1H chemical shift and NOE data indicate that Na+ and Mg2+ continue to interact with heparin in the same manner, even upon deprotonation of the carboxylic acid group; however, there is a site-specific contribution to the binding of Ca2+, Zn2+ and La3+ under these conditions. Acid dissociation constants for heparin carboxylic acid groups and heparin-metal binding constants were determined from the pH dependence of 1H chemical shifts and 23Na spin-lattice (T1) relaxation times. Equilibrium constants for exchange of M2+ for heparin-bound Na+ were obtained from 23Na T1 data. The acid dissociation constants show a strong dependence on Na+ concentration due to the polyelectrolyte character of heparin.

Binding of the growth factor glycyl-L-histidyl-L-lysine by heparin.

Evidence is presented that the growth factor glycyl-histidyl-lysine (GHK) binds to heparin, and the interaction has been characterized by [1H]NMR spectroscopy. 1H chemical shifts indicate that GHK interacts with both the carboxylic acid and the carboxylate forms of heparin. The chemical shift data are consistent with a weak delocalized binding of the triprotonated (ImH+, GlyNH3+, LysNH3+) form of GHK by the carboxylic acid form of heparin. As the pD is increased and the carboxylic acid groups are titrated, chemical shift data indicate that ammonium groups of GHK are hydrogen bonded to heparin carboxylate groups, while the histidyl imidazolium ring occupies the imidazolium-binding site of heparin. Evidence for site-specific binding includes displacement of chemical shift titration curves for heparin to lower pD, increased shielding of specific heparin protons by the imidazolium ring current and displacement of chemical shift titration curves for GHK to higher pD. Specific binding constants were determined for binding of the (ImH+, GlyNH3+), LysNH3+) forms of GHK by the carboxylate form of heparin from chemical shift vs. pD titration data.

Nuclear magnetic resonance study of the thioltransferase-catalyzed glutathione/glutathione disulfide interchange reaction.

The kinetics of the thioltransferase-catalyzed symmetrical glutathione/glutathione disulfide (GSH/GSSG) interchange reaction have been studied by 1H-nuclear magnetic resonance spectroscopy. Kinetic parameters were determined by analysis of exchange-broadened multiplet patterns and by the inversion-magnetization transfer method using concentrations of GSH, GSSG and pig liver thioltransferase similar to intracellular concentrations. The rate constant for the reaction of GSSG with thioltransferase to form a thioltransferase-glutathione mixed disulfide and GSH was estimated to be > or = 7.1(+/- 0.4).10(5) M-1 s-1. This reaction is proposed to be the first step in the mechanism by which the activity of some proteins is modulated by the thioltransferase-catalyzed formation of protein-glutathione mixed disulfides. The rate constant for the reaction of GSSG with thioltransferase is 4-5 orders of magnitude larger than rate constants for the analogous reaction of the thiolate groups of a variety of small molecules with GSSG. The symmetrical gamma-L-glutamyl-L-cysteine/gamma-L-glutamyl-L-cysteine disulfide (GCSH/GCSSCG), L-cysteinyl-glycine/L-cysteinyl-glycine disulfide (CGSH/CGSSGC) and cysteine/cystine (CSH/CSSC) thiol/disulfide interchange reactions were also studied as models for the GSH/GSSG interchange reaction. The GCSH/GCSSCG interchange reaction was found to be catalyzed by thioltransferase, and the rate constant for the reaction of GCSSCG with thioltransferase was estimated to be > or = 5.7(+/- 1.7).10(4) M-1 s-1. In contrast, the CGSH/CGSSGC and CSH/CSSC interchange reactions were found to be slow on the NMR time-scale for the conditions used in this research, both in the absence and presence of thioltransferase. The results suggest that the gamma-L-glutamyl-L-cysteinyl moiety of GSSG and of GSH-containing mixed disulfides is essential for their recognition by thioltransferase.

Characterization of the thiol/disulfide chemistry of neurohypophyseal peptide hormones by high-performance liquid chromatography.

Methodology is described for characterization of the kinetics and equilibria of thiol/disulfide interchange reactions of the disulfide bonds in the neurohypophyseal peptide hormones arginine vasopressin and oxytocin and the related peptides pressinoic acid and tocinoic acid. Thiol/disulfide interchange reaction mixtures are analyzed by reversed-phase high-performance liquid chromatography. The effect of mobile-phase composition and pH on the HPLC capacity factors for the native disulfide and reduced dithiol forms of each peptide was examined. In each case, the capacity factor decreases as the acetonitrile content of the mobile phase increases. For each disulfide/dithiol peptide pair, the capacity factor is larger for the dithiol form of the peptide, indicating that the hydrophobic side chains of the linear peptide are more accessible for interaction with the hydrophobic stationary phase. To illustrate application of the methodology, rate and equilibrium constants are reported for the thiol/disulfide interchange reactions of cysteine with arginine vasopressin at pH 7.0. Cysteine reacts with arginine vasopressin to form two mixed disulfides, which in turn react with another molecule of cysteine to give the dithiol form of arginine vasopressin and cystine. Rate and equilibrium constants were determined for each step by analysis of reaction mixtures by HPLC. The results are compared to rate and equilibrium constants for reaction of cysteine with oxidized glutathione.

Nuclear magnetic resonance studies of the binding of captopril and penicillamine by serum albumin.

The metabolism of the thiol-containing drugs penicillamine (beta,beta-dimethylcysteine) and captopril (D-3-mercapto-2-methylpropanoyl-L-proline) involves the formation of mixed disulfides, including mixed disulfides with serum albumin. The reactions of penicillamine and captopril with serum albumin in aqueous solution and in intact human blood plasma have been studied by 500 MHz 1H NMR spectroscopy. Penicillamine was found to react rapidly at the albumin-cysteine mixed disulfide bond to form penicillamine-cysteine mixed disulfide and to react more slowly at other albumin disulfide bonds. The amino acid cysteine was found to react with albumin by the same two pathways. In contrast, captopril rapidly associates with albumin to form noncovalent albumin-captopril complexes. Exchange of captopril between its free and noncovalently bound forms takes place on the NMR time scale. On a longer time scale, captopril reacts with albumin by thiol/disulfide interchange reactions. Noncovalently bound captopril displaced lactate from its albumin binding sites, both in aqueous solution and in human plasma. The results demonstrate that 1H NMR is a useful method for characterizing the state of drug molecules in human plasma and for detecting and monitoring perturbations by drugs of delicately balanced binding equilibria involving endogenous small molecules and macromolecules in plasma.

Indirect detection of mercury-199 nuclear magnetic resonance spectra of methylmercury complexes.

Measurement of 199Hg NMR spectra of methylmercury species by the heteronuclear multiple quantum coherence (HMQC) indirect proton detection method and its application to the study of the solution chemistry of CH3HgII-thiol ligand complexes are described. A sensitivity enhancement factor of 16 is obtained for measurement of the 199Hg NMR spectrum of a 4 mM solution of the CH3HgII-glutathione complex by the indirect detection method, as compared to a theoretical enhancement of 74. The less-than-theoretical enhancement is attributed to loss of signal by relaxation during the HMQC pulse sequence. Longitudinal relaxation of the 199Hg in CH3HgII-thiolate complexes is fast at the field strength used in this research (11.7 T) due to efficient relaxation by the chemical shift anisotropy mechanism. This in turn causes the transverse relaxation rate for the 199Hg spin-coupled methyl protons to be fast due to efficient relaxation by another mechanism, scalar relaxation of the second kind. Depending on the rate of exchange of CH3HgII among its complexed forms, the 199Hg line width can also include a large contribution from exchange broadening. In such cases, it is shown that extremely broad 199Hg resonances, which would be difficult to detect by direction observation, can be observed by the indirect detection method. For example, 199Hg resonances with exchange-broadened line widths up to 8000 Hz are observed by the indirect detection method for CH3HgII in mixtures of CH3HgOD and the CH3HgII-mercaptoethanol or CH3HgII-glutathione complex. The chemical shift of 199Hg in CH3HgII-thiolate ligand complexes is found to be extremely sensitive to the nature of the thiolate ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

1H-nuclear magnetic resonance study of the oxidation/reduction chemistry of penicillamine in intact human erythrocytes.

The oxidation/reduction chemistry of penicillamine in human erythrocytes was characterized directly in intact erythrocytes by 1H-NMR spectroscopy. Spectra were measured by the Carr-Purcell-Meiboom-Gill pulse sequence to selectively eliminate interfering resonances from hemoglobin and membrane protons and from the intracellular water. Glucose-free penicillamine-containing erythrocytes were subjected to oxidative stress by titration with t-butyl hydroperoxide. The t-butyl hydroperoxide rapidly crosses the erythrocyte membrane and reacts with glutathione (GSH) and penicillamine (PSH), with the PSH being oxidized to penicillamine-glutathione mixed disulfide (PSSG) as indicated by the appearance of characteristic resonances in the high resolution 1H-NMR spectrum. Extracellular PSH is oxidized to penicillamine disulfide (PSSP) when t-butyl hydroperoxide is added to the cells and thereafter in amounts dependent on the oxygenation state of the sample. Following addition of glucose, both the oxidized glutathione and the PSSG are rapidly reduced at comparable rates. The results of additional experiments using erythrocyte lysate and of kinetic experiments on solutions containing PSSG and/or GSH, NADPH and glutathione reductase suggest that the predominant mechanism for reduction of PSSG is by a thiol-disulfide exchange reaction with GSH to form PSH and GSSG, which in turn undergoes enzyme-catalyzed reduction by NADPH.

A quantitative study of the complexation of cadmium in hemolyzed human erythrocytes by 1H NMR spectroscopy.

The stability of complexes formed by Cd2+ in hemolyzed human erythrocytes was studied by spin-echo 1H NMR spectroscopy. Changes in resonances for the carbon-bonded protons of glutathione (GSH) upon addition of the ethylenediaminetetraacetic acid complex of Cd2+ (Cd(EDTA)2-) and the appearance of resonances for Mg(EDTA)2- indicate that the Cd(EDTA)2- complex dissociates in hemolyzed erythrocytes with the formation of Cd(GSH)x and Mg(EDTA)2- complexes. A semiquantitative estimate of the overall stability constant for the complexation of Cd2+ in hemolyzed erythrocytes was obtained from spin-echo 1H NMR data. The stability constant is consistent with the majority of the Cd2+ in erythrocytes present as Cd(SG)2(2-). A conditional equilibrium constant was also determined for the complexation of Mg2+ by ligands in hemolyzed human erythrocytes.

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands.

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).

Determination of homocysteine, penicillamine, and their symmetrical and mixed disulfides by liquid chromatography with electrochemical detection.

A method for the determination of D-penicillamine, homocysteine, homocystine, penicillamine-homocysteine mixed disulfide, and penicillamine disulfide in human plasma and urine is described. The method involves separation of the various thiols and disulfides by high-performance liquid chromatography with detection by a dual Hg/Au amalgam electrochemical detector. D-Penicillamine and homocysteine are detected at the downstream electrode; the disulfides are first reduced to thiols at the upstream electrode and then the thiols are detected at the downstream electrode. Hydrodynamic voltammograms were measured for the various thiols and disulfides to determine optimum settings for the electrochemical detector, and the effect of mobile phase parameters on retention times was studied to optimize the separation. A convenient method for the preparation of calibration solutions of penicillamine-homocysteine mixed disulfide by thiol/disulfide exchange with standardization of the solution by H NMR spectroscopy is described. Detection limits are below the concentrations of homocystine and penicillamine-homocysteine mixed disulfide reported to be present in the plasma and urine, respectively, of homocystinuric patients under treatment with D-penicillamine.