Dipyridyl Disulfide Research Articles

Evidence for the chemical activation of essential cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans.

Nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans which catalyzes the irreversible oxidation of D-glyceraldehyde-3 phosphate (D-G3P) into 3-phosphoglycerate (3-PGA) in the presence of NADP belongs to the aldehyde dehydrogenase (ALDH) superfamily. Oxidation of D-G3P into 3-PGA by GAPN involves the formation of a covalent enzyme intermediate via the nucleophilic attack of the invariant Cys-302. Titration of Cys-302 in the apo-enzyme by two different kinetic probes, iodoacetamide and 2,2'-dipyridyl disulfide, shows a pK(app) of 8.5 and a chemical reactivity surprisingly low compared to a reactive and accessible thiolate. Binding of NADP causes a strong increase of the reactivity of Cys-302-which is time dependent-with a pK(app) shift from 8.5 to 6.1. Concomitant with the increase in the Cys-302 reactivity, an additional protein fluorescence quenching is observed. These data suggest that cofactor binding induces at least a local conformational rearrangement within the active site. The efficiency of the rearrangement depends on the structure of the cofactors and on the protonation of an amino acid with a pK(app)( )()of 5.7. The rate of the rearrangement also strongly increases when temperature decreases. The data on the conformational rearrangement also reveal an amino acid with a pK(app) of 7.6 whose deprotonation increases the reactivity of the thiolate of Cys-302 by a 3-fold factor. The nature of the amino acid involved-which should be located close to Cys-302 in the holo-active form-is likely the invariant Glu-268. Changing Glu-268 into Ala or Cys-302 into Ala leads to mutants in which the rearrangement is only efficient in the presence of saturating concentrations of both NADP and G3P. The structural aspects of the conformational rearrangement occurring during the catalytic process in the wild-type GAPN should include at least reorientation of both Cys-302 and Glu-268 side chains and repositioning of the nicotinamide ring of the cofactor to permit the chemical activation of Cys-302 and the formation of an efficient ternary complex. Thus, it is likely that the conformation of the active site in the reported X-ray structures of ALDHs determined so far in the presence of cofactor, in which the side chains of Cys-302 and Glu-268 are 6.7 A apart from each other, does not represent the biological active form.

2,2′-Bispyridyl disulfide rapidly induces intramolecular disulfide bonds in peptides

A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2′-bispyridyl disulfide (2,2′-dipyridyl disulfide or 2,2′-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.

A new fluorogenic reagent for carboxylic acids, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH), derived from an empirical method for predicting fluorescence characteristics

In our previous studies, we found a relationship between the fluorescence characteristics (the fluorescence intensity, the maximum excitation and emission wavelengths) and Hammett substituent constants (ςp) of the substituent groups at the 4- and 7-positions of benzofurazan compounds, which enabled us to predict the fluorescence characteristics of the benzofurazan compounds. This prompted us to design a new fluorogenic derivatization reagent having a benzofurazan skeleton for carboxylic acids along this line of thought. Accordingly, the fluorogenic reagent, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) was synthesized. The reagent itself fluoresced very weakly but fluoresced strongly after the reaction with carboxylic acids. AABD-SH reacted with carboxylic acids within 5 min at room temperature in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide. The derivatives of five carboxylic acids were separated on a reversed phase column and detected fluorimetrically at 524 nm with excitation at 368 nm. The detection limits were 10–20 fmol (signal-to-noise ratio of 3).

A Simple Preparation of 3-(2-Pyridyldithio)-Propionic Acid

The two-step synthesis of 3-(2-pyridyldithio)propionic acid is described, which includes the chlorination of 2,2′-dipyridyl disulfide with sulfuryl chloride and the reaction of the formed 2-pyridylsulfenyl chloride with 3-mercaptopropionic acid.

Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions.

Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine disulfide (Cys73-Cys120). A highly reactive aromatic disulfide, 2,2'-dipyridyl disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed disulfide protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of conformational fluctuation in ovalbumin was investigated at pH 2.2 using the mixed disulfide derivatives of the cystine-intact and cystine-reduced protein forms. In the presence of a high concentration of urea, both the mixed disulfide derivatives underwent rapid cysteine sulfhydryl/mixed disulfide exchanges, thereby releasing the quantitative amount of 2-thiopyridone. A peptide mapping analysis for disulfide-forming cysteines revealed that this release was mostly accounted for by the nucleophile attack on the Cys367-mixed disulfide by the nearest cysteine residue in the primary structure, Cys382. At the acidic pH, the exchange reaction was practically restricted to the cysteine sulfhydryl/mixed disulfide exchanges; no other exchange reaction, such as the cysteine sulfhydryl/cystine disulfide exchange reaction, was detected. In the absence of urea, the cystine-reduced form, but not the cystine-intact form, underwent significant sulfhydryl/mixed disulfide exchange reactions at a physiological temperature, as determined by the release of 2-thiopyridone. A kinetic analysis for the generation of disulfide-forming cysteines with Cys367 at 37 degreesC revealed that the rate for the intrachain exchange reaction was quite different for the five cysteine sulfhydryls. The effective concentrations of the five cysteine sulfhydryls relative to the Cys367-mixed disulfide were determined by using three related model reactions: the obtained values were 11.4, 4.6, 15.2, 5.9, and 8.9 microM for Cys11, Cys30, Cys73, Cys120, and Cys382, respectively. Implications of the effective concentrations for the conformational state of acidic ovalbumin are discussed.

Electron Paramagnetic Resonance and Oxygen Binding Studies of α-Nitrosyl Hemoglobin: A NOVEL OXYGEN CARRIER HAVING NO-ASSISTED ALLOSTERIC FUNCTIONS

alpha-Nitrosyl hemoglobin, alpha(Fe-NO)2beta(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. alpha-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its beta-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the alpha(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, alpha(Fe-NO)2beta(Fe)2 is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of alpha-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysbeta93 toward 4, 4'-dipyridyl disulfide of several alpha-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, alpha-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the alpha-subunits. It is concluded that the NO bound to the heme sites of the alpha-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions.

Screening system for urease inhibitors using 13C-NMR.

Urease inhibitors are candidate drugs to treat infection with the human pathogen, Helicobacter pylori, which produces a potent urease [urea amidohydrolase; EC 3.5.1.5]. We developed a screening system based on 13C-NMR measurement of the time course of decrease in the signal of 13C-urea in the presence of urease. The effect on urease activity of known inhibitors, hydroxamic acids, L-ascorbic acid, 2,2'-dipyridyl disulfide and ninhydrin, was speedily and conveniently measured by this method.

Kinetics and mechanism of the nitrosation of 2-mercaptopyridine [pyridine-2(1H )-thione

2-Mercaptopyridine (MP) reacts rapidly with nitrous acid in mildly acid aqueous solution (via the thione tautomer) to give an unstable S-nitroso ion (SNO+) in a reversible process with an equilibrium constant (KN) of ca. 1 × 105 dm6 mol–2. SNO+ is readily detected by two peaks in the UV spectrum at 295 and 240 nm with extinction coefficients 9600 and 9300 dm3 mol–1 cm–1 respectively. MP is regenerated when the solution is made alkaline. Kinetic measurements made on the nitrosation reaction give a value of 8200 dm6 mol–2 s–1 for the third order rate constant k3 (defined by rate = k3 [MP] [HNO2] [H+]), which is close to that believed to be the diffusion-controlled limit for attack by NO+ (or H2NO2+). As expected there is marked catalysis by Cl– and Br–, and analysis of the kinetic results obtained from variation of measured rate constants with [halide ion] gave values of 3.5 × 109 and 3.7 × 109 dm3 mol–1 s–1 respectively for the bimolecular rate constants for NOCl and NOBr reactions with MP, again values close to the diffusion limit. The same experimental results also yielded values of 30 and 2400 dm3 mol–1 s–1 for the second order rate constants, for the reverse process of Cl– and Br– reaction with SNO+. Values for KN of 1.3 × 105 and 7.9 × 104 dm6 mol–2 were obtained from the halide catalysed reactions. In acid solution SNO+ decomposed to the disulfide (2,2′-dipyridyl disulfide) and NO (measured with a NO-electrode). Quenching of SNO+ at pH 7.4 gave UV spectroscopic evidence for the neutral deprotonated form (SNO) of SNO+ and there was a transformation to give mainly MP together with some disulfide. There was clear evidence that SNO+ (and maybe SNO) can act as an efficient nitrosating species: addition of the thiol N-acetylcysteine (at pH 6.15) resulted in the almost instantaneous decomposition of SNO. Addition of N-methylaniline (NMA) to an acidified solution of SNO+ resulted in quantitative N-methyl-N-nitrosoaniline formation and kinetic measurements of the nitrosation of NMA in the presence of MP showed marked catalysis at low [MP], which disappeared at higher [MP]. These results are quantitatively consistent with nitrosation via SNO+: catalysis disappears at higher [MP] when the nitrous acid is virtually completely converted to SNO+. A value of 1.7 × 105 dm3 mol–1 s–1 was obtained for the bimolecular rate constant for reaction of SNO+ with the free base form of NMA. MP is thus an excellent catalyst for electrophilic nitrosation. Under somewhat different conditions SNO+ can then act as a source of HNO2/NO2–, NO or NO+. The chemistry reported in this paper bears many similarities to that involved in the nitrosation of thioureas, and subsequent reactions of the S-nitrosothiouronium ions.

Early- and Late-Lanthanide Pyridinethiolates: Synthesis, Redox Stability, and Structure.

The early and late lanthanides form stable complexes with the pyridinethiolate (2-S-NC(5)H(4), or SPy) ligands. The Ce compound Ce(SPy)(3) is relatively insoluble in neutral organic donor solvents such as THF or pyridine but can be solubilized by the addition of [PEt(4)][SPy] to form the orange homoleptic cerium thiolate [PEt(4)][Ce(SPy)(4)] (1). Low-temperature structural characterization of 1 showed that the complex is isostructural with the known Eu(III) derivative. Further oxidation of Ce(III) with dipyridyl disulfide does not occur. Molecular 1 is colored due to a low-energy f(1)-to-d(1) promotion. As the size of the lanthanide ion decreases, the solubility of neutral Ln(SPy)(3) appears to increase. Colorless [PEt(4)][Ln(SPy)(4)] (Ln = Ho (2), Tm (3)) can also be isolated by fractional crystallization, and the compounds are isostructural with the Ce and Eu derivatives. The neutral complexes of Ho and Tm are also slightly soluble in acetonitrile and dimethoxyethane and very soluble in pyridine. Both divalent and trivalent Yb complexes of the pyridinethiolate ligand dissolve in and crystallize from pyridine. Divalent Yb(SPy)(2) crystallizes as the pentagonal bipyramidal molecule (py)(3)Yb(SPy)(2) (4). One pyridine nitrogen and the four donor atoms of the two pyridinethiolate ligands are bound in equatorial positions, and two neutral pyridine ligands occupy the axial sites. The Yb(III) compound crystallizes readily from pyridine as molecular 8-coordinate (py)(2)Yb(SPy)(3) (5). Compounds 4 and 5 are intensely colored; 4 has a visible Yb(II)-to-pyridine charge transfer excitation that is virtually identical in energy to the analogous excitation in SmI(2)(py)(4), while 5 has a visible S-to-Yb charge transfer absorption. Crystal data (Mo Kalpha, 153(5) K) are as follows: 1, monoclinic space group P2/n, a = 15.118(6) Å, b = 16.117(4) Å, c = 26.443(7) Å, beta = 90.14(3) degrees, Z = 4; 4, monoclinic space group Cc, a = 10.588(1) Å, b = 16.810(3) Å, c = 14.833(5) Å, beta = 109.12(2) degrees, Z = 4; 5, monoclinic space group P2(1)/n, a = 9.672(2) Å, b = 16.293(4) Å, c = 19.214(3) Å, beta = 101.51(2) degrees, Z = 4.

A classical enzyme active center motif lacks catalytic competence until modulated electrostatically.

The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).

4- N, N-Dimethylaminosulfonyl-7- N-(2-aminoethyl)amino-benzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography

A new fluorescent reagent for carboxylic acids, 4- N, N-dimethylaminosulfonyl-7- N-(2-aminoethyl) amino-2, l,3-benzoxadiazole (DBD-ED) was synthesized from 4- N, N-dimethylaminosulfonyl-7-chloro-2,1,3-benzoxadiazole (DBD-Cl) and 1,2-diaminoethane. DBD-Cl is also a new compound described in this work for the first time. The applicability of DBD-ED as a precolumn derivatization reagent in liquid chromatography (LC) was examined. DBD-ED reacted with saturated fatty acids and non-steroidal anti-inflammatory drugs (NSAIDs) in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide at room temperature for 30 min for NSAIDs and 1 h for fatty acids to give fluorescent products. The reaction solutions were subjected to LC and the derivatized products were detected spectrofluorimetrically at 560 nm with excitation at 450 nm. The detection limits were in the fmol range

Correlation of the Structural Decomposition and Performance of Pyridinethiolate Surface Modifiers at Gold Electrodes for the Facilitation of Cytochrome c Heterogeneous Electron-Transfer Reactions

This paper describes the results of an electrochemical and spectroscopic (infrared reflection and X-ray photoelectron spectroscopies) investigation of the modified gold electrode surfaces prepared from dilute ethanolic solutions of 4-mercaptopyridine (PySH) and 4,4‘-dipyridyl disulfide (PySS). Both precursors have been used extensively as facilitators for the electron transfer of redox proteins like cytochrome c (cyt c). During the course of an investigation of the interfacial architectures formed from the two different precursors, a previously unreported structural instability in the adlayers was discovered. This instability manifests itself as a decrease in the ability of the modified surfaces to facilitate the electron transfer of cyt c that correlates with an increase of the immersion time in the precursor solutions. Results are presented that delineate the decrease in facilitator performance and probe the structural changes resulting in the decrease in performance. Together, the electrochemical and surface spectroscopic findings reveal that the modified surfaces spontaneously decompose to yield an adlayer composed largely of adsorbed atomic and oligomeric sulfur, an adlayer that we found to be ineffective in the facilitation of the electron transfer reaction of cyt c. The implications of these findings on the use of this type of modifier to studies of electron transfer reactions of redox proteins and to issues of the general structural stability of organosulfur-based monolayers are briefly discussed.

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization.

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzox adi azole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid liquid extraction following the derivatization. The conjugate was analyzed by ion-pair-HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.

Electrochemical investigation of binding sites of plantacyanin: blue, copper-containing protein of plants

The binding site structure of plantacyanin (plant blue Cu-protein) was studied using voltammetry at the electrodes modified with sulphur-containing compounds (thioglycolic acid, dipyridyl disulfide, cysteamine, β-mercaptoethanol). The scanning of the functional groups of the modifying layer was used to reveal the most efficient modifications of the electrode and to determine the character of the binding groups of the protein. A comparison of the electron exchange characteristics of plantacyanin at the different electrodes with that of the other proteins of the known binding site structure (plastocyanin, cytochrome c, cytochrome c553, high potential ion-sulfur protein) was carried out. By means of this comparison and also using literary data about plantacyanin tertiary structure, the localization of the protein binding sites and their hydrophobicity were evaluated. Two poles was suggested to exist with a high permeability to electrons. One of these binding sites is of a high hydrophobicity (4.1–6.7 kj mol −1). The other is hydrophilic and contains several charged amino groups. The problem of how to search the partners of plantacyanin in the electron transport chain was discussed.

Characterization of the Electrostatic Perturbation of a Catalytic Site (Cys)-S –/(His)-Im +H Ion-pair in One Type of Serine Proteinase Architecture by Kinetic and Computational Studies on Chemically Mutated Subtilisin Variants

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN′, to produce (Cys)-S −/(His)-Im +H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific, time dependent inhibitors, 2,2′-dipyridyl disulfide and 4,4′-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2′-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S −/(His63)-Im +H ion-pair is produced by protonic dissociation with p K a4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (p K a10.2). In the BPN′ enzyme, the analogous, (Cys221)-S −/(His64)-Im +H ion-pair is produced by protonic dissociation with p K a5.1 and its reactivity is affected by an ionization with p K a3.5 in addition to the deprotonation of His64 (p K a≥10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the p K adifference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 Å of the catalytic site His residue are included. The p K adifference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN′ enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the p K ashift. The additional kinetically influential p K aof 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

Rapid kinetic studies and structural determination of a cysteine proteinase mutant imply that residue 158 in caricain has a major effect upon the ability of the active site histidine to protonate a dipyridyl probe.

Cysteine proteinases are endopeptidases whose catalytic activity depends upon the nucleophilicity of the active site cysteine thiol group. An ion pair forms with an active site histidine. The presence in some cysteine proteinases of an aspartic acid close to the ion pair has been used as evidence of a "catalytic triad" as found in the serine proteinases. In these enzymes, the correct alignment of serine, histidine, and aspartate residues controls catalysis. However, the absence of the homologous aspartate residue in the mammalian cysteine proteinases cathepsins B and H argues against this pivotal role for aspartic acid. Instead, an Asn, physically close to the histidine in cysteine proteinases, has been proposed as a member of the catalytic triad. Protein engineering is being used to investigate these questions. In this study, the Asp158Glu mutant of the plant cysteine proteinase caricain was analyzed by stopped-flow rapid kinetics. The probe that was used was 2,2'-dipyridyl disulfide (2 PDS), and the profile of k versus pH gave results more closely allied to a small molecule active site model than the normal profile with cysteine proteinases. Multiple pKa's identified in the profile are as follows: pK1 = 3.4 (Cys 25), pK2 = 3.6, pK3 = 7.0, and pK4 = 8.6 (His 158). The structure of the enzyme with the bound inhibitor E64 was solved (R factor of 19.3%). Although the distance between the imadazolium and the surrounding charged amino acids is only slightly changed in the mutant, the reduced steady state activity and narrower pH range can be related to changes in the hydrogen-bonding capacity of the imadazolium.

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds.

Plastid pyruvate kinase (PKp) activity and anti-(castor oil seed [COS] PKp) immunoglobulin G immunoreactive polypeptides were recovered in the stroma but not from envelope membranes of purified COS leukoplasts that had been subfractionated by sucrose density gradient centrifugation. The PKp was highly purified from isolated leukoplasts using anion-exchange and ADP-agarose chromatographies. Proteolysis of PKp was almost entirely eliminated by including 2,2[prime]-dipyridyl disulfide in purification buffers. The final preparation contained 63.5-kD ([alpha] subunit) and 54-kD ([beta] subunit) polypeptides that stained for protein and cross-reacted with anti-(COS PKp) immunoglobulin G with similar intensities. These two polypeptides co-eluted following gel-filtration chromatography and co-migrated during nondenaturing isoelectric focusing-polyacrylamide gel electrophoresis. The enzyme's native Mr was estimated to be 334,000. This PKp thus appears to exist as an [alpha]3[beta]3-heterohexamer. Comparison of the respective N-terminal sequences of the [alpha] and [beta] subunits with the deduced amino acid sequences for several PKp cDNAs indicated that (a) the [alpha] and [beta] subunits are encoded by COS genes previously designated as PKpA and PKpG, respectively, and (b) respective transit peptides of 4.8- and 5.5-kD are cleaved from the [alpha] and [beta] subunit preproteins following their translocation into the leukoplast.

Separation and Quantitation of CytochromecOxidase Subunits by Mono-Q Fast Protein Liquid Chromatography and C18 Reverse-Phase High-Performance Liquid Chromatography

Mono-Q fast protein liquid chromatography (FPLC) combined with C18 reverse-phase HPLC was used for quantitative subunit analysis of bovine heart cytochromecoxidase, a multisubunit membrane complex. By this approach normal cytochromecoxidase preparations were shown to be a mixture of enzyme that has all 13 subunits and complexes that are missing 1–3 subunits. A distinct advantage of this procedure is that homogeneous 13- or 11-subunit enzyme can be easily isolated from heterogeneous cytochromecoxidase mixtures. The method involves: (1) separation of complexes that are depleted of subunits using Mono-Q FPLC and (2) quantitative subunit analysis of the purified complexes by C18 reverse-phase HPLC with a water/acetonitrile gradient in 0.1% trifluoroacetic acid. The approach has four distinct advantages over other methods of analysis, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) or C4 reverse-phase HPLC. (1) The reproducible yield and the baseline resolution between each eluting subunit permits quantitative determination of the subunit content with an accuracy of ± 5%. (2) Subunits that are very difficult to separate by SDS–PAGE, e.g., subunits VIa, VIb, and VIc, are completely resolved by this system. (3) The combination of Mono-Q purification and C18 reverse-phase HPLC analysis permits an accurate assessment of both homogeneity and subunit content. (4) The quantitative nature of the reverse-phase HPLC system also makes it a powerful method for analyzing the specificity and extent of chemical modification of specific subunits as is shown by the difference in reac tivity of subunit VIa towardN-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate and 4,4′-dipyridyl disulfide.

Synthesis and characterization of cationic pyridine-2-thiolate complexes of lanthanoid(III): crystal structures of pentagonal bipyramidal [Ln(SC 5H 4N) 2(hmpa) 3]I (Ln = Sm, Yb; hmpa = hexamethylphosphoric triamide)

The cationic thiolate complexes of general formula [Ln(SC 5H 4N) 2(hmpa) 3]I ( 1; Ln = Pr, Nd, Sm, Eu, Er, Yb; hmpa = hexamethylphosphoric triamide) were prepared in modest yields by the reaction of metallic lanthanoids with 2,2′-dipyridyl disulfide and iodine in the presence of HMPA. The structure of 1 is a pentagonal bipyramidal seven-coordinated geometry consisting of two apical HMPA together with two chelating pyridine-2-thiolate ligands and one HMPA ligand in the pentagonal plane, which was confirmed by X-ray analysis for the samarium complex 1c (monoclinic, space group P2 1 c with a = 16.614(3), b = 13.350(4), c = 21.302(3)Å, β = 100.14(1)°, V = 4650(1)Å 3, Z = 4, R = 0.039 for 4247 reflections with I > 3 σ-( I) and 442 parameters) and the ytterbium complex 1f (monoclinic, space group C2 with a = 19.909(4), b = 11.140(3), c = 21.273(3)Å, β = 105.20(1)°, V = 4553(1)Å 3, Z = 4, R = 0.038 for 4746 reflections with I > 3 σ( I) and 442 parameters).

A Procedure for the Determination of Monothiols in the Presence of Dithiothreitol - An Improved Assay for the Reduction of Disulfides

4,4′-Dipyridyl disulfide (4-PDS) and sodium arsenite have been used to develop a rapid and sensitive assay for monothiols in the presence of dithiothreitol (DTT). The procedure is similar to that of Zahler and Cleland ( J. Biol. Chem. 243, 716-719, 1968) but involves the use of a lower pH and 4-PDS, which is more effective than 5,5′-dithiobis-(2-nitrobenzoic acid) at low pH. Background reactions that interfere with the determination of slow reacting thiol groups are much reduced in the new assay procedure. The reduction of oxidized glutathione by DTT at three different pH values and a partial and the complete reduction of bovine serum albumin by DTT have been characterized in order to demonstrate the use of this procedure. In the first two cases monothiol groups were readily detected without interference from DTT. Lower than expected numbers of thiol groups in the case of three reduced and denatured model proteins appeared to reflect their partial complexation by arsenite. Equilibrium and kinetic constants for the formation and dissociation of the DTT-arsenite complex from pH 5 to 8 are presented.