Reversed-phase high-performance liquid chromatography behavior of chaotropic counteranions.

The retention behavior of inorganic liophilic anions in reversed-phase HPLC columns was studied. Usually, the addition of these ions to the mobile phase influences the retention of protonated basic analytes similar to the effect of amphiphilic ions (ion-pairing agents). The nature of this influence is the subject of this paper. HPLC retention of perchlorate (ClO4-), tetrafluoroborate (BF4-), and hexafluorophosphate (PF6-) ions was studied on six columns with different bonded phases including alkyl, phenyl and perfluorophenyl phases. The effect of the mobile phase ionic strength on the retention of liophilic ions was investigated. The influence of the type of organic modifier, acetonitrile and methanol, on the retention of inorganic ions was also studied and interpreted on the basis of adsorption from solutions. Semi-empirical expression is suggested for the description of the retention profile of studied liophilic ions versus the eluent composition. Significant retention of these ions is observed in acetonitrile-water eluents. Multilayer-type adsorption of the acetonitrile on the reversed-phase surface and its strong dispersive (or pi-pi) interactions with liophilic ions are responsible for significant retention of these ions. This accumulation of liophilic ions in the adsorbed layer on the surface of reversed-phase material introduces an electrostatic component in the retention of protonated basic analytes.

Interpretation of the excess adsorption isotherms of organic eluent components on the surface of reversed-phase phenyl modified adsorbents.

The adsorption of three organic eluent components (acetonitrile, methanol, and tetrahydrofuran) from water were measured on four phenyl-type bonded phases using the minor disturbance method. The thicknesses of organic layer enriched above the phenyl-type bonded ligands were assessed and interpreted. Acetonitrile and tetrahydrofuran showed multilayer formation while methanol showed monomolecular adsorption. These results were compared to those obtained on alkyl bonded phases.

Characterization of phenyl-type HPLC adsorbents.

A set of different phenyl-modified HPLC adsorbents were characterized in terms of their surface area, pore volume, and bonded phase volume using low temperature nitrogen adsorption (LTNA). Adsorbents pore volume and interparticle volume were also measured using HPLC. Comparison of the pore volumes assessed with LTNA and HPLC suggests a compact molecular arrangement for all bonded phases studied. Simple and effective method for determination of the exact mass of adsorbent and total surface area in the column is suggested.

Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitochondrial complex III. Comparison with similar findings for analogous bacterial [2Fe-2S] proteins.

Yeast mitochondrial complex III contains a subunit with a [2Fe-2S] cluster (the Rieske center) that has unusual physical and chemical properties. For apparently similar centers isolated from bacteria, it has been shown by electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) measurements that these [2Fe-2S] centers are coordinated by at least one and probably two nitrogen ligands. This work describes similar ENDOR and ESEEM studies on the intact mitochondrial complex. We find that this [2Fe-2S] cluster exhibits ESEEM and ENDOR properties that appear to be indistinguishable from those observed with the isolated bacterial systems. Furthermore, changes in EPR lineshape that occur as complex III is progressively reduced are not accompanied by any changes in the nitrogen coupling parameters. This spectroscopic evidence for nitrogen coordination is supported by published sequence data on four Rieske iron-sulfur subunits. It seems likely that this is a general characteristic of such [2Fe-2S] redox active centers.

Effect of counter-anion concentration on retention in high-performance liquid chromatography of protonated basic analytes.

The influence of acid and salt concentration in the mobile phase on the retention of basic analytes has been studied. An increase in the retention of fully protonated analytes with increasing the concentration of inorganic additives was found. The addition of salt, such as perchlorate, trifluoroacetate, and phosphate, leads to the increase of retention for fully protonated analytes while mobile phase pH remains constant. The observed effect was attributed to the interaction of protonated analytes with the counter-anion of acid or salt, which leads to the disruption of the analyte solvation shell and the increase of its hydrophobicity and corresponding increase of retention. A mathematical model for the description of the influence of counter-anion concentration on analyte retention is proposed.

Effect of the eluent pH and acidic modifiers in high-performance liquid chromatography retention of basic analytes.

The retention of ionogenic bases in liquid chromatography is strongly dependent upon the pH of the mobile phase. Chromatographic behavior of a series of substituted aniline and pyridine basic compounds has been studied on C18 bonded silica using acetonitrile-water (10:90) as the eluent with different pHs and at various concentrations of the acidic modifier counter anions. The effect of different acidic modifiers on solute retention over a pH range from 1.3 to 8.6 was studied. Ionized basic compounds showed increased retention with a decrease of the mobile phase pH. This increase in retention was attributed to the interaction with counter anions of the acidic modifiers. The increase in retention is dependent on the nature of the counter anion and its concentration in the mobile phase. It was shown that altering the concentration of counter anion of the acidic modifier allows the optimization of the selectivity between basic compounds as well as for neutral and acidic compounds.

Interpretation of the excess adsorption isotherms of organic eluent components on the surface of reversed-phase adsorbents. Effect on the analyte retention.

The excess adsorption isotherms of acetonitrile, methanol and tetrahydrofuran from water on reversed-phase packings were studied, using 10 different columns packed with C1-C6, C8, C10, C12, and C18 monomeric phases, bonded on the same type of silica. The interpretation of isotherms on the basis of the theory of excess adsorption shows significant accumulation of the organic eluent component on the adsorbent surface on the top of "collapsed" bonded layer. The accumulated amount was shown to be practically independent of the length of alkyl chains bonded to the silica surface. A model that describes analyte retention on a reversed-phase column from a binary mobile phase is developed. The retention mechanism involves a combination of analyte distribution between the eluent and organic adsorbed layer, followed by analyte adsorption on the surface of the bonded phase. A general retention equation for the model is derived and methods for independent measurements of the involved parameters are suggested. The theory was tested by direct measurement of analyte retention from the eluents of varied composition and comparison of the values obtained with those theoretically calculated values. Experimental and theoretically calculated values are in good agreement.

Geometry of chemically modified silica.

The effect of alkyl chain length on adsorbent pore volume and void volume of HPLC columns is described. The results provide evidence that alkyl chains attached on silica surface are densely packed. A correlation of a decrease of pore volume with an increase of the alkyl modifier chain length was found. Effective molecular volume of bonded chains was found to be similar to the molecular volume of corresponding liquid alkanes. An absence of noticeable penetration of acetonitrile, methanol, or tetrahydrofuran molecules between bonded chains at any water-organic eluent composition was found.

Kinetic analysis and subambient temperature on-line on-column derivatization of an active aldehyde.

The chromatographic analysis of aldehydes under typical reversed-phase conditions may be a challenging task due to an equilibrium process leading to the formation of a gem diol species regardless of acidic or basic conditions. Initially, a reversed-phase HPLC gradient elution was developed to determine the amount of a n acetylenic aldehyde in a reaction mixture. Significant fronting was observed under acidic and basic conditions even at -5 degrees C. In order to circumvent this problem, a reversed-phase HPLC gradient method on a C18 column at subambient temperature was developed using diethylamine as a mobile phase additive for on-line on-column derivatization of the aldehyde moiety. The on-line on-column reaction rate for the derivatization of the aldehyde with diethylamine was determined as a function of column temperature. An Arrhenius plot was constructed and the activation energy was calculated. The chromatographic behavior of the derivatized acetylenic aldehyde and products formed in-situ in the chromatographic system were studied at various temperatures ranging from -10 to 60 degrees C. It was found that the reaction products could be controlled by adjusting the column temperature. Different reaction pathways were identified as a function of temperature. The products and the reaction pathways were characterized by NMR, LC-MS and UV spectra.

Characterization of moisture-sensitive raw materials with simple spectroscopic techniques.

The quality of raw materials used in a synthetic process needs to be properly controlled in order to ensure optimal reaction conversion and desired quality of the resulting product. For air and water sensitive raw materials, quantitative analysis can be a challenging task. Spectroscopic techniques possess advantages of simple operation, fast analysis, low consumable costs and high sample throughput for the analysis of reactive raw materials. Three case studies utilizing spectroscopic analysis for air and water sensitive materials are discussed. First, FT-IR spectroscopy was utilized to determine the amount of residual acetic acid in acetic anhydride key raw material. Acetic anhydride was used in a methylenation reaction where the presence of residual acetic acid could quench a base used in the reaction, leading to incomplete conversion. A simple, one-frequency calibration method was developed to quantify acetic acid in acetic anhydride (2-35wt.%). Next, a novel near infrared reflectance spectroscopy (NIRS) method was used to determine the concentration of diisobutyl aluminum hydride (DIBAL-H) in toluene. DIBAL-H is a highly reactive and moisture-sensitive reagent used as a key raw material for the reduction of an active intermediate. A calibration method based on one-frequency was also developed to determine the concentration of DIBAL-H in toluene (0-1.5mole/L). Finally, a NIRS method based on partial least squares regression (PLS) was developed to quantify p-toluenesulfonic acid in p-toluenesulfonic anhydride, which is not amenable to chromatographic analysis.

Iron as a bound secondary electron donor in modified bacterial reaction centers.

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.

Effects of nucleotides on the protein ligands to metals at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase.

We have identified the most probable protein ligands at the catalytic M3 and noncatalytic M2 metal-binding sites in the spinach chloroplast F1-ATPase (CF1) and here propose possible residues in the protein sequence for these ligands in latent CF1 in the absence of nucleotide. The changes in the metal ligands at these sites upon binding of nucleotide to the N2 and N3 sites and upon activation of latent CF1 provide a possible molecular basis for inhibition of ATPase activity by free metal, for the lack of activity in the latent state, and for the gating mechanism of the ATPase H+ pump. To these ends, the Mg2+ analogue, vanadyl (VIV = O)2+, was used as a paramagnetic probe at the M2 and M3 metal-binding sites. EPR and ESEEM spectra of VO2+ were obtained, and simulations of the full EPR spectra imply the ligand sets at the different metal-binding sites. When VO2+ is added to CF1 in the absence of ATP, the most likely set of ligands at the M2 site are 1 ROH (alpha T176), 2 H2O, and 1 RCOO- (alpha D269 or alpha D270), where the suggested amino acid designations of the residues are given in parentheses according to the mitochondrial sequence. Evidence suggests a possible axial nitrogen ligand at this site (alpha K175). When the M2 site is filled by addition of VO2+ and ATP, the metal binds as a second species in which N2-bound ATP and M2-bound VO2+ form a monodentate complex with concomitant exchange of the equatorial protein ligands by 3 H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordination of nucleotides to metals at the M2 and M3 metal-binding sites of spinach chloroplast F1-ATPase.

Vanadyl (VIV=O)2+ was used as a paramagnetic probe at the M2 and M3 metal-binding sites of the spinach chloroplast F1-ATPase (CF1) in order to detect interaction of the metals with nucleotides. The M2 site can exist in two forms in the presence of ATP. When ATP and VO2+ are added in a 1.5:1 ratio to CF1, the VO2+ EPR spectrum is identical to that of CF1-VO2+ in the absence of ATP. When the M2 site is filled by the addition of ATP and VO2+ in a 3:1 ratio, the VO2+ binds to M2 in a second form with equatorial coordination to a single phosphate. The treatments required to deplete CF1 of the monodentate VO2+(-)nucleotide complex indicate that the VO2+ is coordinated to the ATP at the nonacatalytic N2 site. The presence of uncomplexed nucleotide appears to induce formation of the second form, possibly via ATP binding to the N3 site. This change in coordination at the M2 noncatalytic site may regulate the ATPase activity of CF1. The M3 site also exists in two forms: (i) in latent CF1, no phosphate coordination is evident; and (ii) after the ATPase has been activated, the EPR line shape is consistent with the two phosphates from ADP at N3 coordinated to the VO2+ at M3. This work establishes a connection between the metal- and nucleotide-binding sites as M2-N2 and M3-N3.

Redox effects on the bacteriochlorophyll a-containing Fenna-Matthews-Olson protein from Chlorobium tepidum.

The BChl a-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacterium Chlorobium tepidum was purified and characterized. Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials. The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added. A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition. A 16 Gauss wide EPR signal with g factor = 2.005 is observed in samples without dithionite. This signal largely disappears upon addition of dithionite. Dithionite induces large reversible changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells. These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer. Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.

Identification of the P-loop lysine as a metal ligand in the absence of nucleotide at catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reinhardtii.

Site-directed mutations were made to the phosphate-binding loop lysine in the beta-subunit of the chloroplast F(1)-ATPase in Chlamydomonas reinhardtii (betaK167) to investigate the participation of this residue in the binding of metal to catalytic site 3 in the absence of nucleotide. The cw-EPR spectra of VO(2+) bound to site 3 of CF(1)-ATPase from wild type and mutants revealed changes in metal ligation resulting from mutations to betaK167. The three-pulse ESEEM spectrum of the wild-type CF(1)-ATPase with VO(2+) bound to site 3 shows an equatorially coordinating (14)N from an amine. The ESEEM spectra of the mutants do not show evidence of an equatorially coordinating amine group. The results presented here show that, in the absence of nucleotide, betaK167 is a ligand to the metal bound at catalytic site 3, suggesting a regulatory role for the P-loop lysine in addition to its known role in catalysis.

Coordination of vanadyl(IV) cation in complexes of S-adenosylmethionine synthetase: multifrequency electron spin echo envelope modulation study.

S-Adenosylmethionine (AdoMet) synthetase requires two freely dissociable divalent cations for activity, and its activity is greatly stimulated by certain monovalent cations (K+, Tl+). Omission of the native Mg2+ cations prevents enzyme-catalyzed reactions, although the substrates and products still bind. Vanadyl (oxovanadium, VO2+) serves as a convenient paramagnetic probe of the substrate-independent, divalent cation binding site in the enzyme. In the present study, multifrequency electron spin echo envelope modulation (ESEEM) is employed to explore the cation's coordination sphere in several functionally relevant complexes. In the substrate complex enzyme.VO2+.ATP.methionine.K+, an equatorially coordinating 14N ligand is found, with Aiso = 4.3 MHz. Selective 15N labeling of the epsilon-amino nitrogens of all lysine residues in the protein reveals that lysine is the source of the ligand. A lysine 14N ligand is also observed in the intermediate complex enzyme.VO2+.AdoMet.PPPi.K+, with Aiso = 4.8 MHz, and in the initial product complex enzyme.VO2+.AdoMet.PPi.K+. In the subsequent product, enzyme.VO2+.AdoMet.K+ (formed upon dissociation of PPi), the methionyl amino nitrogen of AdoMet coordinates VO2+ (Aiso = 5.3 MHz), and the lysine ligand is lost. In each complex, the monovalent cation activator can be changed from K+ to Tl+ or Na+ with no effect on the ESEEM spectra. Combination of the ESEEM data from this study with previous CW data [Markham, G. D. (1984) Biochemistry 23, 470-478] leads to identification of three of the equatorial ligands to VO2+ and places constraints upon the identity of the fourth ligand, in both the substrate and product complexes. A hypothetical outline of changes in the metal coordination scheme during the reaction is presented, based upon these results.

EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168).

Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.

Human malignant melanomas with varying degrees of melanin content in nude mice: MR imaging, histopathology, and electron paramagnetic resonance.

The etiology of the paramagnetic relaxation enhancement seen in malignant melanoma on proton magnetic resonance (MR) images has been the subject of many recent investigations and has been ascribed to iron from associated hemorrhage or chelated metal ions, rather than directly due to melanin. The purpose of this study was to correlate proton relaxation times on MR images in malignant melanomas with histopathologic features (i.e., degree of pigmentation, iron deposition, and necrosis), water content, and electron paramagnetic resonance (EPR) spectra to elucidate the etiology of the relaxation behavior demonstrated by these neoplasms. Cultured cells derived from human malignant melanoma metastases were implanted subcutaneously into nude mice. Twelve separate lesions were evaluated in 10 mice. Magnetic resonance imaging was performed in vivo at 1.9 T using spin echo and inversion recovery acquisitions for the purposes of calculating T1, T2, and proton density [N(H)]. Histopathologic examination was performed on specimens resected immediately after imaging, using hematoxylin/eosin, Prussian blue, and Fontana stains to assess tumor necrosis, and iron and melanin content. Dry/wet weight ratios and EPR spectra were also obtained on resected specimens. Our results indicate that T1 shortening correlates with increasing melanin content and not with increasing iron deposition, EPR-active metallic cations, necrosis, or water content. In fact, a presumably unrelated statistical correlation was found between increased iron and T1 prolongation. The T2 relaxation times did not correlate with the presence of any single factor other than proton density. Although the unique relaxation behavior of nonhemorrhagic malignant melanoma in vivo cannot be traced to a single cause, our data suggest that, contrary to previous investigations, it is strongly influenced by the presence of melanin rather than iron or other naturally occurring paramagnetic ions.

Influence of the protein environment on the properties of a tyrosyl radical in reaction centers from Rhodobacter sphaeroides.

The influence of the local environment on the formation of a tyrosyl radical was investigated in modified photosynthetic reaction centers from Rhodobacter sphaeroides. The reaction centers contain a tyrosine residue placed approximately 10 A from a highly oxidizing bacteriochlorophyll dimer. Measurements by both optical and electron paramagnetic resonance spectroscopy revealed spectral features that are assigned as arising primarily from an oxidized bacteriochlorophyll dimer at low pH values and from a tyrosyl radical at high pH values, with a well-defined transition that occurred with a pK(a) of 6.9. A model based on the wild-type structure indicated that the Tyr at M164 is likely to form a hydrogen bond with His M193 and to interact weakly with Glu M173. Substitution of Tyr or Glu for His at M193 increased the pK(a) for the transition from 6.9 to 8.9, while substitution of Gln for His M193 resulted in a higher pK(a) value. Substitution of Glu M173 with Gln resulted in loss of the partial formation of the tyrosyl that occurs in the other mutants at low pH values. The results are interpreted in terms of the ability of the residues to act as proton acceptors for the oxidized tyrosine, with the pK(a) values reflecting those of either the putative proton acceptor or the tyrosine, in accord with general models of amino acid radicals.