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PubMed1983

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase.

A new thermotropic phase transition, at -30 degrees C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at -60 degrees C. The thermotropic phase transition at -30 degrees C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30 degrees C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.

Dimyristoylphosphatidylcholine↗

PubMed1985

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin.

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidylglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97-108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175-178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigitated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.

Calorimetry, Differential Scanning↗

PubMed1985

Phase 1 and Phase 2 drug metabolism in isolated epidermal cells from adult hairless mice and in whole human hair follicles.

A sensitive fluorimetric assay to determine both Phase 1 (oxidation) and Phase 2 (conjugation) drug metabolism in epidermal cells isolated from hairless mice, using ethoxycoumarin as a model substrate, is described. Ethoxycoumarin was metabolized by isolated epidermal cells via dealkylation to 7-hydroxycoumarin (7-OHC) and subsequent conjugation. Phase 1 metabolites were extracted in ether from the aqueous incubation media, back extracted into sodium hydroxide and determined fluorimetrically. Conjugated metabolites remaining in the aqueous phase were hydrolysed by the action of beta-glucuronidase and extracted and determined in a similar manner. The production of free 7-OHC by isolated epidermal cells was biphasic at all substrate concentrations tested, exhibiting an initial linear increase followed by a plateau phase. The plateau phase was attributable to the conjugation of 7-OHC produced in situ. Metabolism was inhibited by SKF 525A, carbon monoxide, and alpha-naphthoflavone. Endogenous supplies of reducing equivalents in the form of NADPH were adequate to attain maximal rates of metabolism. With human hair follicles both Phase 1 and Phase 2 activity was detectable in 7 out of 11 subjects. The assay has the advantages of being sensitive, producing single defined metabolites from both Phase 1 and Phase 2 metabolism; is readily adaptable to human skin samples.

Animals↗

PubMed1986

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between L alpha and HII phases.

Inverted cubic and isotropic phases have been observed in phospholipid and glycolipid systems. These phases exhibit characteristic morphologies in freeze-fracture electron micrographs, isotropic 31P-NMR resonances and (in some cases) cubic X-ray diffraction patterns. It is proposed here that these phases may form from the same intermediates that are involved in lamellar/inverted hexagonal (L alpha/HII) phase transitions, and that it is possible that these cubic and isotropic phases are metastable. According to a kinetic theory of L alpha/HII phase transitions, intermediates in such transitions can form structures known as interlamellar attachments (ILAs). It is shown that ILAs should form in large numbers during L alpha/HII transitions in systems like those reported to form inverted cubic or isotropic structures. ILAs cannot readily assemble into either the HII phase or well-ordered arrays of L alpha phase bilayers, and represent a kinetic trap for intermediates in L alpha/HII transitions (although it is possible that they are marginally more stable in a thermodynamic sense than the L alpha phase in a small temperature range below TH). It is also shown that arrays of ILAs should form metastable arrays with the same morphology and isotropic 31P-NMR resonances that are observed in isotropic and inverted cubic states. In particular, under some circumstances ILAs will assemble into a structure identical to the bicontinuous inverted cubic phase previously described in monoglycerides and very similar in morphology to structures observed in phospholipid systems. Finally, since isotropic and cubic states form from ILAs, which also can mediate fusion of unilamellar vesicles, unilamellar vesicles should fuse to at least some extent under the same conditions in which multilamellar samples of the same lipid form isotropic or inverted cubic states. This correlation has been observed.

Glycolipids↗

PubMed1987

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro.

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.

Animals↗

PubMed1995

Retention and selectivity of flavanones on homopolypeptide-bonded stationary phases in both normal- and reversed-phase liquid chromatography.

Three linear polymers of repeating amino acid units, or homopolypeptides, have been individually covalently bonded to microparticulate silica and evaluated for liquid chromatographic separations. The retention and selectivity of seven flavanones were investigated on these stationary phases and a structurally similar, commercially available reference stationary phase, Chiraspher. All three of the homopolypeptide stationary phases retain solutes in the normal-phase mode. The aromatic-containing homopolypeptide stationary phases also retain solutes in the reversed-phase mode. Selectivity values for the flavanones were higher in the normal-phase mode; chiral selectivity was observed for the amphiphilic homopolypeptide stationary phase in the reversed-phase mode. The retention mechanism of each stationary phase is suggested based on the chemical nature and conformation of the corresponding homopolypeptide ligand.

Chromatography, Liquid↗

PubMed1996

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents.

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C18 micropellicular and Vydac C4 and C18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C4 and the Hy-Tech C18 column, respectively.

Acetonitriles↗

PubMed1984

C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q.

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.

Antigen-Antibody Complex↗

PubMed1994

cAMP contributes to mossy fiber LTP by initiating both a covalently mediated early phase and macromolecular synthesis-dependent late phase.

Memory storage has a short-term phase that depends on preexisting proteins and a long-term phase that requires new protein and RNA synthesis. Hippocampal long-term potentiation (LTP) is thought to contribute to memory storage. Consistent with this idea, a cellular representation of these phases has been demonstrated in NMDA receptor-dependent LTP. By contrast, little is known about the NMDA receptor-independent LTP of the mossy fiber pathway. We find that mossy fiber LTP also has phases. Only late phase is blocked by protein and RNA synthesis inhibitors, but both phases are blocked by inhibitors of cAMP-dependent protein kinase, and both are stimulated by forskolin and Sp-cAMPS. During early phase, paired-pulse facilitation is occluded. This occlusion decays with the onset of late phase, consistent with its using a different mechanism. Thus, although Schaffer collateral and mossy fiber pathways use very different mechanisms for early phase, both use a cAMP-mediated mechanism for late phase.

Animals↗

PubMed1996

Chromatographic behaviour in reversed-phase high-performance liquid chromatography with micellar and submicellar mobile phases: effects of the organic modifier.

Continuing our earlier study of the retention behaviour in reversed-phase systems with aqueous mobile phases containing surfactants in concentrations lower (submicellar systems) and higher (micellar systems) than the critical micellar concentration (CMC), we investigated the chromatographic behaviour of various non-ionic solutes in mixed aqueous-organic micellar and submicellar mobile phases and their dependence on the methanol concentration. CMC values were measured for two cationic surfactant and one anionic surfactant in mixed aqueous-methanolic solvents, and were found to increase slightly with increasing methanol concentration. Depending on the character of the surfactant, a limiting concentration of methanol was found, above which micelles do not occur anymore. Sorption isotherms of the surfactants on an octylsilica gel column were measured as a function of the concentration of methanol in aqueous-methanolic solvents. A modified Langmuir equation was used to describe the distribution of the surfactants between the stationary and the mobile phases in the concentration range below CMC. The retention of several polar solutes was measured on an octylsilica gel column both in micellar and submicellar mobile phases containing methanol. The dependencies of the capacity factors of the solutes studied on the concentration of methanol in the mobile phase can be suitably described by the same form of equation as that conventionally used for aqueous-organic mobile phases that do not contain surfactants, but the slopes of the dependencies for a given solute are different in the two ranges of surfactant concentrations. The ratio of the two slopes is controlled by the interaction with micelles and is approximately equal to, below or above 1, depending on whether the solutes do or do not associate with the micelles, or are repulsed from them. Simultaneous control of the concentrations of the organic solvent and of the surfactant in the mobile phase can be used for fine tuning the selectivity of separation as a complement to commonly used adjusting concentrations of two organic solvents in ternary aqueous-organic mobile phases. These effects are illustrated by practical examples of submicellar HPLC with mobile phases containing methanol.

Adsorption↗

PubMed1997

Phase transition between hexagonal II (H[II]) and liquid-crystalline phase induced by interaction between solvents and segments of the membrane surface of dioleoylphosphatidylethanolamine.

We have investigated effects of several water-soluble organic solvents such as acetone, acetonitrile, and ethanol, which also have high solubility in alkane, on the structure and phase behavior of dioleoylphosphatidylethanolamine (DOPE) dispersion. X-ray diffraction data indicated that a phase transition from hexagonal II (H[II]) to liquid-crystalline (L alpha) phase in DOPE dispersion, occurred at 13% (v/v) acetone in H2O at 20 degrees C. The temperature of the L alpha-H(II) phase transition of DOPE dispersion increased with an increase in acetone concentration, and it was 37 degrees C at 20% (v/v) acetone. These results indicated that acetone stabilized L alpha phase relative to H(II) phase. Similar results were obtained in interactions of DOPE dispersions in H2O with acetonitrile or ethanol. X-ray diffraction data indicated that the H(II)-L alpha phase transition occurred at 9.0% (v/v) acetonitrile or at 9.8% (v/v) ethanol in water at 20 degrees C. The L alpha-H(II) phase transition temperature of DOPE dispersion increased with an increase in acetonitrile or ethanol concentration, and it was 66 degrees C at 20% (v/v) acetonitrile. Substitution of H2O by D2O (deuterium oxide) increased their threshold concentrations of the H(II)-L alpha phase transition induced by these organic solvents. A mechanism of these phase transitions and the effect of the substitution of H2O by D2O is proposed and discussed; an interaction free energy between solvents and the hydrophobic segments of the alkyl chains in the membrane surface, and also a packing parameter of the phospholipid may be main factors to explain these phenomena reasonably.

Acetone↗

PubMed2003

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography.

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C18 bonded silica stationary phase and an acetonitrile-water mixture mobile phase, the sorption process of PEO to the stationary phase showed deltaH(o) > 0 and deltaS(o) > 0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy-entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile-water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: deltaH(o) < 0 and deltaS(o) < 0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy-entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.

Chromatography, Liquid↗

PubMed1997

Description and prediction of retention in normal-phase high-performance liquid chromatography with binary and ternary mobile phase.

The suitabilities of several earlier reported models for description and prediction of retention in normal-phase systems with mobile phases comprised of two organic solvents-a polar and a non-polar one-were tested on the chromatographic behaviour of phenylurea herbicides and alkyl-, aryl- and nitrophenols as sample solutes with a silica-gel column and 2-propanol, n-heptane and dioxane as mobile phase components. With few exceptions, the data obtained from the best-fit three-parameter retention equation differ from the experimental capacity factors at less than 0.1, or 2%, and for most compounds the fit is better than 1%. Of the two two-parameter equations derived from theoretical models, one failed to describe the retention behaviour. The fit of the other two-parameter equation to the retention data of phenols is slightly inferior than with the three-parameter equation. The differences between the fitted data and the experimental capacity factors of substituted phenylureas were < or = 0.2, or 5%. Three-parameter equations were suggested to describe the retention behaviour in three-component mobile phases either at a constant sum or at a constant ratio of the two stronger solvents in ternary mobile phases. To fit the three-component data at any combination of concentrations of the three solvents with the error comparable to that in binary mobile phases, a nine-parameter equation is necessary. Suitability of these equations to describe the experimental behaviour of substituted phenylureas and phenols was verified in ternary mobile phases with various concentrations of 2-propanol, dioxane and n-heptane. Finally, a method was suggested for the prediction of retention in ternary mobile phases with varying concentration ratios of the two polar solvents from the parameters of best-fit equations in binary mobile phases and a single experimental capacity factor in a ternary mobile phase. For most capacity factors, the error of prediction was lower than 0.2, or 5%. Dried solvents were used to improve the reproducibility of the results and the temperature was controlled to +/-0.1 degree C in all experiments. With these precautions, differences between the original retention volumes and the data from repeated experiments measured after ten months of use of the column in the system tested were less than 0.2 ml for 85% of the values compared.

1-Propanol↗

PubMed1998

Purification of recombinant apolipoprotein A-1Milano expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation.

A method for purification of recombinant apolipoprotein A1 in aqueous two-phase systems has been studied. A mutant of apolipoprotein A-1, the Milano variant, was expressed in E. coli. Phase systems containing ethylene oxide (EO)-propylene oxide (PO) random copolymers have been used. These polymers are thermoseparating and have the ability to separate into one water-rich and one polymer-rich phase when heated above a critical temperature i.e. the cloud point. The filtrate from an E. coli fermentation was added to a primary aqueous two-phase system composed of an EO-PO copolymer and Reppal, which is an inexpensive hydroxypropyl starch. Apolipoprotein A-1 was partitioned to the top EO-PO copolymer phase and contaminating proteins to the bottom starch phase. The phase diagrams for Reppal PES 100-EO50PO50 (Ucon) and Reppal PES 100-EO30PO70 were determined. The effect on partitioning, when changing parameters such as polymer concentration, type of polymer, protein concentration, pH, salt concentration and volume ratio, were studied. Studies on E. coli DNA partitioning showed that DNA could be partitioned strongly to the bottom phase. An optimal system was scaled up from 5 g to 5 kg with similar degrees of purification, i.e. 2.5 and 2.7 and yields of 79% and 82% respectively. Furthermore temperature-induced phase formation was used for separation of apolipoprotein A-1 from the copolymer by raising the temperature above the copolymer cloud point; thus, recovering protein in a 'clean' water phase.

Apolipoprotein A-I↗

PubMed2000

Stationary-phase contribution of 1-propanol organic modifier to changes in sorption of 1-hexanol on an ODS-bonded phase

Using the reversed-phase bonded-phase HPLC packing Partisil-10 ODS-3, sorption isotherms have been measured for the alcohols 1-propanol (PrOH) and 1-hexanol (HexOH), and as well, a simultaneous sorption curve for the two alcohols has been measured from solutions containing a low and constant concentration of HexOH as sample with increasing concentrations of PrOH as organic modifier. The mobile-phase effect of PrOH is quantified by solution-phase activity coefficients obtained either from vapor/solution equilibrium measurements or from cloud point measurements. Since sorbed alcohols are located at the ODS/solution interface, the stationary-phase effect of PrOH is modeled in terms of three processes: (i) competition for space; (ii) decrease of space required per mole with increasing concentration of sorbed PrOH; and (iii) change of free energy of sorption with increasing concentration of sorbed PrOH. The model yields excellent fits to the isotherms and to the simultaneous sorption curve. Comparison of the model-fitting parameters for the simultaneous sorption curve with those for the PrOH isotherm confirms that the stationary-phase effect of PrOH on HexOH is due exclusively to processes i and ii. Sorbed PrOH causes rearrangement of the C18 chains of the ODS phase. For volume percent PrOH less than 15% in the mobile phase, the effect of PrOH on sample sorption is nearly exclusively in the stationary phase. Between 15 and 30%, both mobile- and stationary-phase effects are important.

Journal Article↗

PubMed1986

Role of head group structure in the phase behavior of amino phospholipids. 1. Hydrated and dehydrated lamellar phases of saturated phosphatidylethanolamine analogues.

Analogues of dimyristoylphosphatidylethanolamine (DMPE) have been prepared with head groups modified by N-alkylation, alkylation of carbon 2 of the ethanolamine group, or interposition of extra methylene segments between the phosphoryl and amino groups. The phases formed by these lipids in aqueous dispersions have been examined by high-sensitivity differential scanning calorimetry and Raman spectroscopy. All of the DMPE analogues examined, excepting N-methyl-DMPE but including N-ethyl-DMPE, form hydrated gel phases that are metastable with respect to a dehydrated "high-melting" solid phase that has been observed previously for DMPE itself. The properties and the conditions of formation of this high-melting phase are qualitatively distinct from those of the "subgel" phase, which is observed for dipalmitoylphosphatidylcholine and for some of the DMPE analogues examined in this study. The high-melting phases of different DMPE analogues all exhibit similarly tight packing of the acyl chains, which however do not pack according to a single type of subcell that can be universally and specifically associated with this phase. Increasing the size of the PE head group invariably decreases the melting temperature of the hydrated gel phase, even when the normal hydrogen-bonding capability of the head group is preserved. By contrast, addition of larger alkyl substituents to either the amino group or carbon 2 of the ethanolamine moiety substantially increases the transition temperature of the high-melting solid phase, indicating that the contributions of the head group to the energies of the hydrated gel and the high-melting phases are fundamentally different. Our results suggest that the head group structural requirements for a neutral phospholipid to form stable hydrated bilayers are rather stringent, a fact that may explain the overwhelming predominance of only a few such head group structures in most natural membranes.

Calorimetry, Differential Scanning↗

PubMed1981

Quantitative solid-phase Edman degradation for evaluation of extended solid-phase peptide synthesis.

Quantitative solid-phase Edman degradation was used for the amino acid sequence analysis of synthetic peptidyl-resins prepared by the Merrifield solid-phase procedure. A model peptide, Ala-[3H]Pro-Ala-Gly-Phe-Ala-Gly-, was synthesized on a solid support and was sequenced to measure the efficiency of the solid-phase sequencing protocol used. An average of 92% of the first four residues was removed from the peptidyl-resin as indicated by subtractive amino acid analysis. Quantitation of the radioactive proline residue at cycle 2 revealed that it was efficiently recovered both from the acid conversion procedure (99%) and also following high-pressure liquid chromatography of the phenylthiohydantoin (Pth) amino acid (88%). In order to facilitate identification and quantification of the side chain protected Pth amino acids, we prepared these derivatives and characterized them by high-pressure liquid chromatography. Thereafter, by the use of solid-phase Edman degradation as an analytical procedure, the synthesis of residues 2-118 of the heavy-chain variable region (VH) of a homogeneous rabbit antibody was undertaken. At 10-15-residue intervals during the solid-phase synthesis, samples of peptidyl-resin were removed from the synthesis vessel and sequenced. When gross synthetic errors caused by deletion of amino acids residues were detected, the solid-phase synthesis was terminated and restarted by using modified protocols. A 117-residue peptidyl-resin was prepared finally which possessed the desired amino acid sequence as indicated by a series of solid-phase Edman degradation experiments. In the final degradation experiment on the 117-residue peptidyl-resin, a 92% efficiency for the automatic Edman reaction was measured ([3H]Leu, penultimate amino-terminal residue). We have found two advantages for the concurrent use of solid-phase Edman degradation during an extended solid-phase synthesis: (1) on the basis of the level of error due to incomplete incorporation of amino acids, the solid-phase assembly could be terminated in favor of restarting the synthesis, hence avoiding further work on a defective product and (2) direct verification of incorporation of amino acids, which during acid hydrolysis are destroyed (Cys, Trp) or are deamidated (Asn, Gln), is possible by high-pressure liquid chromatography of the corresponding Pth derivatives.

Amino Acid Sequence↗

PubMed2001

Protonation of platinated adenine nucleobases. Gas phase vs condensed phase picture.

Protonation of adenine carrying a Pt(II) moiety either at N7, N3, or N1 is possible in solution, but the site of protonation is influenced by the location of the Pt(II) electrophile and to some extent also by the overall charge of the metal entity (+2, +1, 0, -1), hence the other ligands (NH(3), Cl(-), OH(-)) bound to Pt(II). Quantum chemical calculations based on density functional theory (DFT) have been carried out for intrinsic protonation energies of adenine complexes carrying the following Pt(II) species at either of the three ring N atoms: [Pt(NH(3))(3)](2+) (1), trans- [Pt(NH(3))(2)Cl](+) (2a), cis-[Pt(NH(3))(2)Cl](+) (2b), trans-[Pt(NH(3))(2)Cl(2)] (3a), cis-[Pt(NH(3))Cl(2)] (3b), [PtCl(3)](-) (4), trans-[Pt(NH(3))(2)OH](+) (5a), cis-[Pt(NH(3))(2)(OH)](+) (5b), trans-[Pt(NH(3))(OH)(2)] (6a), cis-[Pt(NH(3))(OH)(2)] (6b), and [Pt(OH)(3)](-) (7). The data have been compared with results derived from solution studies (water) and X-ray crystallography, whenever available. The electrostatic effects associated with the charge of the metal entity have the major influence on the calculated intrinsic (gas phase) proton affinities, unlike the condensed phase data. Nevertheless, the relative gas phase trends correlate surprisingly well with condensed phase data; i.e., variation of the pK(a) values measured in solution is consistent with the calculated gas phase protonation energies. In addition to a systematic study of the ring proton affinities, proton transfer processes within the platinated adenine species were often observed when investigating Pt adducts with OH(-) ligands, and they are discussed in more detail. To the best of our knowledge, this is the first study attempting to find a systematic correlation between gas phase and condensed phase data on protonation of metalated nucleobases. The gas phase data provide a very useful complement to the condensed phase and X-ray experiments, showing that the gas phase studies are capable of valuable predictions and contribute to our understanding of the solvent and counterion effects on metal-assisted proton shift processes.

Adenine Nucleotides↗