bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors influencing breakthrough volumes when using sorption traps at high flow rates
Authors: Laurens J. B. , Rohwer Egmond R.
Published: May 2005

The effects of the sample flow‐rate and bulk and bulk sample composition on the breakthrough behaviour of H2 in small sorption traps were studied. The information is required for the bulk thermal properties of the sample affect the temperature inside the cooled traps and therefore also the breakthrough volume. It was also found that the brakthrough volume, measured downstream of a packed trap, can qctually increase with volume flow rate.
Characterization of complex hydrocarbon mixtures using on‐line coupling of size‐exclusion chromatography and normal‐phase liquid chromatography to high‐resolution gas chromatography
Authors: Blomberg Jan , Mes Edwin P. C. , Schoenmakers Peter J. , van der Does Jan J. B.
Published: May 2005

An on‐line coupling of size‐exclusion Chromatography (SEC), normal‐phase liquid Chromatography (NPLC), and gas Chromatography (GC) for the characterization of complex hydrocarbon mixtures is described. The hyphenated system separates according to size, polarity, and boiling point. The use of size exclusion as the first separation step allows for the direct injection of complex (“dirty”) samples withont prior clean‐up. SEC‐NPLC coupling was realized using an on‐line solvent evaporator based on fully concurrent solvent evaporation (FCSE) using a modified loop‐type interface, vapor exit and co‐solvent trapping. Complete reconcentration of the analytes was realized by the introduction of a cryogenic cold trap. For the subsequent hydrocarbon group‐type separation an ammo‐silica column with n‐heptane as eluent was used. The NPLC‐GC coupling was based on an on‐column interface using partially concurrent solvent evaporation (PCSE) and an early vapor exit. Initial results obtained on the analysis of a residue from the atmospheric crude‐oil distillation (a so‐called long residue) are presented as an example of the enormous separation power of the SEC‐NPLC‐GC system. The application of the system for quantitative analysis has not yet been studied.
Determination of p,p′‐DDE and PCBs in adipose tissues using high‐performance liquid chromatography coupled on‐line to capillary GC – the EURAMIC study
Authors: Gort Steven M. , van der Hoff G. Rene , Baumann Robert A. , van Zoonen Piet , Martin‐Moreno Jose M. , van't Veer Piet
Published: May 2005

Long‐term exposure to fat‐soluble xenobiotics is assessed by the concentration of DDE [1,1‐dichloro‐2,2‐bis(p‐chlorophenyl)ethyl‐ene], the persistent metabolite of DDT, in subcutaneous fat, aspirated from the buttocks of breast cancer patients and age‐matched controls, from five European centers collaborating in a case‐control study on breast cancer. In such studies using sample material of living subjects only small amounts of samples can be made available for analysis. In this particular study the only sample material available for the analysis of DDE were aliquots of the aspirates that were originally analyzed for fatty acids. Due to the small sample quantities available, e.g. aliquots of 200–800 p. 1, on‐line LC‐GC is most convenient because a major part of the sample can be used in the analytical procedure. In the LC‐GC procedure 50 μl of sample was injected on the LC column resulting in a 180 μl fraction containing the analytes of interest. The LC fraction was transferred to a capillary GC with electron capture detector by means of partially concurrent solvent evaporation. This way, sample handling is minimized thus reducing losses and preventing contamination. The feasibility of the on‐line LC‐GC system for the analysis of DDE is demonstrated with the analysis of 634 adipose tissue extracts in different series. The validity of this approach, using samples already analyzed for another parameter, with LC‐GC is clearly demonstrated by the fact that in over 97% of the samples DDE can be quantified. Thus rendering a meaningful data‐set for further epidemiological evaluation. The DDE levels found ranged from 0.99–3.13 μg/g adipose tissue.
Cyclodextrin‐modified micellar electrokinetic chromatography for separation of norgestrel enantiomers
Authors: Liu Yu , Gu Junling , Fu Ruonong
Published: May 2005

A simple method for the separation of highly hydrophobic neutral enantiomers by cyclodextrin‐modified micellar electrokinetic chro‐matography (MEKC) is presented and strongly supported by experiments. The separation depends on the ratio between the concentrations of cyclodextrin (CD) and sodium dodecylsulfate (SDS) micelles and there is an optimum value of the ratio for the separation of the enantiomers of norgestrel and 4‐androstene‐3,17‐dione. At the optimum value of the ratio, obtained by adjusting the absolute concentrations of CD and SDS. The electrophoretic mobility difference between two enantiomers can be maintained nearly constant.
Signal enhancement in capillary electrophoresis by using a sleeve cell arrangement for optical detection
Authors: Djordjevic Nebojsa M. , Widder Martin , Kuhn Keinhard
Published: May 2005

This study comparts signal enhancement and efficiency in CZK employing three modes of detection: the sleeve cell (a simple method for creating a region of extended path length for absorption detection), the bubble cell (extended light path capillaries), and on‐column detection in 75 μm i.d. capillary. Flow profile in the sleeve cell was monitored under a microscope. An abrupt change in capillary diameter in the sleeve cell region (from 50 μm to 220 μm) did not produce extensive band broadening. The sleeve‐cell detection arrangement delivered a 3.5 fold increase in corrected peak area when compared with an oil‐column detection in 75 μm i.d. column.
Determination of chlorinated and brominated micropollutants by capillary gas chromatography coupled with on‐column radio frequency plasma atomic emission detection
Authors: Asp Tone Normann , Pedersen‐Bjergaard Stig , Greibrokk Tyge
Published: May 2005

Capillary gas chromatography (GC) combined with on‐column radio frequency plasma atomic emission detection was evaluated for the determination of polychlorinated and polybrominated biphenyls (PCBs and PBBs). Quantitation was possible utilizing a single chlorine or bromine calibration curve based on a randomly selected reference compound, because the signal per ng of halogen ranged within 17 % for 29 congeners. Combined with an internal standard to correct for potential plasma quenching from matrix components, this type of universal quantitation represented a sub‐stantial simplification of current calibration procedures. In combi‐nation with relatively low detection limits (1–5 pg/s of halogen), the present work suggested that GC, coupled with on‐column atomic emission detection is a promising technique for the determination of halogenated micropollutants.
Enantioselective separations of chiral molecules by μ‐HPLC and SFC on microbore and packed microcapillary columns
Authors: D'Acquarica Ilaria , Gasparrini Francesco , Giannoli Barbara , Misiti Domenico , Villani Claudio , Mapelli Gian Pietro
Published: May 2005

New results obtained with microbore or microcapillary columns packed with DACH‐DNB CSP (spherical silica, 5 μm) using conventional LC eluents or supercritical fluids are reported. Examples of direct chromatographic separations of complex mixtures of stereo‐isomers on: a) long, packed microbore or microcapillary columns and b) several columns coupled in series are shown; in the latter case, a home‐made calculation procedure (Simulation for Multi‐Column Chromatography–SMCC) was used to quickly select the optimal conditions (kind of stationary phases and relative columns length).
Unusual fatty acids in compositae: γ‐Linolenic acid in Saussurea spp. seed oils
Authors: Tsevegsüren Nanzad , Aitzetmüller Kurt , Vosmann Klaus
Published: May 2005

The plant family Compositae is known to produce a set of unusual fattly acids in their seed oil. Saussurea, a genus of the Compositae is less studied in respect to the fatty acid compsition of their seed oil. Only Saussurea candicans was reported to contain crepenynic acid (33%) as seed oil component.

In continuation of our exploration of the portential of wild oil seeds, fatty acids in seed oils of seven Saussurea species (S. amara, S. salicifolia, S. lipschitzii, S. pseudoalpina, S. pricei, S. parviflora, and S. dorogostaiskii) growing in Mongolia a were investigated by means of capillary GLC on capiallary columns of different selectivity (Silar 5 CP and BPX 70).

γ‐Linolenic acid was found at levels up to 11% of the consitituent fatty acids of Saussurea spp. seed oils. This is the first time that γ‐Linolenic acid has been found in members of the plant family Compositae. Moreover, the number, position and configuration of the double bonds in γ‐linolenic acid and that of other fatty acids was additionally confirmed by silver ion thin layer chromatography and infrared spectroscopy. The occurence and distribution of γ‐linolenic acid, which has found considerable interest for pharmaceutical and dietary use, may be of chemotaxonomical significance in the plant family Compositae.
Enantiodifferentiation of α‐ketols in sherry by one‐ and two‐dimensional HRGC techniques
Authors: Häring Dietmar , König Thorsten , Withopf Barbara , Herderich Markus , Schreier Peter
Published: May 2005

The enantiomeric distrubution of solerone (5‐oxo‐4‐hexanolide) 1, ethyl 4‐hydroxy‐ 5‐oxohexanoate 2, ethyl 5‐hydroxy‐4‐oxohexanoate 3, 4‐oxo‐5‐hexanolide 4, and solerol (5‐hydroxy‐4‐hexanolide) 5 in sherry wines was determined by several HRGC techniques. While gas chromatography‐mass spectrometry on chiral cyclodextrin phases (chiral GC‐MS) prevented any racemization of α‐ketols 2 and 3 caused by keto‐enol tautomerization during analysis, multidimensional gas chromatography MDGC (coupled either by “live‐T” switching or by “moving column stream switching” MCSS) led to rearranged constitutional‐and stereoisomers. The stereochemical results are discussed regarding the biogenesis of sherry constituents 1–5.
Influence of the lateral alkoxy chain length on the gas chromatographic properties of five liquid crystals
Authors: Perez F. , Berdagué P. , Courtieu J. , Bayle J. P. , Boudah S. , Guermouche M. H.
Published: May 2005

The gas Chromatographic properties of five laterally substituted liquid crystals are the subject of a comparative study. These liquid crystals belong to the homologous series: 2‐alkoxy‐3‐methyl‐4‐(4‐ethoxybenzoyl‐oxy)‐4′‐(4‐trans‐n‐pentyclcyclohexanecarboxyloxy) azobenzene, referred to as ALn, where n, the carbon number in the lateral alkoxy chain, is equal to 4, 6, 8, 10, or 12 carbon atoms. Their thermal properties were established by differential scanning calorimetry (DSC). It appears that the introduction of a lateral alkoxy chain affects the nematic temperature range which decreases when the carbon number increases. The chromatographic separation abilities of AL4, AL6, AL8, AL10, and AL12 were studied using capillary glass columns. The five liquid cystals are efficient before and after solid‐nematic or nematic‐liquid transitions; however, the plate numbers are higher in the nematic state. Interesting analytical properties were noted in various fields: iisomeric separation of alkanes, aromatics, polyaromatics, volatile aroma compounds, and cis and trans isomers. However, the chain length does not exert any great influence on the separation abilities.
Development of methods for testing barriers in food packaging materials
Authors: Aurela Birgit , Tapanila Tarja , Osmonen Riitta‐Maija , Söderhjelm Lliva
Published: May 2005

In some countries recycled materials are not allowed to come into contact with food without a protective layer such as plastic or virgin fiber. The purpose of this kind of barrier is to reduce migration of substances form beyond the barrier to the food. Two methods for studying a functional barrier are described. Both utilize migration cells in which one surface of the test specimen (for example a paper plate) is exposed to the food simulant. The first method involves the addition of indidcatsor substances to the non‐food contact layer. The second method is to measure the migration of substances originally present in the layers beyond the functional barrier layer. Several kinds of barrier used in commercial papaer plates were studied and differences between them were found.
Compehensive two‐dimensional gas chromatography (GC×GC) and its applicability to the characterization of complex (petrochemical) mixtures
Authors: Blomberg Jan , Schoenmakers Peter J. , Beens Jan , Tijssen Robert
Published: May 2005

In general, petrochemical products contain only a limited number of chemical classes of compounds (sample dimensionality). The enormous number of individual components within these classes, however, soon puts limitations upon a single chromatographic technique when it comes to adequate characterization of these products. Comprehensive two‐dimensional gas chromatography (GC×GC) clearly opens the possibility of estimating the composition of hydrocarbon mixtures in a far more detailed fashion than hitherto possible. Although the emphasis of papers of GCxGC thus far almost exclusively applies to the unsurpassed peak‐capacity, in the oil industry there is a need for characterization, rather than for analyzing all the individual compounds. In principle a GCxGC system can provide an almost perfect match between its intrinsic properties and the dimensionality of oil samples. To establish the applicability of GCxGC towards petrochemical analytical challenges, a commercially aavailable prototype instrument was subjected to an exhaustive characterization of a typical hydrocarbon precess stream and a fast characterization of a light gas oil. Although there are no fundamental limitations towards the quantitative aspects of a GCxGC system, this paper confines itself to qualitative results only. Quantitative aspects of GCxGC will be published in a forthcoming paper.
Theory of fast capillary gas chromatography. Part 1: Column efficiency
Authors: Blumberg Leonid M.
Published: May 2005

Typically, fast GC requires high column pressure drop. Under that condition, known relations do not provide a complete description of dependence of [apparent] plate height, ĥ, on average velocity, u, of carrier gas.

A complete descriptions for ĥ vs. u is derived and analyzed. It is shown that, under the high pressure drop, ĥ = B/u2 + C1 u2 + C2 u. This is substantially different from the Van Deemter equation ĥ = B/u + Cu for the low pressure drop. Analytical expressions for the minimum plate height and for the respective optimum u under the high pressure drop are derived and analyzed.
Investigations on wine bouquet components by solid‐phase microextration ‐capillary gas chromatography (SMPE‐CGC) using different fibers
Authors: De la Calle García Demetrio , Reichenbächer Manfred , Danzer Klaus , Hurlbeck Christian , Bartzsch Christine , Feller Karl‐Heinz
Published: May 2005

The extraction of wine bouquet components with solid‐phase microextraction (SPME) using different fibers was studied. Polyacrylate fibers (PA‐85) achieve the most complete bouquet profiles. About ninety substances were identified by capillary gas chromatography‐MS and their retention factors were calculated to allow peak identification in capillary‐GC‐FID chromatograms. The terpene profiles of different wines were recorded by SPME (PA‐85)‐CGC‐FID.
Analysis and use of the serum albumin binding domains of streptococcal protein G
Authors: Nygren Per‐Åke , Eliasson Margareta , Abrahmsén Lars , Uhlén Mathias , Palmcrantz Elisabeth
Published: May 2005

Streptococcal protein G is an IgG‐binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG‐binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG‐ and albumin‐binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity Chromatography, using IgG or HSA immobilized on Sepharose, Showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin‐binding. The structure of this part of the proteins suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one‐step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.
Conformational homologies among cytokines: Interleukins and colony stimulating factors
Authors: Parry David A. D. , Minasian Elizabeth , Leach Sydney J.
Published: May 2005

Some 30 cytokine amino acid sequences (mainly interleukins, colony stimulating factors and tumor necrosis factors) have been examined for evidence of secondary structure as well as longer‐range interactions of a type likely to lead to stable α‐helical bundles. Most, though not all, of the cytokines examined have a high predicted α‐helical content (40–60%) and quasi‐repeating heptads containing i/i+3 apolar periodicities. This major subset of the cytokines is predicted to be characterized by molecules in which 4‐α‐helical bundles with an average length of 25Å are the most marked conformational features. Based on these conclusions, we suggest structures for huG‐CSF, huGM‐CSF and muIL‐5 in which defined loop segments at the ends of helical bundles are the most likely sites for binding and recognition by specific cell receptors. As such, they provide a means for testing or refining the three working models we have defined, using currently available methods of site‐directed substitution and deletion mutagenesis, as well as synthetic peptides corresponding to the proposed loop sequences and the use of monoclonal antibodies of defined epitopic specificity. The structure arrived at for huGM‐CSF is consistent with the limited data currently available concerning the residues which are important for binding and activity.
Molecular basis for potentiation of bleomycin‐mediated degradation of DNA by polyamines. Experimental and molecular mechanical studies
Authors: Strekowski Lucjan , Harden Donald B. , Wydra Roman L. , Stewart Kent D. , Wilson W. David
Published: May 2005

The bleomycin‐mediated degradation of DNA is stimulated (amplified) by certain DNA binding compounds, such as polyamines, that distort the double helix. Computer modelling studies suggest that putrescine (1), spermidine (2), and spermine (3) bind preferentially on the floor of the major groove of (dGdC)5·(dGdC)5. This interaction results in a bend of the oligomer helix toward the major groove and enlargement of the minor groove, both effects being in the order 1 < 2 < 3. These polyamine‐induced distoritions, as obtained from theoretical studies, parallel the experimental values of the amplification activites of 1–3 in the bleomycin‐mediated degradation of poly(dGdC)·poly(dGdC). The amplification mechanism of non‐competitive binding of amplifier molecules in the major groove, and bleomycin in the minor groove, is proposed. It is suggested that the amplifier‐induced conformational changes of the DNA helix increase affinity of the activated bleomycin complex toward the DNA minor groove and, consequently, result in an increased efficiency of the bleomycin‐mediated degradation of the helix.
Preparation of a functional, highly selective polymer by molecular imprinting. A demonstration with L‐p‐aminophenylalanine anilide as a template molecule allowing multiple points of attachment
Authors: Moradian Aydin , Mosbach Klaus
Published: May 2005

The technique of molecular imprinting was tested using various phenylalanine derivatives. It could be shown that polymers obtained by imprinting with L‐p‐aminophenylalanine anilide gave best recognition of the original L‐form, with separation factors (α) above 8, on applying racemic mixtures to the polymer obtained and present in a HPLC column. We believe that these results are due to multiple points of attachment that are possible between print or template (guest) molecules and the formed polymer (host).
Sequence‐selective, pH‐dependent binding to DNA of benzophenanthridine alkaloids
Authors: Bajaj Narinder P. S. , McLean Michael J. , Waring Michael J. , Smekal Emil
Published: May 2005

The sequence selectivity associated with binding to DNA of three alkaloids belonging to the benzophenanthridine family has been analysed by DNase I footprinting, and the results were compared with those obtained from an analysis of the behaviour of the standard intercalator, ethidium bromide. Like the ethidium, the benzophenanthridine compunds appear to bind best to regions of mixed nucleotide sequence, especially those containing alternating purines and pyrimidines, although there are some notable differences in behaviour. There is also a marked lack of binding to sequences such as (AT)n, where n⩾3. The binding to DNA of the benzophenanthridines is specifically related to the hydrogen ion concentration of the medium, in that the DNase I footprints are considerably enhanced when the reaction is performed at a pH below 7.0. We discuss these results in terms of a greater preponderance of the intercalating species being present at lower pH.
Conformational constraints: Nonpeptide β‐turn mimics
Authors: Ball Jonathan B. , Alewood Paul F.
Published: May 2005

The β‐turn, which has also been referred to as the β‐bend, β‐loop or reverse turn, has been implicated as an important site for molecular recognition in many biologically active peptides and in globular proteins. This small secondary structure therefore makes an attractive target for mimicry by a conformational constraint, because a peptide which is constrained in a biologically active conformation can display a number of advantages over the parent substrate. The less peptide‐like such a constraint is, the more potential there is to maximize these advantages. A decade has passed since the first (and highly successful) attempt to mimic the β‐turn with a nonpeptide conformational constraint was disclosed by Freidinger et al. (1980). Since this report, rapidly growing interest in the field of nonpeptide β‐turn mimics has seen a variety of experimental approaches and a mixed bag of results. It is attempted in this review, not only to summarize and critically analyse these approaches, but also to touch on the compexities associated with the conformational mimicry of such a diverse structure as the β‐turn.
27 amino acid residues can be deleted from the N‐terminus of human lymphotoxin without impairment of its cytotoxic activity
Authors: Nishikawa Satoshi , Matsuo Noriyuki , Isaka Yoshitaka , Uesugi Seiichi , Miura Kazunobu , Ohtsuka Eiko , Takeda Ken , Konno Kunio , Ikehara Morio
Published: May 2005

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N‐terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LTδ37N, in which 37 residues were absent from the N‐terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LTδ28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LTδ27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.
Antibody affinity measurements
Authors: Azimzadeh A. , Van Regenmortel M. H. V.
Published: May 2005

The use of antibodies in immunoaffinity separations represents one of the most specific methods for purifying substances of biological interest. Since the binding affinity of antibody greatly influences its behavior in such separations, it is often important to know the value of the antibody affinity expressed as an equilibrium constant K. The present review discussed the equations used in the quantitative analysis of antigen/antibody interactions and describes currently used experimental methods for measuring K values. Advantages and shortcomings of the solution phase and solid phase approaches used for measuring antibody affinity are discussed.
New developments in affinity chromatography
Authors: Lowe Christopher R. , Burton Steven J. , Burton Nicolas , Stewart David J. , Purvis Duncan R. , Pitfield Ian , Eapen Saji
Published: May 2005

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.
Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver
Authors: Spacciapoli Peter , Thurlow David L.
Published: May 2005

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidylransferase from rabbit liver was studied using 12 tRNAs, previously treated with the chemical modifier diethylpyrocarbonate (DEP). Such chemically modified tRNAs were labeled with 32P by nucleotidyltransferase, using α‐[32P]ATP as a cosubstrate. A carbethoxylated purine at position 57 in the Ψ‐loop interfered with recognition of the tRNA in all instances. DEP‐modified purines at other positions (58 in the Ψ‐loop, 52 or 53 in the Ψ‐stem, and 71–73 in the acceptor stem), also interfered with the interaction, but in only a few tRNAs. The mammalian enzyme was more similar to the homologous enzyme from yeast than that from bacteria, in its requirements for chemically unmodified purines. The extent of exclusion of modified bases from 32P‐labeled material diminished as the concentration of enzyme increased, demonstrating that interference was not due to the inability of the chemically altered tRNA to refold into a recognizable conformation. The degree of purification of the enzyme did not affect the identity of bases that inhibited the reaction when modified.
X‐ray crystal structure of the bovine α‐chymotrypsin/eglin c complex at 2.6 Å resolution
Authors: Bolognesi Martino , Pugliese Luisa , Gatti Giuseppina , Frigerio Francesco , Coda Alessandro , Antolini Luciano , Schnebli Hans Peter , Menegatti Enea , Amiconi Gino , Ascenzi Paolo
Published: May 2005

The crystal structure of the molecular complex formed by bovine α‐chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 Å resolution were employed to determine the structure by molecular replacement techniques. Bovine α‐chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 Å resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine α‐chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine α‐chymotrypsin between subsites P3 and P′2; Leu 45I acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic‐ and secretory‐ inhibitors are found around the scissile peptide bond.
Binding of the neuroleptic drug haloperidol to a monoclonal antibody: Refinement of the binding site molecular model using canonical structures
Authors: Anchin Jerry M. , Subramaniam Shankar , Linthicum D. Scott
Published: May 2005

The ligand binding site of a monoclonal antibody (185), which binds the neuroleptic drug haloperidol, has been modelled using canonical structures and energy minimization techniques. This refined modelling protocol has allowed us to predict the variable region loop conformation. Three key residues, H:50(W), H:100a(D) and L:96(Y) appear to create the basis of the electrostatic, π–π stacking interactions and hydrogen bonding required for the high affinity binding site characteristics present in this antibody. The use of computer‐aided graphics techniques and appropriate three‐dimensional modelling permits inspection of the predicted molecular recognition features of the ligand binding site.
Cell interactions of enkephalin/polypeptide conjugates
Authors: Tsukada Motohiko , Sasaki‐Yagi Yoshiko , Kimura Shunsaku , Imanishi Yukio
Published: May 2005

Enkephalin molecules were bound to poly(Lys) (poly‐K) or poly(Ala‐Lys‐Ala‐Leu) (poly‐A) and their interactions with NG108‐15 cells, platelets, erythrocytes and fibroblast cells were investigated. A fluorescent probe, rhodamine, also was bound to the conjugates for monitoring interactions with these cells. Observations by fluorescence microscopy revealed that NG108‐15 cells, and fibroblast cells were labelled by the conjugates, whereas erythrocytes were not. Since polypeptides without enkephalin moieties were only weakly adsorbed on the cells, it was concluded that the enkephalin/polypeptide conjugates were bound specifically to receptors on the cells membrane. Interestingly, when the enkephalin/poly‐K conjugate was bound to NG108‐15 and fibroblast cells, fluorescent patches appeared on the membrane. Such patch formation was not clearly observed with an enkephalin/rhodamine or enkephalin/poly‐A conjugate. In the case of fibroblast cells, the fluorescence converged to a large cluster, which was ultimately internalized. The results suggest that clustering of the receptors in cell membranes in influenced by the carrier polymer presumably due to cross‐linking of the receptors and/or the effect of the cationic polypeptides.
A 500 MHz proton NMR study of stacking interactions: Binding of tripeptide Lys‐Tyr‐Lys to tetradeoxynucleotide d‐GpCpGpC
Authors: Barthwal R. , Mujeeb A. , Kukreti S. , Gupta A. , Govil G.
Published: May 2005

The complete sequential assignment and conformation of d‐GpCpGpC in D2O has been determined from ID NMR spectra at 285–320 K and room temperature 2D‐COSY and NOESY spectra. The tetradeoxynucleotide exists primarily as a right handed double helix at 285 K, having Tm as 314 K. On binding to a tripeptide Lys‐Tyr‐Lys in a concentration equimolar to tetranucleotide duplex, the Tyr ring protons shift uplifted by 0.14 ppm at 285 K. The increase in Tm on binding suggests stabilization of duplex. The existence of intermolecular NOEs between C4 sugar protons and Tyr αC and Lys αC protons give direct evidence of proximity of Tyr residue to the C4 base of d‐GpCpGpC. The conformation of d‐GpCpGpC remains unchanged on binding. The observed results are interpreted in terms of preferential stacking of aromatic ring of Tyr residue with proximal base‐pair of d‐GpCpGpC, stabilized by electostatic interaction of Lysine side chains with backbone phosphates. This is in contrast to intercalculation of aromatic dyes within base‐pairs resulting in a change in sugar conformation at the binding site.
‘Hot spot’ amino acid distribution in Ha‐ras oncogene product p21: Relationship to guanine binding site
Authors: Cosic Irena , Hearn Milton T. W.
Published: May 2005

Although the rules which describes the atomic basis of structure–function relationships of proteins have yet to be deciphered, they are nevertheless coded within the framework of the amino acid and nucleotide sequence. The objectives of the present investigations were to document a composite, new approach for the evaluation of the structure–function dependencies of proteins based on the analysis of the informational content of the primary amino acid sequence as well as the topological and functional regions of a protein. This approach is validated with the example of the p21 Ha‐ras oncogene family of proteins. Using this approach is validated with the example of the p21 Ha‐ras oncogene family of proteins. Using this approach, amino acids crucial for p21 transforming activity have been identified and these amino acid residue assignments compared with experimental data.
A strategy for efficient characterization of macromolecular heteroassociations via measurement of sedimentation equilibrium
Authors: Hsu Carol S. , Minton Allen P.
Published: May 2005

A method is proposed for the selection of experimental conditions for sedimentation equilibrium experiments that will provide maximal information about the values of equilibrium association constants within a given scheme for heteroassociation of two solute components. A discriminator function is proposed that indicates the sensitivity of the experimentally observed gradient or gradients to alternations in the underlying association constants. The value of this function is plotted or tabulated as a function of the concentrations of the two components, over a broad range of solution compositions. It is suggested that experiments performed with loadings compositions corresponding to large absolute values of the discriminator function will yield the most information with respect to determination of the underlying association constants. This method was tested by predicting optimal conditions for three different types of sedimentation equilibrium experiments: (i) measurement of total (natural) solute absorbance; (ii) measurement of individual component gradients via measurement of tracer absorbance; and (iii) global analysis of multiple experiments. Experimental data resulting from sedimentation equilibrium experiments carried out under the specified conditions were simulated by addition of realistic levels of random error to calculated equilibrium gradients. The simulated data were then analyzed exactly as real experimental data, i. e. without prior knowledge of the underlying association constants. It was found that the highest accuracy and precision in determination of heteroassociation constants are obtained by global analysis of multiple experiments performed using significantly different loading compositions, each of which is selected from ‘sensitive’ regions of the discriminator map.
Operational aspects of antibody affinity constants measured by liquid‐phase and solid‐phase assays
Authors: Azimzadeh A. , Pellequer J. L. , Van Regenmortel M. H. V.
Published: May 2005

The association constant of monoclonal antibodies (MAbs) to tobacco mosaic virus has been determined in solution and solid‐phase binding assays. The ElISA equilibrium titration method developed by Friguet et al. (1985) was found to be suitable for large antigens such as viruses. In the case of intact IgG antibody, it gave equilibrium constant (K) values ca 30% lower than those obtained by classical solution‐phase assay while in the case of Fab', the same values were obtained in both assays. Solid‐phase binding assays gave higher K values than solution‐phase assays by a factor which varied with the Mab tested (1.5‐ to 5.4‐fold higher). Furhtermore, in solution‐phase assay, K values were found to depend on the antibody concentration used in the assay. These results confirm the operational nature of antibody affinity constants and indicate that in order to compare the affinity of different Mabs in a meaningful way, it is necessary to use a single technique under standardized conditions.
Design of novel cationic ligands for the purification of trypsin‐like proteases by affinity chromatography
Authors: Burton Nicolas P. , Lowe Christopher R.
Published: May 2005

A number on new cationic ligands have been designed and synthesized for the selective resolution an purification of the trypszin‐like proteases. A series of ligands based on 4‐[2′‐methyl‐4′‐(2″,4″‐dichloro‐1″,3″,5″‐triazin‐6‐ylamino) phenylazo]benzamidine were able to bind to trypsin and the trypsin‐like proteases, thrombin and urokinase, but bound pancreatic kallikrein only weakly. Ligands possessing a second cationic group (either 4‐aminophenyltrimethylammonium or 4‐aminobenzamidine) substituted onto the triazine ring displayed higher affinities than the parent compound for trypsin in solution but bound the enzyme weakly or not at all after immobilization. In contrast, these bis‐cationic ligands bound pancreatic kallikrein in solution ad following immobilization. The presence of the second cationic group was crucial, since its replacement by neutral or anionic groups led to loss of affinity for pancreatic kallikrein. One of the bis‐cationic ligands was used to purify pancreatic kallikrein 9.5‐fold from a crude pancreatic extract in 79% yield, to generate a product 99.9% free of contaminating trypsin activity.
Using a neural network to identify potential HLA‐DR1 binding sites within proteins
Authors: Bisset Leslie R , Fierz Walter
Published: May 2005

The presentation by antigen‐presenting cells of immunodominant peptide segments in association with major histocompatibility complex (MHC) encoded proteins is fundamental to the efficacy of a specific immune response. One approach used to identify immunodominant segments within proteins has involved the development of predictive algorithms which utilize amino acid sequence data to identify structural characteristics or motifs associated within vivoantigenicity. The parallel‐computing technique termed ‘neural networking’ has recently been shown to be remarkably efficient at addressing the problem of pattern recognition and can be applied to predict protein secondary structure attributes directly from amino acid sequence data. In order to examine the potential of a neural network to generalize peptide structural feature related to binding within class II MHC‐encoded proteins, we have trained a neural network to determine whether or not any given amino acid of a protein is part of a peptide segment capable of binding to HLA‐DR1. We report that a neural network trained on a data base consisting of peptide segments known to bind to HLA‐DR1 is able to generalize features relating to HLA‐DR1‐binding capacity (r= 0.17 andp= 0.0001).
Kinetic measurements of molecular interactions by spectrofluorometry
Authors: Voss Edward W.
Published: May 2005

The kinetics of antibody–antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti‐fluorescein antibodies are featured in the review as model proteins to explore fluorescence‐based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti‐fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k1) and dissociation (k2). The near diffusion‐limited k1 rates and the prolonged k2 rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti‐metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformational substates.
Peptide vaccines incorporating a ‘promiscuous’ T‐cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity
Authors: Kaumaya Pravin T. P. , Kobs‐Conrad Susan , Seo Young Hoon , Lee Hyosil , Vanbuskirk Anne M. , Feng Ningguo , Sheridan John F. , Stevens Vernon
Published: May 2005

An ideal peptide vaccine should contain both B‐ and T‐cell epitopes. Recognition of antigen by B cells is highly dependent on the three‐dimensional conformation of the antigen whereas T cells recognize antigen only after it has been processed to release a peptide fragment which is bound to the major histocompatibility complex (MHC) class II molecule. However, T cells provide ‘help’ to B cells displaying the same processed, MHC‐restricted from of the antigen, demonstrating that the T‐cell response to a protein antigen is under genetic control. Thus, strategies for co‐inclusion of T cell ‘helper’ epitopes with the B‐cell determinant elicit immune responses that are in most cases genetically restricted to only one or a few alleles of the MHC with limited activity across divergent MHC class II haplotypes. This genetically restricted T cell stimulatory activity of peptides is a serious obstacle and consequently such constructs would be of limited practical value as a vaccine targeted to a majority of an outbred population. In the study described here, we have engineered tow peptides to encompass the sequences from the universally immunogenic tetanus toxoid (TT) epitope and the contraceptive vaccine candidate lactate dehydrogenase C4 (LDH‐C4). We demonstrate the feasibility of using ‘promiscuous’ T‐Cell epitopes colinearly constructed with a defined B‐cell epitope to induce high titer antipeptide IgG antibodies specific for native protein antigen LDH‐C4 in several inbred strains of mice, outbred mice and rabbits. There appears to be a strong correlation between the capacity for the hybrid peptides to be stimulatory for the corresponding T cells in C57BL/6 (H‐2b) and C3H/HeJ (H‐2k) mice and their ability to be immunogenic. This correlation, however, appears to break down in H‐2d strains of mice since no antibodies were detected in BALB/c and barely detectable levels of antibodies in B10.D2 although activated T cells were detectable. Conversely, high titers of antipeptide antibodies are elicited in some strains (B10.BR) (H‐2k); C57BL/10 (H‐2k) without detectable IL‐2 responses. Finally, we show that a determinant which was previously restricted to H‐2k can be rendered immunogenic in H‐2b with the ‘promiscuous’ TT epitope. Thus, certain haplotype‐restricted immune responses can be bypassed, setting forth the ground work for the design of a universal vaccine by broadening the effective response in a larger number of individuals typically of the genetically diverse outbred human population.
Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection
Authors: Okuda Kenji , Kaneko Tamiko , Yamakawa Tadashi , Tanaka Shunichi , Shigematsu Takashi , Yamamoto Akihiro , Hamajima Kenji , Nakajima Kiichiro , Kawamoto Susumu , Phanuphak Praphan
Published: May 2005

We synthesized one V3 peptide each from HTLV‐IIIB, Thai A and Thai B, conjugating then to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP‐30). These peptide were then coupled to 8‐lysine copolymers using N‐succinimidyl maleimido carboxylate (Mr = ca 60 000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptide were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti‐peptide antibodies to each V3loop peptide and the HGP‐30 peptide. Strong inhibition of CD4+ dependent cell fusion was obtained with these antisera when IIIb, Thai A and Thai B strains of human immunodeficiency virus (HIV) were used. Strong anti‐fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti‐HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL‐2 production in lymphocytes was observed in p24 assay systems using these immunized antisera. Activation of IL‐2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV‐1.
Enzymology in vivo using NMR and molecular genetics
Authors: Brindle Kevin M. , Fulton Alexandra M. , Williams Simon‐Peter
Published: May 2005

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non‐invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo
Glycolytic enzyme–tubulin interactions: Role of tubulin carboxy terminals
Authors: Volkar K. Warren , Knull Harvey R.
Published: May 2005

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of α‐or β‐tubulin by cleaving a peptide fragment from the C‐terminals. Generation of α′β′‐tubulin, which is cleaved at both the α‐ and β‐subunit terminals, and αβ′‐tubulin, which is cleaved at the β′‐subunit C‐terminal, have already been reported. In this work an isotype, α′β‐tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of α led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3‐phosphate dehydrogenase. When the α‐subunit C‐terminal was intact, inhibition was greatest. These results imply that the C‐terminal of the tubulin α‐subunit is subunit is responsible for interactions with glycolytic enzymes.
Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement
Authors: Nielsen Peter E. , Egholm Michael , Buchardt Ole
Published: May 2005

The binding of PNA (peptide nucleic acid) T2CT2CT4‐LysNH2 to the double‐stranded DNA target 5′ ‐A2GA2GA4 was studied by KMnO4 and dimethylsulfate (DMS) probing. It is found that upon sequence‐specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N‐7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 that at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C+G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA·DNA‐PNA triplex.