bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calculation of modified capacity factor in micellar electrokinetic chromatography
Authors: Idei Miklós , Kiss Eva , Kéri György 
Published: May 2005

AbstractIn addition to the expression, k′ = (tm−to)/to (1–tm/tmc), we propose the expression k″ = (tm−to)/(tmc−to) to calculate the capacity factor in micellar electrokinetic chromatography (MEKC), where tm, to, and tmc are the migration time of the analyte, the flow marker, and the micelles, respectively. The k′ and k″ values that were obtained from simulated data as well as from MEKC analysis of different peptides (in 100 mM sodium dodecyl sulfate/0.1 N sodium borate buffer at pH 11.0) were calculated and compared. The k″ value is equal to zero for an analyte remaining in the aqueous phase whereas it is equal to one for an analyte always staying in the micellar phase. By applying k″ a finite capacity factor can be obtained for an analyte, indicating its partition between the two moving phases (aqueous and micellar) even in those cases when tm equals tmc. The slope of the curve k″ as a function of tm is constant through the whole migration window and therefore peak compression does not occur when applying k″ to calculate the capacity factor. A given difference in k″ corresponds the same difference in migration times and this value does not depend on the position within the migration window. Since k″ is a normalized paramete it is easy to evaluate the significance of a given difference in capacity factor or to estimate the relative position of an analyte with a given capacity factor in the migration window by applying k″. Therefore, k″ seems to be an adequate parameter to calculate the capacity factor in MEKC and, similar to k′, it also refers to the hydrophobicity of the analyte.
Separation and isolation of subcellular‐size particles by electrophoresis in polymer solution using the commercial scanning apparatus
Authors: Chang Huang‐Tung , Zakharov Sergey F. , Chrambach Andreas 
Published: May 2005

AbstractElectrophoresis of fluorescently labeled rat liver microsomes and polystyrene carboxylates of 10 and 30 nm radius was conducted in buffered 10–15% polyvinylpyrrolidone (Mr = 106) solutions, using a horizontal gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE‐1000, LabIntelligence). Banding, constant migration rates and Ferguson plots were obtained in these polymer solutions. The major microsome band detected by the automated scan was located visually on the gel by means of its fluorescein label and was isolated by volumetric withdrawal; recovery was monitored by scanning and ascertained to be near quantitative after three consecutive steps, in each of which 30 μL were withdrawn. This preparative method promises to be generally applicable to particles that are too large to enter into gels.
Protein degradation in human pure pancreatic juice analyzed by two‐dimensional gel electrophoresis
Authors: Furui Toshifumi , Ikeda Miyuki , Chao‐Ming Liao , Okita Kiwamu , Nakamura Kazuyuki 
Published: May 2005

AbstractTwo‐dimensional gel electrophoresis (2‐DE) was used to study protein degradation in human pure pancreatic juice (PPJ) which was collected at 5 min intervals for 20 min by selective endoscopic cannulation of the main pancreatic duct. In PPJ collected from healthy subjects no significant degradation was observed by incubating PPJ at 37°C up to 6 h. By further incubation for 24 h, glycoprotein‐1, procarboxypeptidase A‐1 and lipase were nearly completely degraded, while α‐amylase and procarboxypeptidase B‐1 were not degraded under these conditions; α‐amylase became labile in the presence of 1 mM ethylene diaminetetraacetic acid (EDTA) or 10 mM phenyl methyl sulfonyl fluoride (PMSF). Protein degradation was observed by 2‐DE of an initial fraction of PPJ collected from patients with chronic calcific pancreatitis (CCP). The 2‐DE patterns of subsequent fractions resembled those of PPJ from healthy subjects. The mixture of the last fraction with the initial fraction showed significant protein degradation, inhibited by adding aprotinin. Furthermore, the extent of protein degradation correlated with the dilatation of the main pancreatic duct as a consequence of intraductal stagnation of pancreatic juice. These findings demonstrate that protein degradation in PPJ is accelerated by intraductal activation of serine proteases in the case of patients with CCP. 2‐DE of PPJ from patients with CCP provides useful information for the evaluation of intraductal activation of zymogens and the progress of chronic pancreatitis.
Structural analysis and identification of gel‐purified proteins, available in the femtomole range, using a novel computer program for peptide sequence assignment, by matrix‐assisted laser desorption ionization — reflectron time‐of‐fligh — mass spectrometry
Authors: Gevaert Kris , Verschelde Jean‐Luc , Puype Magda , Van Damme Jozef , Goethals Marc , de Boeck Stefaan , Vandekerckhove Joël 
Published: May 2005

AbstractA procedure is described for structural characterization and identification of proteins, purified by either one‐ or two‐dimensional gel electrophoresis in the low picomole to femtomole range. The purified proteins are first detected in the primary gels by the sensitive reverse staining procedure described by Fernandez‐Patron et al. (Anal. Biochem. 1995, 224, 203–211) and consecutively reeluted from combined gel pieces and concentrated in the tip of a Pasteur pipette in a secondary gel matrix consisting of either sodium dodecyl sulfatepolyacrylamide or agarose. The concentrated proteins are in‐matrix‐digested and the resulting peptides are separated by reverse‐phase high performance liquid chromatography (HPLC) combined with microsequencing or analyzed by matrix‐assisted laser desorption ionization — time of flight — mass spectrometry. Protein identification is based on sequence homology or on the peptide mass pattern. The matching peptide sequences can additionally be verified by matching their measured post‐source decay spectra with the calculated fragmentation patterns of the isobaric candidate peptides appearing on the search list. This is done by a computer program referred to as MassFrag, described in this paper. We demonstrate that it is possible to identify protein that are only available in the femtomole range and whose sequences are stored in nonredundant protein databases or nucleotide and expressed sequence tag databases.
Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers
Authors: Bossi Alessandra , Righetti Pier Giorgio , Visco Carlo , Breme Umberto , Mauriello Maurizio , Valsasina Barbara , Orsini Gaetano , Wenisch Elisabeth 
Published: May 2005

AbstractOn isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full‐length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N‐terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C‐terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val‐Ser at the N‐terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post‐synthetic modifications often encountered in r‐DNA proteins.
Protease‐catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix‐assisted laser desorption/ionization mass spectrometry
Authors: Schnölzer Martina , Jedrzejewski Paul , Lehmann Wolf D. 
Published: May 2005

AbstractProteins were digested in normal and highly 18O‐enriched water using proteases commonly employed for protein sequencing. The extent of 18O incorporation into the resulting peptide fragments was characterized by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The endoproteinases trypsin, Lys‐C and Glu‐C incorporate two atoms of 18O, resulting in a mass shift of +4 D for the peptide fragments. This indicates that, following proteolytic cleavage, peptide products continue to interact with these proteases and undergo repeated binding/hydrolysis cycles, resulting in complete equilibration of both oxygens in the carboxy terminus of the fragments with oxygen from solvent water. In contrast, chymotrypsin and Asp‐N incorporate only one atom of 18O, resulting in a mass shift of +2 D, indicating that after the cleavage step these proteases do not accept the peptides as substrates. In addition, it was found that the proteases trypsin, Glu‐C, and Lys‐C exhibit minor or nontypical sequence specificities, resulting in unexpected peptide fragments. These fragments incorporate only one 18O atom, indicating that they do not undergo further binding/hydrolysis cycles with the enzyme. Thus, it is possible to discriminate between enzyme‐typical peptide fragments with mass shifts of +4 D and nontypical fragments with mass shifts of only +2 D. Based on these observations, protein digest strategies are described for the generation of 1:1 ion doublets spaced either by 2 or 4 D. In addition, the C‐terminus of a protein can be identified by the absence of an ion doublet in the corresponding peptide fragment. In protein sequencing by mass spectrometry, digest protocols generating ion doublets provide the most clear‐cut analytical results for the recognition of ion series in ESI‐MS/MS and MALDI post‐source decay (PSD) product ion specta. Only the mass spectrometric fragment ions of a C‐terminal series show ion doublets spaced either by 2 or 4 D, whereas the fragment ions belonging to an N‐terminal series remain unshifted. This assignment unequivocally reveals the direction of the identified sequence.
Intrinsic curvature of plasmid DNAs analyzed by polyacrylamide gel electrophoresis
Authors: Strutz Kurt , Stellwagen Nancy C. 
Published: May 2005

AbstractThe electrophoretic mobility of two small DNA plasmids, pUC19 and Litmus 28, linerarized by digestion with a variety of single‐cut restriction enzymes, has been studied. The permuted sequence isomers migrate with identical mobilities in agarose gels, as expected, but exhibit different mobilities in large‐pore polyacrylamide gels, suggesting that the parent plasmids contain sequencespecific sites of curvature and/or anisotropic flexibility. Both plasmids contain apparent bend centers near their origin(s) of replication; pUC19 also has a major apparent bend center near the promoter of the ampicillin resistance gene. These apparent bend centers are observed under a variety of experimental conditions, suggesting that they correspond to sites of stable curvature in the parent plasmids. Both plasmids also contain minor bend centers that are observed under a sub‐set of electrophoretic conditions and disappear when divalent cations are added to the solution, suggesting that these apparent bend centers may correspond to localized regions of variable flexibility.
Dynamic symmetry breaking in a model of polymer reptation
Authors: Aalberts Daniel P. , Van Leeuwen J. M. J. 
Published: May 2005

AbstractA numerical study of the transition between oriented and nonoriented polymers in a model for reptation is presented. An electric field biases a charged polymer to drift in the direction of its leading end. At low fields this bias is not enough to orient the polymer, but at larger fields it succeeds in orienting the polymer with a definite head and tail. The resulting motion depends on the relative magnitudes of the field and the chain length. For a given chain length, the field plays the role of inverse temperature in this transition. The critical field between these “phases” is found to be a non‐trivial power of the length of the chain. This scaling extends over two full decades in the coupling parameter and the simulations performed involved 2 × 109 Monte Carlo steps per data point.
Theory of gel electrophoresis of DNA: Orientational effects
Authors: Subbotin Andrei V. , Semenov Alexander N. 
Published: May 2005

AbstractSome aspects of the reptation model of gel electrophoresis of DNA, with emphasis on the orientational effects, are considered. A master equation for the reptational dynamics with the end boundary conditions is proposed. Based on this equation, the orientation and dynamics of the persistent‐chain are investigated. In particular, the role of trap conformations for this chain, which essentially reduce the chain mobility, was analyzed. A transient orientation of the chain during pulsed field gel electrophoresis is also discussed.
Cyclic migration of DNA in gels: DNA stretching and electrophoretic mobility
Authors: Åkerman Björn 
Published: May 2005

AbstractAvailable data from spectroscopic and microscopy studies of electrophoretic orientation of long DNA (above 40 kbp) in agarose gels is analyzed on the basis of the fact that the migration in constant fields is cyclic in nature. Defining a cycle period as the time between two consecutive compact states, a simple model is used to obtain data on the average time period (〈T〉) and the step length (〈L〉) of the migration cycle from spectroscopic measurements of the dynamics of helix orientation and center‐of‐mass velocity. Furthermore, the degree of orientation is used to analyze tube‐orientation and DNA stretching contributions to 〈L〉 and 〈T〉. Finally, the average electrophoretic velocity v = 〈L〉/〈T〉 is analyzed in terms of 〈L〉 and 〈T〉 for different DNA sizes (Lc), field strengths (E), and gel concentrations (A). The main results of the analysis are: (i) the increase and saturation of the electrophoretic mobility with increasing E is mainly governed by 〈L〉 via the degree of DNA stretching, (ii) DNA molecules of different sizes migrate with the same velocity because 〈L〉 and 〈T〉 both increase approximately linearly with Lc, and (iii) migration in a denser gel is slower mainly because 〈T〉 increases, while the step length is approximately constant. Assuming the charge Q of DNA is the same as in free solution, these results suggest that the reason the fundamental reptation equation for the electrophoretic mobility μ = (Q/ζ) < (hx/Lt)2 > also applies in the presence of strong fluctuations in the tube length Lt and end‐to‐end distance hx, is that the friction coefficient ζ for motion along the tube is lower the more stretched the DNA is.
Pulsed‐field electrophoresis in microlithographic arrays
Authors: Duke Thomas A. J. , Austin Robert H. , Cox Edward C. , Chan Shirley S. 
Published: May 2005

AbstractTransverse pulsed‐field electrophoresis of DNA has been conducted in a silicon array engineered by optical lithography and the motion of individual molecules observed by fluorescence microscopy. In strong fields, the molecules can be maintained in highly stretched, linear conformations. When the field is switched through an obtuse angle, they head off in the new direction led by what was formerly their tail end. This backtracking gives rise to fractionation that is linear with molecular weight. A simple prescription exists for choosing the field parameters to obtain a particular range of separation. Since the molecular motions are much more uniform than those that occur in a gel, it is anticipated that the arrays will permit more efficient fractionation than traditional pulsed‐field gel electrophoresis. Arrays suitably scaled down in size may be useful for pulsed‐field sequencing.
Theory of solitary waves in electrophoresis
Authors: Kist Tarso B. L. 
Published: May 2005

AbstractA theoretical study about the generation of solitary waves (SW) in electrophoresis and capillary electrophoresis is performed. Two models that use velocity terms in Fick's first law and absorption and decaying terms in Fick's second law are presented. These models give the time evolution of the band or zone shape in electrophoresis and capillary electrophoresis in the presence of electromagnetic radiation. In particular, the effect of electrophoretic mobility changes of the analytes due to radiation excitation is included in the present models. The analytes are represented either by three‐level or four‐level systems. It is shown that the resulting system of coupled nonlinear partial differential equations governing the spatial motion of these bands over time exhibits SW solutions for some values of the equation parameters. We analyze the conditions in which these SWs, which propagate with constant velocity, constant area, constant standard deviation, and without change of form, are generated. The results of the present models are compared with those from a previous two‐level model (Phys. Rev. Lett. 1995, 75, 1210–1213). The velocities of these SWs are calculated analytically. The time evolution of their standard deviations is shown. The numerical integration of a two‐component electrophoresis run shows higher resolutions under some conditions. The expected practical difficulties which may be encountered when observing this phenomenon are discussed. Some practical difficulties that are likely to limit its useful application in electrophoresis and capillary electrophoresis are mentioned.
Effect of temperature and viscosity of sieving medium on electrophoretic behavior of sodium dodecyl sulfate‐proteins on capillary electrophoresis in presence of pullulan
Authors: Nakatani Manabu , Shibukawa Akimasa , Nakagawa Terumichi 
Published: May 2005

AbstractPullulan was used as a sieving medium in high‐performance capillary gel electrophoresis to study the effect of temperature and viscosity on electrophoretic behavior of sodium dodecyl sulfate (SDS)‐proteins with molecular masses in the range of 14 400–116 000 Da. The migration time decreased with increasing capillary temperature. Equations were derived relating mobility of SDS‐proteins to capillary temperature and viscosity of pullulan solution. Linear relationships were obtained from experiments between logarithm of mobility and reciprocal temperature and between double logarithmic values of mobility and viscosity of pullulan solution. The experimental results agree well with the equations.
Vancomycin as a chiral selector in capillary electrophoresis: An appraisal of advantages and limitations
Authors: Vespalec Radim , Billiet Hugo A. H. , Frank Johannes , Boček Petr 
Published: May 2005

AbstractThe properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3–5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 102 L/mol, as that found for β‐cyclodextrin (β‐CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 × 102 L/mol, is one order of magnitude higher than that of enantiomers separated with β‐CD. Analytically convenient mobility differences up to 1–2 × 109 m2V−1S−1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250 000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin‐analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.
Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with Immobiline membranes
Authors: Bossi Alessandra , Righetti Pier Giorgio , Riva Ernesto , Zerilli Luigi 
Published: May 2005

AbstractThe purification of small glycopeptides (a Hepta‐Tyr of the teicoplanin family, exhibiting broad activity against highly glycopeptide‐resistant enterococci) by isoelectric focusing in multicompartment electrolyzers with buffering, isoelectric membranes, is described. The main obstacle to such a preparative technique, in common with all focusing methodologies, is the poor solubility of the analyte at the pI value with resultant precipitation and coprecipitation of all impurities with the main fraction. A good solubilizing power was obtained in hydro‐organic solvents, particularly a mixture of 6 M urea and 20–25% trifluoroethanol. Best results, however, were obtained with mixtures of 8 M urea and zwitterionic detergents, notably the 3‐[(3‐cholamidopropyl)dimethylammonia]‐1‐propanesulfonate (CHAPS) family. A unique behavior of the peptide was found in concentration gradients of CHAPS: solubility increases up to 3.5% CHAPS, but the curve shows a maximum and then solubility decreases again at 5% CHAPS. In mixtures of 8 M urea and 3.5% CHAPS, sample loads of 500 up to 1000 mg Hepta‐Tyr could be purified in a single run, with recoveries > 90% and purity in excess of 99%. The main glycopeptide fraction (pI 8.56) was collected into an isoelectric trap delimited by pI 8.46 and pI 8.65 membranes. Attempts at purifying the glycopeptide by most known reversed phase‐high performance liquid chromatography (RP‐HPLC) techniques failed completely.
Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock
Authors: Yasuda Toshihiro , Takeshita Haruo , Sawazaki Kazumi , Iida Reiko , Kishi Koichiro 
Published: May 2005

AbstractThe forensic application of the DNase I polymorphism for individualization of used socks is described. We devised a technique that combines a simple extraction, method, including a partial purification step, using a Phenyl‐Sepharose CL‐4B column, isoelectric focusing and activity staining, for DNase I typing from socks. As a result, DNase I types were determined from socks kept at room temperature for a month or more in all of the samples tested. The DNase I types determined from socks exactly matched those determined from corresponding sweat and urine samples. By contrast, AB0 groupings could not be determined from these samples, using the conventional serological method. Therefore, we have shown that the DNase I polymorphism is the first genetic marker found to be well suited for individualization of used socks.