Heat-induced conformational transition of cytochrome c observed by temperature gradient gel electrophoresis at acidic pH

Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change deltaHvh and transition temperature Tm), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).     

Phase transitions of polycrystalline [Fe(H2O)6](ClO4)3 and [Cr(H2O)6](ClO4)3 studied by DSC

The effects of heat treatment on soymilk protein denaturation were studied by differential scanning calorimetry (DSC) and electrophoresis. Transition behavior of soymilk was studied by DSC. Three endotherms were found in DSC heating curves; the transition observed at around 70°C is attributed to the denaturation of 7S (b-conglycinin) and the transition at around 90°C is to 11S (glycinin). The denaturation temperature increased with the increasing soymilk protein content. The change of electrophoretic patterns after heat treatments indicated that soy proteins were dissociated into subunits, some of which coalesced. When the heating temperature is below their denaturation temperature, the protein fractions cannot completely be denatured even after heat exposure for extended periods of time. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electrophoretic mobility of partially denatured DNA in a gel: qualitative and semiquantitative differences between bubbles and split ends

Partially melted DNA is known to exhibit an abrupt decrease of electrophoretic mobility in a gel. Although this is the main phenomenon exploited in TGGE/DGGE (temperature gradient gel electrophoresis/denaturing gradient gel electrophoresis), not much is known about the physical processes responsible for the blocking. While there is a commonly used formula for the reduced mobility based on the theory of branched polymers, it does not discriminate between denatured domains bounded on one (split end) or two sides (bubble). To better understand how the blocking occurs in both of these cases, a coarse-grained model of DNA gel electrophoresis is simulated using Langevin Dynamics. The simulations reveal that the low-field mobility is much more sensitive to denatured domains located at the ends of a DNA fragment. A denatured domain occurring at the center of a fragment indeed reduces the mobility, but at a much lower rate.     

Anthracycline-dependent heat-induced transition from positive to negative supercoiled DNA

The conformational stability of individual DNA topoisomers depends on the concentration of DNA intercalating drugs. To study the DNA-drug interaction, we used ethidium bromide (EtBr) and negative supercoiled pUC19 as a model system. The effects of two anthracyclines widely used in cancer therapy, daunorubicin (Dau) and doxorubicin (Doxo), and EtBr were compared. In spite of their different chemical structures and intercalation mode, all intercalating agents show similar effects on the conformational stability of supercoiled DNA. Our observations show that the studied intercalators have at least two main effects on the supercoiled DNA: (i) they decrease the level of negative supercoiling and, at certain concentrations, they may induce positive supercoiling in DNA; (ii) a temperature increase can cause a recovery of negative supercoiling in DNA. The conformational stability of plasmid DNA-drug complexes has been investigated by temperature gradient gel electrophoresis (TGGE). We demonstrate the suitability of TGGE for this purpose, because it offers a global view on DNA-drug complexes over a continuous range of temperature. Images of DNA plasmids adsorbed onto a substrate at different temperatures and drug concentrations were acquired by atomic force microscopy (AFM), allowing us to distinguish directly the conformation chirality assumed by the plasmid under different conditions confirming TGGE results. Our detection system allows to characterize unknown drugs and to determine their intercalating properties.     

盐酸胍诱导的变性卵清溶菌酶分子重折叠过程及其各稳定构象态的分布和过渡

采用变性和非变性电泳、 高效凝胶排阻色谱、 内源荧光发射光谱和荧光相图以及生物活性测定等方法, 研究了盐酸胍诱导的变性卵清溶菌酶分子的重折叠过程及此过程中卵清溶菌酶分子各稳定构象态的分布和过渡. 结果表明, 当复性液中盐酸胍浓度分别约为5.0和2.4 mol/L时, 变性卵清溶菌酶分子的重折叠过程各存在1个稳定折叠中间态, 重折叠过程符合"四态模型". 在卵清溶菌酶分子四态重折叠过程基础上, 结合盐酸胍与卵清溶菌酶分子之间的缔合-解离平衡, 给出了一个定量描述变性剂诱导的蛋白质分子复性过程中蛋白质分子复性率随溶液中变性剂浓度变化的方程. 该方程包含2个特征折叠参数, 一个是蛋白质分子从一个稳定构象态过渡到另一个稳定构象态的热力学过渡平衡常数k; 另一个是在此过程中平均每个蛋白质分子所结合的变性剂分子数目m. 通过这2个特征折叠参数能够定量描述盐酸胍诱导的变性卵清溶菌酶完全去折叠态、 折叠中间态和天然态分子随复性液中盐酸胍浓度变化的分布和过渡情况.     

Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

Two-dimensional electrophoresis of human parotid salivary proteins from normal and connective tissue disorder subjects using immobilised pH gradients.

Two-dimensional electrophoretic analysis of human salivary proteins using immobilised pH gradients in the first dimension, thin-layer gradient horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second, and modified staining procedures has resulted in a substantial improvement in their resolution. Unlike carrier ampholyte-based techniques, immobilised pH gradients prevent the loss of proteins of pI greater than 8; accordingly, basic components, including basic proline-rich proteins, can now be resolved. A two-dimensional map showing the locations and identities of most of the major proteins has been constructed. Narrow-range pH gradients can be constructed to give increased resolution of proteins of particular interest. By means of a pH 3.5-5.0 gradient, the abnormal salivary proteins associated with connective tissue disorders were found to be a highly heterogeneous group of pI approximately 3.75-4.75 and Mr approximately 32,000; although low levels occurred in some normal individuals, there was less heterogeneity (pI approximately 3.75-4.25). The technique should form a base for future structural, functional, and clinical studies on human salivary proteins.     

Determination of the Molecular Weight of Extracellular Ribonuclease Isoenzymes from Aspergillus Niger in Crude Extracts by Thin Layer Gel Filtration and Polyacrylamide Gel Electrophoresis

Abstract

The extracellular ribonucleases from Asperqillus niger culture medium were fractionated according to their molecular weight by thin layer gel filtration through Sephadex G100 superfine and the enzyme activity was detected by a standard staining technique on a replica print paper. Another replica paper was laid onto the top of a polyacrylamide gel and the absorbed proteins were separated by electrophoresis. By comparing the electrophoretic pattern with that of a control not subjected to gel filtration, the molecular weight of each isoenzyme in the crude extract could be determined. Gel electrophoresis however, is only used to establish the correspondence between the original electrophoretic pattern of the isoenzymes in the crude preparation and that detected on the replica print paper taken after the thin layer gel filtration run. There was good agreement between the values obtained for the crude and purified enzymes.     

Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.     

Separation of the isoenzymes of glyoxalase I from human red blood cells by electrophoresis and isoelectric focusing on polyacrylamide gel and by ion exchange chromatography.

Methods have been devised for the separation of the isoenzymes of glyoxalase I(S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from human red blood cells by electrophoresis and electrofocusing on polyacrylamide gel slabs. Three different staining methods were used for the location of the enzyme. Three electrophoretic phenotypes of the enzyme were resolved, the fast and slow types with one band and the intermediate type with three glyoxalase I activity bands. In gel electrofocusing (pH gradient 3.5-9.5) two glyoxalase I activity bands were found for all electrophoretic types. In electrofocusing on gel with a narrow pH gradient, at least four separate enzyme components were resolved for the fast and slow electrophoretic types and at least six components for the intermediate type. The phenotypes could be distinguished correspondingly to the electrophoretic results. Preparative separation of the isoenzymes were achieved by ion exchange chromatography on DEAE-Sephacel but gel chromatography on Sephadex G-100 gave the same elution volume for all enzyme phenotypes. This corresponds to an apparent molecular weight of about 47 000.     

Transitions of serum albumin in patients with glomerulosclerosis 'in vivo' characterization by electrophoretic titration curves

HSA functions as a physiological transporter of solutes and small molecules that induce structural transitions 'in vitro'. Analysis of these transitions requires prior purification of HSA that could introduce bias due to conformational changes. We utilized electrophoretic titration curves to describe a neutral to acid (N-A) transition of HSA directly in sera of seven patients with active focal segmental glomerulosclerosis (FSGS). The divergent electrophoretic profile of HSA was characterized by a shift in the range of pHs between 4.5 and 7.5 with an average variation of free electrophoretic mobility corresponding to loss of 1 positive charge in the pKa protonation range of histidyl residues and should involve domain I of HSA. 'In-gel' determination by maleimide-PEO2-biotin of free SH 34 of domain I showed inaccessibility of the dye at this site in pathological HSA and alkylation with the same complex induced N-A transition in normal HSA. Potential binders of free imidazoles such as Ca++ and/or of SH 34 such as NO were excluded on the basis of direct titration and studies on binding stimulation. This is the first report describing a transition of HSA directly 'in vivo', and the utilization of electrophoretic titration curves was critical to this purpose. This transition appears to be specific to FSGS and is unrelated to the nephrotic syndrome, Ca++ and NO binding. Spectroscopic analysis will elucidate the structural implication.     

Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay

Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process. Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.     

A rapid (less than 10 minute) electrophoresis method for identification of wheat varieties

Conventional procedures for electrophoretic identification of grain samples according to variety are too slow to permit checking at the time of delivery. The method described permits electrophoretic identification within an hour. It involves extraction of gliadin proteins from crushed grain with 6% urea solution or ethylene glycol, cathodic electrophoresis for 9 min at 300 V in a Micrograd gel (MG 315 from Gradipore Ltd, Sydney, Australia) using sodium lactate buffer (pH 3.1), and staining in Gradipore (at about 50 degrees C). Distinction between a set of Australian varieties was similar to that obtainable with the Australian Standard Procedure.     

Purification of α-amylases using magnetic alginate beads

Magnetic alginate beads were used to purify alpha-amylases from porcine pancreas, starchzyme, BAN 240L (a commercial purification from Bacillus subtilis), and wheat germ. The beads bound a significant level of alpha-amylase activity from porcine pancreas, BAN 240L, and wheat germ. In each case, the enzyme activity could be eluted by using 1.0 M maltose, a known competitive inhibitor of alpha-amylase. In the case of BAN 240L, 3.6-fold purification with 72% recovery of activity was observed. In the case of wheat germ enzyme, starting from the crude extract, 48-fold purification with 70% activity recovery was observed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis also indicated considerable purification in the latter case.     

DSC Analysis of foodborne bacteria

Differential scanning calorimetry (DSC) is applicable to studying the thermal properties of bacteria when treated with heat, cold, or antibiotics. Foodborne pathogens are inactivated by heat, and denaturation transitions observed by DSC indicate potential sites of cellular injury. Ribosomes, which are the sites for messenger RNA translation, are one critical component of thermal damage as evidenced by characteristic denaturation transitions in the 66-74°C range. These transitions disappear when cells of Clostridium perfringens are subjected to heat, suggesting structural or conformational changes to ribosomal proteins, and when cells of Listeria monocytogenes are cold-shocked by refrigeration, indicating ribosomal dissociation. DSC can be used to show that refrigeration followed by heat treatment improves the killing of dangerous microorganisms.     

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.     

Simultaneous, two-color fluorescence detection of total protein profiles and beta-glucuronidase activity in polyacrylamide gel

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.     

Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer

This study demonstrates structural and conformational characterization of proteins by nanoflow electrospray ionization (nanoESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer (Micromass, Manchester, England). Model peptides were successfully sequenced at the 35 attomole (amol) level, and peptides derived from a tryptic in-gel digest of 25 femtomole (fmol) bovine serum albumin (BSA) were successfully sequenced. The results demonstrated that the MS/MS sensitivity of the Q-TOF clearly surpassed the detection limit of the silver stain. A silver destaining step greatly improved the mass analysis of peptides derived from in-gel digests. Interestingly, sequence analysis revealed BSA residue 424 (tyrosine) as a potential chlorination site. In addition, a modified procedure was successfully used to extract and measure the masses of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-resolved proteins in the 10-68.5 kDa range. The Q-TOF was also used to monitor conformational changes of proteins. These experiments demonstrated an acid-induced denaturation of BSA in the pH 3-4 range, and heat-induced unfolding of cytochrome c between 50 and 60 degrees C. Finally, Zn2+ binding was demonstrated for the carbonic anhydrase apoprotein. In summary, the wide range of applications and the high quality of the experimental data made the Q-TOF mass spectrometer a powerful analytical tool for protein characterization.     

Membrane proteins in thin films

Molecular functions and structural changes of membrane proteins in an aqueous environment can be elucidated by reaction-induced FTIR difference spectroscopy upon photolysis of caged compounds. The achieved detection of IR band changes even due to single amino acid residues is, however, only possible in the presence of very high protein concentrations, implying that a low water content must be present. In general, the films are formed by controlled dehydration of membrane protein suspensions at reduced pressure and low temperature. For the retention of enzymatic activity of Na,K-ATPase, for example, a cosolvent such as glycerol is required. In order to interprete the results obtained by FTIR spectroscopy, it is important to know whether essential properties of the proteins such as hydration are changed upon film formation. Therefore, a differential scanning calorimetry (DSC) study has been carried out with purified Na,K-ATPase and Ca-ATPase in suspension, in form of pellets obtained by high-speed ultracentrifugation and in thin films. As relevant thermoanalytical properties, the endothermic denaturation transitions of the proteins have been studied. For Na,K-ATPase in the presence of 20% glycerol as cosolvent, a single, comparatively narrow endothermic and irreversible denaturation transition with a denaturation enthalpy of about 1.7 MJ mol⁻¹ and transition temperatures of about 65 and 70°C is found in concentrated suspension and in the state of the pellet, respectively. In the case of thin films suitable for IR spectroscopy, a characteristic change is observed in a reproducible manner. The enthalpy change of the remaining transition around 70°C is reduced but an additional transition at about 77°C is observed. Based on control experiments, the new high temperature transition is attributed to a partially dehydrated state of the protein. Furthermore, a comparatively broad endothermic transition around 20°C is found under conditions of high protein concentrations (film), which is tentatively assigned to a transition of the lipid environment of this integral membrane protein. Similar results are found for Ca-ATPase films. In the absence of glycerol, the deoxycholate treated enzyme in suspension exhibits a narrow endothermic main transition at 52°C with a denaturation enthalpy around 0.9 MJ mol⁻¹. For the film of this protein, two almost equally large endothermic transitions are found at 59 and 77°C. Also here, the data are characteristic of partial protein dehydration. These results show clearly that DSC can easily be applied in a sensitive manner to control and characterize the integrity and hydration properties of concentrated protein samples in thin films.