Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes

A comparison of HSQC and HMQC pulse schemes for recording (1)H[bond](13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. (1)H[bond](13)C correlation spectra are recorded on U-[(15)N,(2)H], Ile delta 1-[(13)C,(1)H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5 degrees C, and of (ii) the protease ClpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5 degrees C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.     

Separation/enrichment of the low-content high molecular weight natural protein using protein-imprinted polymers with ARPCs

We introduce a new method for separation/enrichment of the low-content cellular protein in high mo-lecular weight on the basis of molecular imprinting. The template protein, bacterial cloned immu-noglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. The assemblies of proteins and ARPCs were adsorbed by porous polyme...     

Synthesis of high molecular weight polystyrene using AGET ATRP under high pressure

High molecular weight polystyrene (PS) was synthesized by ATRP. Under atmospheric pressure (1 bar), PS with M_n up to 200,000 was prepared using either ARGET or ICAR ATRP. Under high pressure (6 kbar), higher molecular weight PS could be obtained due to accelerated radical propagation and diminished radical termination in polymerization of styrene. Therefore, it was possible to synthesize PS with M_n > 1,000,000 and M_w/M_n < 1.25 using AGET ATRP under a pressure of 6 kbar at room temperature. This is the highest molecular weight linear PS prepared by a controlled radical polymerization.     

Molecular weight determination of high molecular mass (glyco)proteins using CGE-on-a-chip, planar SDS-PAGE and MALDI-TOF-MS

The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1?MDa.     

Steam-cracking of high molecular weight paraffins

Solid paraffins were steam-cracked in order to imitate the pyrolysis of low polymerization grade polyethylene. The behavior of the paraffins in steam-cracking corresponds to that of gasolines due to the similar C/H ratio.     

An energy efficient and facile synthesis of high molecular weight polyesters using ketenes

A facile, ketene-based strategy for the synthesis of polyesters from stable Meldrum's acid monomers has been developed which overcomes many issues associated with traditional step-growth procedures. A significant increase in polymerization efficiency is observed with only 10 min reaction time at 220 °C being needed to obtain high molecular weight polymers.     

MALDI mass spectrometry analysis of high molecular weight proteins from whole bacterial cells: Pretreatment of samples with surfactants

The use of surfactants as additives in conjunction with on-probe whole cell bacterial protein analysis employing MALDI-TOF-MS is described. Nonionic and zwitterionic surfactants were used to enhance the detection of high molecular weight proteins. Three nonionic, N-octyl-B-D-glactopyranoside, N-decyl-B-D-maltopyranoside, and N-dodecyl-B-D-maltoside, and two zwitterionic surfactants, N,N-dimethyldodecylamine-N-oxide and zwittergent 3-12 were evaluated with five different MALDI matrix systems. New peaks in the mass range of 2 to 80 kDa were produced with all of the various combinations of matrix and surfactant from both whole cell gram-positive and gram-negative bacteria. Ferulic acid used in conjunction with a 1.0 mM solution of N-octyl-B-D-glactopyranoside produced the highest quality spectra with high signal to noise ratios and peaks up to 140 kDa.     

Photodissociation of high molecular weight peptides and proteins in a two-stage linear time-of-flight mass spectrometer

A two-stage linear time-of-flight mass spectrometer is used to investigate the requirements for performance of laser photodissociation of peptide and protein ions. Results are presented that demonstrate that desorption and dissociation laser pulses can be synchronized to irradiate ions that travel at high velocities down the drift tube of a time-of-flight mass spectrometer. For example, 193-nm photodissociation of bovine insulin and doubly charged lysozyme is demonstrated, and laser power studies suggest that dissociation is initiated by the absorption of a single 193-nm photon. These results are encouraging because they suggest that laser photodissociation of high molecular weight proteins can lead to fragmentation on time scales compatible with time-of-flight mass spectrometry.     

Dye-free protein molecular weight markers

Protein molecular weight markers are widely used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Here, we describe novel protein molecular weight markers in which a prestaining procedure is no longer needed. Green fluorescent protein (GFP) is stable and resistant to denaturing agents/conditions. Various histidine-tagged GFP fusion proteins were overexpressed in Escherichia coli and purified by metal affinity chromatography. The minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot under visible light was 62.5 and 125 ng, respectively. Under ultraviolet (UV) ray, the minimal amount of each protein marker needed for analysis in SDS-PAGE and Western blot was half of those amounts used under visible light, respectively. Collectively, the accuracy, sensitivity, ease, economy, and flexibility of our strategy may reinforce the application of GFP in molecular biology.     

Composition and molecular weight analysis of styrene-acrylic copolymers using thermal field-flow fractionation

Thermal field-flow fractionation coupled with online multiangle light scattering, differential refractive index and quasielastic light scattering (ThFFF-MALS/dRI/QELS) was used to simultaneously determine the molecular weight (MW) and composition of polystyrene-poly(n-butyl acrylate) (PS-PBA) and polystyrene-poly(methyl acrylate) (PS-PMA) copolymers. The online measurement of the normal diffusion coefficient (D) by QELS allowed calculation of the copolymer thermal diffusion coefficient (D(T)) of sample components as they eluted from the ThFFF channel. DT was found to be independent of MW for copolymers with similar compositions and dependent on composition for copolymers with similar MW in a non-selective solvent. By using a solvent that is non-selective to both blocks of the copolymer, it was possible to establish a universal calibration plot of DT versus mole fraction of one of the monomer chemistries comprising the copolymer. PS-PBA and PS-PMA linear diblock polymers were determined to vary in composition from 100/0 to 20/80 wt% PS/acrylate and ranged in MWs between 30 and 360 kDa. The analysis of a PS-PBA miktoarm star copolymer revealed a polydisperse material with a weight percent PBA of 50-75% and MW ranging from 100 to 900 kDa. The presented ThFFF-MALS/dRI/QELS method allowed rapid characterization of polymers with MW and chemical distributions in a single analysis.     

Solution properties of high molecular weight polystyrene

A series of polystyrenes with weight-average molecular weight M?_w up to 1.3 × 10⁷ was prepared by anionic polymerization in tetrahydrofuran (THF). Each sample was characterized by gel-permeation chromatography, light scattering, and viscometry. It was found that each sample had an almost symmetrical and very narrow molecular weight distribution (M?_w/M?_n < 1.07). The mean-square unperturbed radius of gyration 〈S²〉₀ was determined in trans-decalin at 20.4°C as 〈S²〉₀ = 7.8₆ × 10^?18M?_w (cm²). The particle scattering factor was well represented by the Debye equation irrespective of solvent in the range of M?_w < 4 × 10⁶, and only a small deviation was observed in benzene at higher molecular weights. The penetration function Ψ ≡ A₂M²/4π^3/2N_A〈S〉^23/2 was found to approach a relatively low asymptotic value of 0.21–0.23 at molecular weights above 2 × 10⁶ in benzene at 30°C, where A₂ is the second virial coefficient and N_A is Avogrado's number. It was also found that the theta temperature in trans-decalin was affected by the nature of polymer samples. A difference of about 3°C in the theta temperature was observed between two series of anionic polystyrenes, one prepared in THF and the other in benzene, but there was practically no difference in unperturbed chain dimension.     

DSC ofγ-irradiated ultra-high molecular weight polyethylene and high density polyethylene of normal molecular weight

The melting and the crystallization of-irradiated (doses: 0–6Mrad) ultra-high molecular weight nascent polyethylene (UHMWPE) and high density nascent polyethylene with normal molecular weight (NMWPE) were investigated by DSC. The heat of melting of the nascent UHMWPE (DSC degree of crystallinity, respectively) increases up to a dose of 3 Mrad, after which it slightly decreases. The heat of the second melting of UHMWPE and of the first and second melting of NMWPE increases slightly up to a dose of 3 Mrad, after which it does not change. The X-ray degree of crystallinity of the nascent non-irradiated and irradiated polymers was 0.62±0.02. The calorimetric crystallinity was compared to the X-ray one. The results show that radiation does not affect the polymer crystallinity, but influences the thermodynamic heat of melting. The increase ofH _m vs. dose in UHMWPE is explained in terms of processes of tie molecule scission within the amorphous regions and on the surface of the crystals, which predominate over crosslinking up to a dose of 3 Mrad. That leads to an increase in the conformational mobility of the molecules and to an increase in the enthalpy, according to Peterlin's formula. The scission of the chains at the points of entangling of the tie molecules leads to a decrease in the temperature and to an increase in the enthalpy of crystallization of UHMWPE vs. dose. In NMWPE these effects are considerably weaker.     

Flocculation by very high molecular weight polymers

Polymer-induced flocculation in which the size of polymer molecules is much greater than that of colloidal particles is investigated. A dynamic analysis is conducted which takes the transient behaviors of the adsorption of particles to a polymer molecule and the particle-particle interactions into account. We show that the number of particles adsorbed to a polymer molecule follows approximately a binomial distribution. An approximate expression for the degree of flocculation of the system under consideration is presented.     

Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.     

Effects of molecular entanglements during electrospray of high molecular weight polymers

Concentration-dependent bimodal size distributions (comprised of single-molecule particles and multimolecule clusters) observed by microscopic examination of particles collected during electrospray (ES) of dilute solutions of high molecular weight polymers suggest that chain entanglement can interfere with the droplet subdivisions believed to be intrinsic to the electrospray process. The feasibility of such interference is discussed in the context of the spray model of Kebarle, along with its potential impact on the ES mass spectrometry of macromolecules.     

Replacement of plasma by high molecular weight substances

In the course of the development work, natural and synthetic polymers were examined to see whether they were suitable for use as plasma substitutes. The criteria of a plasma substitute are discussed in this connection. The most important of the base substances used at present (dextran, modified gelatin, polyvinylpyrrolidone, and hydroxyethyl starch) are described, and a typical example of proven therapeutic value is discussed for each type.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.     

Novel synthesis of high molecular weight polymers with extremely low polydispersity by the unique initiation properties of organo-lanthanide complexes

Organolanthanide(III) complexes such as |(C₅Me5₅)₂SmH|₂ and (C₅Me₅)₂SmMe(THF) were found to initiate the living polymerizations of methyl methacrylate (MMA) to give high molecular weight polymers (M _n > 500 × 10³) with extremely low polydispersity (M _w/M_n = 1.04). The syndiotacticity increased up to 95.2% by lowering the temperature to −95°C. The molecular structure of the 1:2 adduct of (C₅Me₅)₂SmH with MMA determined by X-ray method indicates that this intermediate assumes the 8 membered ring conformation where the Sm atom is bound to MMA in an enolate form and the ester of penultimate MMA is coordinated to the metal. Based upon these results, an anionic coordination mechanism has been proposed for the present reaction. Organolanthanide(II) complexes also exhibit high activity and proceed the living polymerizations. Organolanthanide(III) complexes also initiate the living polymerizations of lactones such as ϵ-caprolactone and δ-valerolactone. The stoichiometric reactions indicate that real active species assumes the alkoxylanthanide(III) form.