Estimation Of Molecular Weight Research Articles

Synthesis and Ring-Opening Polymerization of Cyclic Butylene 2,5-Furandicarboxylate

A new synthetic pathway for the polymerization of furan based polyesters is reported in this work. First, poly(butylene 2,5-furandicarboxylate) cyclic oligoesters (COEs) are chemically synthesized by semi-batch esterification. The structure of the COEs is confirmed by infrared spectroscopy, 1H, and 13C-NMR, while the molecular weight distribution of the COEs is determined by matrix-assisted laser desorption/ionization time of flight mass spectroscopy. The cyclic oligoesters are then successfully polymerized via ring-opening polymerization using tetrakis(2-ethylhexyl)-titanate as catalyst. Differential scanning calorimetry and 1H-NMR analysis unambiguously proves the formation of polymeric species. Both end-group analysis from 1H-NMR spectrum and calculation through Flory–Fox equation give comparable estimates of the number average molecular weight: 5.8 × 103 g mol−1 and 7.8 × 103 g mol−1, respectively.

Overview of Methods for the Direct Molar Mass Determination of Cellulose.

The purpose of this article is to provide the reader with an overview of the methods used to determine the molecular weights of cellulose. Methods that employ direct dissolution of the cellulose polymer are described; hence methods for investigating the molecular weight of cellulose in derivatized states, such as ethers or esters, only form a minor part of this review. Many of the methods described are primarily of historical interest since they have no use in modern cellulose chemistry. However, older methods, such as osmometry or ultracentrifuge experiments, were the first analytical methods used in polymer chemistry and continue to serve as sources of fundamental information (such as the cellulose structure in solution). The first part of the paper reviews methods, either absolute or relative, for the estimation of average molecular weights. Regardless of an absolute or relative approach, the outcome is a molecular weight average (MWA). In the final section, coupling methods are described. The primary benefit of performing a pre-separation step on the molecules is the discovery of the molecular weight distribution (MWD). Here, size exclusion chromatography (SEC) is unquestionably the most powerful and most commonly-applied method in modern laboratories and industrial settings.

Influence of long-chain branching extent in polyethylenes on molecular weight and molecular weight distribution predicted via rheological analysis

This work highlights the potential of rheological analysis as an alternative powerful tool for estimation of molecular weight (MW) and molecular weight distribution (MWD) in terms of long-chain branching (LCB) extent in polyethylenes (PE). Different PE samples with different branching structure (mLLDPE, mVLDPE, LDPE, LLDPE, and LDPE/LLDPE blends) were prepared and their viscoelastic behavior was studied. The results showed that in metallocene samples a suitable agreement existed between the MWDs determined both experimentally and also by rheological data evaluation, while in the rest of samples this agreement was not so good. It seems that the presence of branching in LDPE and LLDPE makes this difference significant, while the absence of branching in metallocene samples caused suitable agreement between the results of predictions and measurements. In blend samples, this significant difference was attributed to existence of branching and also to noncompatibility of polymer components in some blend ratios. The results showed that normalized relaxation time spectrum is a good measure for qualitative comparison of MWDs of various samples. Applying Thimm Kernel function and relaxation modulus data in generalized mixing rule, the calculated MW and MWD of PE samples (without LCB) showed good agreement with the experimental results with high accuracy.

Rheological Consequences of Hydrogen Bonding: Linear Viscoelastic Response of Linear Polyglycerol and Its Permethylated Analogues as a General Model for Hydroxyl-Functional Polymers

Viscoelastic properties of linear, hydroxyl-functional polymers are only little understood with respect to the effect of functional group interactions. Melt rheology and thermal phase transitions of linear polyethers (polyglycerol, linPG-OH) and their methylated analogues (linPG-OMe) in a broad molecular weight range (Mn = 1–100 kg/mol) with low polydispersities (PDI) have been investigated as a general model for hydroxyl-functional polymers with respect to their functionality and hydrogen bond interactions. We provide detailed insight into the rheodynamics of nonentangled and well-entangled polyethers bearing one functional group per monomer unit. Booij–Palmen plots (BBP) revealed failure of the time–temperature superposition principle (TTS) for both types of polymers in the segmental relaxation region, while TTS holds in the terminal relaxation region. The characteristic modulus of linPG-OMe derived from the BBP clearly reflects the transition from the nonentangled to the fully entangled state with increasing molecular weight. Quantitative analysis of these data allows for different estimates of the entanglement molecular weight, which is approximately 14 kg/mol. In case of linPG-OH a lower apparent entanglement molecular weight (8 kg/mol) leads to estimated 36 entanglement interactions in a cube of 10 nm edge length together with 47 association sites in the same volume. This can be determined from the molecular-weight-independent plateau modulus only, which is significantly lower than for linPG-OMe. This is explained as a consequence of the overlay of an entanglement network and an association network created by hydrogen bonding of the OH groups with themselves and with the ether linkages.

On the use of hydroxyl radical kinetics to assess the number-average molecular weight of dissolved organic matter.

Dissolved organic matter (DOM) is involved in numerous environmental processes, and its molecular size is important in many of these processes, such as DOM bioavailability, DOM sorptive capacity, and the formation of disinfection byproducts during water treatment. The size and size distribution of the molecules composing DOM remains an open question. In this contribution, an indirect method to assess the average size of DOM is described, which is based on the reaction of hydroxyl radical (HO(•)) quenching by DOM. HO(•) is often assumed to be relatively unselective, reacting with nearly all organic molecules with similar rate constants. Literature values for HO(•) reaction with organic molecules were surveyed to assess the unselectivity of DOM and to determine a representative quenching rate constant (k(rep) = 5.6 × 10(9) M(-1) s(-1)). This value was used to assess the average molecular weight of various humic and fulvic acid isolates as model DOM, using literature HO(•) quenching constants, kC,DOM. The results obtained by this method were compared with previous estimates of average molecular weight. The average molecular weight (Mn) values obtained with this approach are lower than the Mn measured by other techniques such as size exclusion chromatography (SEC), vapor pressure osmometry (VPO), and flow field fractionation (FFF). This suggests that DOM is an especially good quencher for HO(•), reacting at rates close to the diffusion-control limit. It was further observed that humic acids generally react faster than fulvic acids. The high reactivity of humic acids toward HO(•) is in line with the antioxidant properties of DOM. The benefit of this method is that it provides a firm upper bound on the average molecular weight of DOM, based on the kinetic limits of the HO(•) reaction. The results indicate low average molecular weight values, which is most consistent with the recent understanding of DOM. A possible DOM size distribution is discussed to reconcile the small nature of DOM with the large-molecule behavior observed in other studies.

Hybrid ozonation–microfiltration system for the treatment of surface water using ceramic membrane

The aim of this work was the investigation of performance efficiency of a hybrid system, integrating two processes, i.e. membrane filtration and ozone oxidation, as a potential method for the contaminated surface water quality improvement and the mitigation of membrane fouling. Ozone–oxygen gas mixture at various flow rates (and corresponding ozone concentrations) was introduced into the experimental vessel by gas spargers, located at the bottom of vessel and producing fine gas bubbles. Ozone–oxygen gas flow rates ranged from 0.3L/min to less than 0.05L/min, while ozone amounts in the gas mixture added to the system varied from 6.6mg to less than 1.1mg O3/min of experiment, respectively. Ozonation and ceramic membrane microfiltration were performed simultaneously. Treated water samples were collected and analyzed for major quality parameters, i.e. turbidity, pH, UV254 absorbance and Total Organic Carbon (TOC) content. The permeates were also analyzed with the HPLC technique for the estimation of molecular weights of resulting humic acids oxidation products. The hybrid process of membrane microfiltration–ozonation resulted in substantial reduction of membrane fouling rate for all the examined concentrations and flow rates of ozone, while the quality of permeate varied, depending on the ozone concentration used. With the optimum ozone dosage the quality of permeate for single microfiltration and hybrid processes were similar, while the addition of ozone to the system resulted in a substantial fouling reduction.

A culture-based method for determining the production of secreted protease inhibitors

We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them.

An Approach to the Identification and Characterisation of a Psychrotrophic Lipase Producing <i>Pseudomonas</i> sp ADT3 from Arctic Region

The aim of this research work was to explore psychrotrophic microbes from soil sample of NyAlesund, Svalbard, arctic region and to investigate their potential use as an effective tool for industrial application. A novel psychrotrophic bacterial strain showed good growth on minimal medium containing lipid as the only carbon source. Microbiological characterisation of the isolate showed that it was a gram negative rod. The strain was tested for the production of extracellular lipase enzyme. The enzymes were partially purified by 90% saturated ammonium sulfate and dialysis for desalting. The bacterium was identified as Pseudomonas sp ADT3 by 16S rRNA amplification and sequencing which had been deposited in the NCBI GenBank with accession number JX914667. Phylogenetic tree was also constructed with MEGA5 software and showed the highest level of sequence similarity with Pseudomonas sp. HC3-13 strain. The microorganism had a growth optimum at pH 8.0 and temperature 22°C. Optimization of different parameters e.g. temperature, pH, incubation time, cofactors etc. was performed for the extracellular lipase activity. The hydrolytic activity of the enzyme was enhanced 5 times by Pb2+ but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. For the molecular weight estimation of enzyme SDS-PAGE was done which showed an inducible band of approximately 13.9 KDa. Activity staining and mass spectrometry techniques were also performed.

Dual-Functionalized PAMAM Dendrimers with Improved P-Glycoprotein Inhibition and Tight Junction Modulating Effect

This study aims to surface modify poly(amido amine) or PAMAM dendrimers by sequentially grafting poly(ethylene glycol) or PEG and 4-thiobutylamidine (TBA) so as to reduce PAMAM cytotoxicity while improving the ability of PAMAM to modulate P-glycoprotein (P-gp) efflux and tight junction integrity. Conjugation of functional groups was determined by NMR spectroscopy, FT-IR, thiol group quantification and molecular weight estimation. The yield of the dual-functionalized dendrimers was >80%. The dual-functionalized dendrimer could significantly reduce PAMAM cytotoxicity to <15% as reflected by LDH release in Caco-2 and MDCK/MDR1 cells after 72 h of exposure. Thiolated dendrimers could increase cellular accumulation and permeation of the P-gp substrate R-123, and such effect could be affected by the extent of PEGylation of the dendrimer. Surface-modified PAMAM dendrimers, either by single or dual functionalization, could better modulate tight junction integrity in comparison with unmodified PAMAM, as demonstrated through immunostaining of the tight junction marker ZO-1, permeation of the model compound Lucifer Yellow (LY) and transepithelial electrical resistance (TEER). Of importance, reversible tight junction modulating effect was only observed in the dual-functionalized dendrimers. Collectively, dual functionalization with PEG and TBA represented a promising approach in altering PAMAM dendrimer surface for potential application in oral drug delivery.

Expression, purification, and characterization of full-length bovine leukemia virus Gag protein from bacterial culture

In retroviruses, the Gag protein is a precursor from which the mature proteins matrix, capsid, and nucleocapsid are derived. Gag plays an important structural role in the assembly of virions at the plasma membrane. While Gag proteins from several different retroviruses have been purified for study in vitro, there has yet to be a report of successful purification of deltaretroviral Gag. In this paper, we report the cloning, expression and purification of full-length bovine leukemia virus (BLV) Gag from Escherichia coli using a combination of polyethyleneimine precipitation, ammonium sulfate precipitation, and affinity chromatography. Experiments using size-exclusion chromatography were also performed to analyze the oligomeric state of the Gag protein in solution, and results suggest that it exists primarily as a monomer but may oligomerize into higher-order complexes to a small extent. Molecular weight estimation by comparison of elution volume to a set of protein standards supports the hypothesis that BLV Gag adopts a slightly extended conformation in solution. The results are discussed in comparison to the solution structure and assembly pathways of other retrovirus genera.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Legumes seed storage proteins characterization by SDS-PAGE and Lab-on-a-Chip electrophoresis

This study was conducted to analyze the seed storage proteins of grain legumes cultivars using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Lab-on-a-Chip (LoaC) electrophoresis. Using reference proteins, the relative accuracy and precision in estimating molecular weight was similar when these two methods were compared. However, the LoaC method had a shorter analysis time and higher resolution, demonstrating a significant advantage. The molecular weight estimation of grain legume storage proteins gave very similar results (R2=0.99). Relative quantification of the separated proteins also indicated good agreement of the two techniques (R2=0.81). Both methods can be used to analyze the composition of the legume seed proteins, but the LoaC method is more suited to the separation and detection of proteins, enabling low reagent consumption, easy operation and faster turnaround. The LoaC method seems suitable for legume quality screening and cultivar development where large numbers of samples need to be analyzed.

Two ATPases

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.

Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis

Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a few disaccharides up to at least 25,000 units, reaching molecular weights of 1010(3)kDa. HA has many biological functions, and both its size and tissue concentration play an important role in many physiological and pathological processes. It is relatively easy to determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range of 30-2400kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10min using only 6pg of HA. In addition, the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determinations and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.

A novel PAGE buffer for faster runs and sharper protein bands

Since the mid‐70s Laemmli's protocol based on a Tris‐glycine‐SDS buffer system has been widely used for analyzing protein mixtures by PAGE. Laemmli's buffer system allows clear and accurate molecular weight estimation of proteins in complex samples from a wide variety of sources, but at the cost of long electrophoresis time. Modifications and improvements of this method reducing total run‐time are limited to mainly high priced precast gels.In this work, we demonstrate an improved Laemmli method offering significant cut in run‐time and improved sharpening of protein bands. The patent pending modifications apply to the running buffer system which can be run at higher voltages without generating high heat and impacting gel performance. We show that typical mini‐gels (10 x 10 cm gel) cast with the Laemmli recipe can be run 50 to 60% faster and still maintain a wide separation range similar to porosity gradient gels. In fact, a 10% Laemmli discontinuous mini‐gel will provide ultra‐fine band resolution over a wide molecular weight range (10 to 240 kDa) in less than 30 minutes using this unique running buffer. The need for gradient gels may be over since a uniform concentration gel can now fractionate proteins covering a wide range of molecular masses in half the time.

Humic Acid Molecular Weight Estimation by High‐Performance Size‐Exclusion Chromatography with Ultraviolet Absorbance Detection and Refractive Index Detection

High‐performance size‐exclusion chromatography (HPSEC) with ultraviolet absorbance detection (UVAD) has been widely utilized to estimate the molecular weight (MW) and MW distribution of humic acids (HAs). The MW estimated by UVAD was inherently inaccurate, however, because UVAD set at 254 nm only detects limited HA components, and the molar absorptivities of different HA constituents are not equal. The objective of this study was to evaluate the refractive index detection (RID)‐based HPSEC method for quantifying the MW of HAs. Five HA samples were quantified with both UVAD and RID. The chromatograms obtained on the two detectors showed that the RID/UVAD response ratios were consistently &gt;10, indicating that RID is more sensitive for the detection of HAs. The chromatograms obtained with RID had three peaks compared with two peaks shown on UVAD chromatograms because RID detected a late‐eluting peak (F3) that was not shown on the UVAD chromatograms. Comparison of the RID/UVAD response ratio showed that the highest MW HA fraction (F1) and lowest MW HA fraction (F3) have higher RID/UVAD response ratios, whereas the medium HA fraction (F2) had a lower RID/UVAD response ratio. These suggested that F1 and F3 may have relatively lower contents of UV‐sensitive bonds such as C=C double bonds than F2 Compared with HPSEC‐UVAD chromatograms, the HPSEC‐RID chromatograms resulted in higher weight‐averaged MWs, lower number‐averaged MWs, and higher polydispersivity for the tested HA samples. This study indicated that RID is less selective than UVAD for detection of structurally highly heterogeneous HA molecules and is thus better for characterizing the MW distribution of HA molecules.