High-resolution Size-exclusion Chromatography Research Articles

Natural rubber – Increasing diversity of an irreplaceable renewable resource

This paper discusses the importance of introducing domestic natural rubber production and presents the rediscovery of a rubber-producing species, Scorzonera tau-saghyz or “mountain gum”, originally discovered in 1929 on the Karatau mountains in Kazakhstan. This plant could potentially also be cultivated in the U.S. In this exploratory work, roots (2–5 years old) were harvested on June 16, 2021 from wild strands in the Karatau mountains, Kumantas ridge, and Saraba, Kazakhstan, and processed at the Ohio State University. The rubber extraction method was based on an indigenous method in Kazakhstan to make natural chewing gum. Water extraction followed by purification yielded 16.2 wt% rubber from the dry roots, in comparison with 4–8 wt% from most rubber dandelion (Taraxacum kok-saghyz) plants, also a potential domestic rubber producing plant. High-resolution size exclusion chromatography was used to analyze rubber samples. The molecular weights and gel and oligomer contents were very similar to the rubber from Hevea brasiliensis, the current commercial source of natural rubber. More detailed investigations of this very interesting rubber-producing plant are in progress.

Multifarious analytical capabilities of the UV/Vis protein fluorescence in blood plasma

Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for ∼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods.

Abstract 197: Proteomic Characterization Of Human Brain Lipoproteins Speciated By Size Exclusion Chromatography

Like plasma, the cerebrospinal fluid (CSF) contains lipid-protein complexes called lipoproteins tasked with transporting lipids and protecting against cerebrovascular dysfunction. These brain lipoproteins (BLps) are generated de novo in the central nervous system (CNS), but little is known about them, because they exist in CSF at <0.5% the concentration of lipoproteins in plasma making them difficult to study. BLps are thought to resemble plasma high-density lipoproteins (HDL), a heterogenous population of compositionally distinct particles important for transporting lipid, protecting against oxidative stress, and modulating immune and inflammatory responses in the periphery. Mass spectrometry (LC-MS/MS) studies have shown that 260 different proteins can interact with different HDLs in the periphery and specific combinations of these proteins cluster on the lipid surface to govern particle function. In the current study, we developed a lipoprotein profiling technology that overcomes the BLp abundance problem in CSF to capture BLp subspecies with unprecedented sensitivity and resolution. Using high-resolution size exclusion chromatography, we identified at least 10 distinctly sized BLp populations in human CSF with a majority of the particles eluting in size ranges between plasma low-density lipoproteins (LDL) and HDL. BLps were isolated from fractionated CSF using a lipid-binding resin. Proteomic analysis of the particles using LC-MS/MS identified 299 different lipid-associated proteins across fractions with 189 of those proteins not currently reported on the human HDL Proteome Watch. Co-migration analysis further revealed at least 27 unique protein communities across the fractions indicating that multiple distinct subspecies exist within the same sized subfractions. DAVID functional analysis shows that the communities are highly enriched in proteins important in the immune response and neurometabolic functions such as neuron generation and development. In summary, our study shows that similar to plasma HDL, BLps are a highly speciated population of compositionally distinct particles but are enriched in CNS-derived proteins that are essential for maintaining cognitive health.

Apolipoprotein A-I modulates HDL particle size in the absence of apolipoprotein A-II

Human high-density lipoproteins (HDLs) are a complex mixture of structurally related nanoparticles that perform distinct physiological functions. We previously showed that human HDL containing apolipoprotein A-I (APOA1) but not apolipoprotein A-II (APOA2), designated LpA-I, is composed primarily of two discretely sized populations. Here, we isolated these particles directly from human plasma by antibody affinity chromatography, separated them by high-resolution size-exclusion chromatography and performed a deep molecular characterization of each species. The large and small LpA-I populations were spherical with mean diameters of 109 Å and 91 Å, respectively. Unexpectedly, isotope dilution MS/MS with [15N]-APOA1 in concert with quantitation of particle concentration by calibrated ion mobility analysis demonstrated that the large particles contained fewer APOA1 molecules than the small particles; the stoichiometries were 3.0 and 3.7 molecules of APOA1 per particle, respectively. MS/MS experiments showed that the protein cargo of large LpA-I particles was more diverse. Human HDL and isolated particles containing both APOA1 and APOA2 exhibit a much wider range and variation of particle sizes than LpA-I, indicating that APOA2 is likely the major contributor to HDL size heterogeneity. We propose a ratchet model based on the trefoil structure of APOA1 whereby the helical cage maintaining particle structure has two “settings”—large and small—that accounts for these findings. This understanding of the determinants of HDL particle size and protein cargo distribution serves as a basis for determining the roles of HDL subpopulations in metabolism and disease states.

Diabetes Impairs Cellular Cholesterol Efflux From ABCA1 to Small HDL Particles.

HDL (high-density lipoprotein) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1). The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease risk, but how diabetes mellitus impacts that step is unclear. To test the hypothesis that HDL's cholesterol efflux capacity is impaired in subjects with type 2 diabetes mellitus. We performed a case-control study with 19 subjects with type 2 diabetes mellitus and 20 control subjects. Three sizes of HDL particles, small HDL, medium HDL, and large HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CEC-but not ABCG1 CEC-of small HDL was lower in the subjects with type 2 diabetes mellitus than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of SERPINA1 (serpin family A member 1) in small HDL was also lower in subjects with diabetes mellitus. Enriching small HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic α-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles. The ABCA1 CEC of small HDL is selectively impaired in type 2 diabetes mellitus, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase cardiovascular disease risk in subjects with diabetes mellitus.

Ultratrace Metal Speciation Analysis by Coupling of Sector-Field ICP-MS to High-Resolution Size Exclusion and Reversed-Phase Liquid Chromatography.

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Anti-inflammatory effects of vanadium-binding protein from Halocynthia roretzi in LPS-stimulated RAW264.7 macrophages through NF-κB and MAPK pathways.

Vanadium-binding protein (VBP) was separated from the blood of the fresh sea urchin Halocynthia roretzi through (NH4)2SO4 precipitation, Diethylaminoethyl Sepharose fast-flow ion-exchange chromatography, and Sephacryl S-200 high-resolution size-exclusion chromatography. The protein size and purification yield of VBP were 27 kDa and 5.5%, respectively. VBP exerted anti-inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages by downregulating iNOS expression and inhibiting nitric oxide production. VBP also suppressed the expression of pro-inflammatory mediators such as COX-2, IL-1β, IL-6, and TNF-α. The anti-inflammatory activity of VBP was further demonstrated in the NF-κB and MAPK inflammation pathways, in which VBP inhibited phosphorylation of signaling proteins such as p65, JNK, ERK1/2, and p38. Therefore, VBP from H. roretzi has anti-inflammatory effects and could potentially be used to treat inflammation.

Functional Insights from the Proteomic Inventory of Ovine Forestomach Matrix.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.

The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly

High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed.

Plasminogen promotes cholesterol efflux by the ABCA1 pathway.

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages.

Polysaccharides and Oligosaccharides Produced on Malvar Wines Elaborated with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 Native Yeasts from D.O. "Vinos de Madrid".

Polysaccharides and oligosaccharides released into Malvar white wines elaborated through pure, mixed, and sequential cultures with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 native yeasts from D.O. "Vinos de Madrid" were studied. Both fractions from different white wines were separated by high-resolution size-exclusion chromatography. Glycosyl composition and wine polysaccharide linkages were determined by GC-EI-MS chromatography. Molar-mass distributions were determined by SEC-MALLS, and intrinsic viscosity was determined by differential viscometer. Yeast species and type of inoculation have a significant impact on wine carbohydrate composition and structure. Mannose residues from mannoproteins were significantly predominant in those cultures where T. delbrueckii was present in the fermentation process in comparison with when pure cultures of S. cerevisiae were present in the fermenation process. Galactose residues from polysaccharides rich in arabinose and galactose presented greater values in pure cultures of S. cerevisiae, indicating that S. cerevisiae released fewer mannoproteins than T. delbrueckii. Moreover, we reported structural differences between mannoproteins released by T. delbrueckii CLI 918 and those released by S. cerevisiae CLI 889. These findings help to provide important information about the polysaccharides and oligosaccharides released from the cell walls of Malvar grapes and the carbohydrates released from each yeast species.

Abstract 255: Plasminogen Promotes Cholesterol Efflux by the ABCA1

Background: The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo. Methods: We used genetic and biochemical approaches in mice to identify important mediators of CEC. Results: On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in Apoa1 -/- mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane. Conclusions: HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.

A rapid and simple screening method to identify conditions for enhanced stability of modular vaccine candidates

Virus-like particles (VLPs) and capsomere subunits have been developed as safe and effective vaccine candidates in the fight against infectious diseases. These bioengineered structures are suitable platforms for repetitive high density presentation of foreign epitope modules. However, due to the module’s diverse physicochemical properties, modularisation of capsomeres and VLPs requires tailoring of the physicochemical environment specific to each module to maintain protein stability against aggregation. Here, we report a high-throughput technique for screening buffer components to stabilize capsomeres, based on light scattering analysis. This screening method was applied to modular capsomeres presenting peptide epitopes from the rotavirus spike protein VP8 subunit domain, engineered as a next-generation rotavirus vaccine candidate. Among various additives tested, non-ionic detergents, such as Triton X-100, Tween-80 and Tween-20, were able to stabilize modular capsomeres, either alone or in combination with l-arginine, as confirmed with high-resolution size exclusion chromatography. Results demonstrate that tailoring the nature of the environment surrounding self-assembling proteins using small organic molecules can enhance the bioprocessing of modular vaccine capsomeres. The developed screening method potentially provides a powerful approach for rapid tailoring of processing conditions specific to antigenic modules displayed on next-generation recombinant capsomere and VLP vaccines, for low-cost vaccine delivery at global scale.

Oligosaccharides of Cabernet Sauvignon, Syrah and Monastrell red wines

Wine oligosaccharides were recently characterized and their concentrations, their composition and their roles on different wines remain to be determined. The concentration and composition of oligosaccharides in Cabernet Sauvignon, Syrah and Monastrell wines was studied. Oligosaccharide fractions were isolated by high resolution size-exclusion chromatography. The neutral and acidic sugar composition was determined by gas chromatography. The MS spectra of the oligosaccharides were performed on an AccuTOF mass spectrometer. Molar-mass distributions were determined by coupling size exclusion chromatography with a multi-angle light scattering device (MALLS) and a differential refractive index detector. Results showed significant differences in the oligosaccharidic fraction from Cabernet Sauvignon, Syrah and Monastrell wines. This study shows the influence that the grape variety seems have on the quantity, composition and structure of oligosaccharides in the finished wine. To our knowledge, this is the first report to research the oligosaccharides composition of Cabernet Sauvignon, Syrah and Monastrell wines.

Investigation of purification process stresses on erythropoietin peptide mapping profile.

Background:Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results.Materials and Methods:Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h.Results:No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation.Conclusions:Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance.

UNRAVELING THE MYSTERY OF NATURAL RUBBER BIOSYNTHESIS. PART II: COMPOSITION AND GROWTH OF IN VITRO NATURAL RUBBER USING HIGH-RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY

ABSTRACT The superior properties of natural rubber (cis-1,4-polyisoprene [NR]) are a function of its structure and composition, properties that still remain a mystery and that are irreplaceable by any synthetic rubber. NR from guayule (Parthenium argentatum) has been gaining special interest for its hypoallergenic properties while maintaining superior mechanical properties that are commonly associated with the Brazilian rubber tree (Hevea brasiliensis), the most common source of NR. Techniques exist to isolate washed rubber particles (WRPs) that contain enzymatically active rubber transferase, to study NR biosynthesis, and previous work on the in vitro NR growth in Hevea has demonstrated the presence of around 50 wt% of a low molecular weight ([MW], Mn <10 000 g/mol) fraction. Structural and compositional analyses of this low MW fraction in Hevea are challenging due to the high protein content. We discuss the analysis and composition of guayule latex and WRPs using high-resolution Size Exclusion Chromatography. We also discuss the composition of the soluble fraction of inactive guayule latex using matrix-assisted laser desorption ionization/time of flight mass spectrometry.

Association of Connexin43 with E3 Ubiquitin Ligase TRIM21 Reveals a Mechanism for Gap Junction Phosphodegron Control

Gap junctions (GJs) are sites of direct cell-to-cell communication formed by the connexin (Cx) family of ion channel proteins. The aberrant intercellular communication mediated by GJs is associated with a variety of hereditary and acquired human diseases. GJs utilize a highly interconnected network that is indispensible for synthesis, trafficking and degradation of their constituent proteins. By unbiased proteomic examination and network enrichment, we identified interacting components of the ubiquitin proteasome system associated with Cx43. LC-MS/MS identification and quantification of tryptic peptides from IP materials revealed a variety of interacting candidates, including the E3 ligase TRIM21 and ubiquitin. The interaction of Cx43 with TRIM21 was confirmed by confocal microscopy and coimmunoprecipitation of these proteins from C6 rat glioma and mouse primary astrocyte cultures. To gain a better understanding of this interaction, complexes isolated by high-resolution size-exclusion chromatography revealed signal integration by phosphorylation, ubiquitylation and proteolytic turnover within complexes of Cx43/TRIM21. Cx43/TRIM21 is also responsive to E1 UBE1 and E2 UbcH5a, with the interruption of this activity being an effective inhibitor of in vitro ubiquitin-conjugation. Mathematical models of these complexes demonstrated a mechanism for the switch-like degradation of GJs that were validated in EGF-stimulated cell cultures. Our finding of the interaction of Cx43 with TRIM21 provides mechanisms for the down-regulation of GJ intercellular communication that are known to impact a variety of physiological processes.

Quantitative determination of wine polysaccharides by gas chromatography–mass spectrometry (GC–MS) and size exclusion chromatography (SEC)

Wine polysaccharides play an important role on a number of technological and quality properties of wines and thus several methods have been proposed for their quantification. The present study evaluates the suitability of gas chromatography with mass spectrometry detector (GC–MS) for determining the content of wine monosaccharides and thus polysaccharide families. Factors affecting the yield of polysaccharide precipitation were firstly evaluated and the GC–MS method was characterised and compared with the previously reported GC–FID method. Repeatability and reproducibility values were similar in both methods, with values ranging from 1% to 14%. LODs obtained by MS were below 1.0μg for all monosaccharides and LOQs were below 1.8μg. Moreover, a recovery study of the whole method was carried out and it provided absolute recoveries between 81% and 116% for different wine samples, very good values taking into account the multi-step procedure. Both GC–MS and GC–FID were applied to determine the content of wine polysaccharide families in three wine samples and no significant differences were observed. Finally, high-resolution size exclusion chromatography with refractive index detector (HRSEC-RID) was applied to obtain the molecular weight distributions of the wine polysaccharides and to estimate their global content. The correlation observed between the polysaccharide values obtained with the GC method and the HRSEC-RID method (r=0.746, p<0.05) indicated that the latter could serve as a rapid and simple method to give an estimation of total wine polysaccharides although it can not be used to quantify in a precise way.

UNRAVELING THE MYSTERY OF NATURAL RUBBER BIOSYNTHESIS. PART I: INVESTIGATION OF THE COMPOSITION AND GROWTH OF IN VITRO NATURAL RUBBER USING HIGH RESOLUTION SIZE EXCLUSION CHROMATOGRAPHY

Abstract Monitoring the growth of in vitro natural rubber was accomplished by high resolution size exclusion chromatography, SEC. Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphate monomer in the presence of farnesyl pyrophosphate initiator. The high-resolution SEC was able to detect the formation of new rubber. Changes in the low molecular weight fraction were also detected. Gravimetric analysis revealed ∼30% mass gain after the in vitro synthesis. The overall gel content was found to be reduced, which further supported the formation of new rubber. This is the first report that utilizes high-resolution SEC to monitor the in vitro NR growth without the use of radiolabeling.

Effect of Macerating Enzymes on the Oligosaccharide Profiles of Merlot Red Wines

Commercial pectinase preparations are applied in winemaking to improve wine processing and final quality. These preparations contain pectolytic enzyme activities such as polygalacturonases, pectin esterases, pectin lyases, and rhamnogalacturonases. These enzymes modify the polysaccharide and oligosaccharide composition of wines. The influence of various commercial enzyme preparations on wine oligosaccharide composition was studied, on Merlot wines from the Bordeaux area. Wine oligosaccharides were isolated by high-resolution size-exclusion chromatography on a Superdex-30 HR column. The glycosyl residue and glycosyl linkage compositions of the oligosaccharide fractions obtained were determined. The MS spectra of the Merlot oligosaccharide fractions from control and enzyme-treated wines were recorded on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyzer. Oligosaccharides in the control wines were partly methylated homogalacturonans, corresponding to smooth regions of pectins, whereas those of the enzyme-treated wines were mostly rhamnogalacturonan-like structures linked with neutral lateral chains, arising from the hairy regions. The enzyme preparations used thus cleaved the rhamnogalacturonan backbone of the hairy zones and demethylated and hydrolyzed the smooth regions. Besides, different structures were detected, depending on the enzyme preparation used, indicating that they contained rhamnogalacturonase activities with different specificities. The oligosaccharide profiles can serve as a marker of enzymatic treatments.