Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup.

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

Irreversible hardening of a colloidal gel under shear: The smart response of natural rubber latex gels.

Natural rubber is obtained by processing natural rubber latex, a liquid colloidal suspension that rapidly gels after exudation from the tree. We prepared such gels by acidification, in a large range of particle volume fractions, and investigated their rheological properties. We show that natural rubber latex gels exhibit a unique behavior of irreversible strain hardening: when subjected to a large enough strain, the elastic modulus increases irreversibly. Hardening proceeds over a large range of deformations in such a way that the material maintains an elastic modulus close to, or slightly higher than the imposed shear stress. Local displacements inside the gel are investigated by ultrasound imaging coupled to oscillatory rheometry, together with a Fourier decomposition of the oscillatory response of the material during hardening. Our observations suggest that hardening is associated with irreversible local rearrangements of the fractal structure, which occur homogeneously throughout the sample.

Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis latex.

Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to ß-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles.

Comparative study of the mesostructure of natural and synthetic polyisoprene by size exclusion chromatography-multi-angle light scattering and asymmetrical flow field flow fractionation-multi-angle light scattering.

This paper presents results from the first analyses of the mesostructure of natural rubber (NR) by asymmetrical flow field flow fractionation (AF4). The results are compared with those obtained by size exclusion chromatography (SEC) in terms of average molar masses, radius of gyration and insoluble part (or gel quantity). Comparable results were obtained for the sample not containing gel. Conversely, for samples with gel, significant differences were found due to the presence of microaggregates. Contrary to SEC, AF4 fractionation enables partial fractionation of polyisoprene chains and microaggregates in a single run without preliminary treatment. The results presented here also highlight the special structure (very compact spheres) of microaggregates in NR compared to chemical crosslinked microaggregates in synthetic polyisoprene. The advantages and drawbacks of both techniques for analysing NR samples are also discussed.

Characterization of natural rubber using size-exclusion chromatography with online multi-angle light scattering. Study of the phenomenon behind the abnormal elution profile.

Natural and synthetic poly(cis-1,4-isoprene) were characterized by size-exclusion chromatography coupled with an online multi-angle light scattering detector (SEC-MALS). Unlike synthetic poly(cis-1,4-isoprene) (SR), natural rubber (NR) samples showed anomalous elution profiles. The beginning of elution was very similar to SR but, after a certain elution volume, the molar masses of the eluting macromolecules increased with elution volume instead of continuing to decrease, which resulted in an upturn curve profile. Adding tetrabutylammonium bromide (TBABr) to THF (solvent and mobile phase) removed this phenomenon. In addition, using different concentrations of TBABr showed that TBABr had two simultaneous actions. TBABr reduced the abnormal elution profiles and the quantity of aggregates (insoluble part or gel). These results mean that the main phenomenon involved in abnormal elution was delayed entities adsorbing on the column packing. Their delayed elution was responsible for the artificial increase in molar masses, especially at high elution volumes. The results obtained suggest that these entities are very compact and have a sphere-like structure.

Enhanced solvent extraction of polar lipids associated with rubber particles from Hevea brasiliensis.

Biochemical studies of lipids bound to rubber particles have been complicated due to the solubility of polyisoprene chains in most extracting solvents and the rather delicate nature of polar lipids that are often denatured when traditional solvent extraction techniques are employed. In this paper, we describe a traditional technique and accompanying solvents that permit optimal extraction of rubber particle bound lipids. The technique, which is validated after characterizing the lipid extracts by elemental analysis, silica column adsorption and thin layer chromatography, appeared more suitable for extracting total lipids with optimal glycolipid and phospholipid contents. This technique is proposed as an alternative to traditional extraction methods used for solid natural rubber as it offers advantages with respect to ease of application, extract quality, extraction yields and reproducibility.

Michael reactions carried out using a bench-top flow system.

The Michael reaction between methyl 1-oxoindan-2-carboxylate and methyl vinyl ketone was achieved successfully by pumping solutions of the reactants in toluene through a fluid bed of Amberlyst A21 at 50 degrees C. The use of a fluid bed reactor is attractive as it allows gel-type beads, i.e. the type of bead used in most studies of polymer-supported (PS) organic reactions, to be used satisfactorily in a flow system. When polymer-supported cinchonidine was used in place of Amberlyst A21, the Michael product was obtained in high yield with an enantiomeric excess (ee) of 51%. This % ee is comparable to that achieved when the reaction was catalysed by cinchonidine itself.