Alginate Binding Research Articles

Alginate binding enhances the structural stability and potentiates the lytic activity of bacteriophage endolysin's partially folded conformation

Polysaccharide polymers are increasingly being used as chaperon-like macromolecules in assisting protein folding of unfolded protein molecules. They interact with unfolded or partially folded proteins in a charge and conformation specific manner that results in the formation of stable protein-polysaccharide complexes. In most of the cases, the complex formation of protein-polysaccharide is driven via non-covalent interactions that have found to endorse the activity of proteins. T4L (18.7 kDa) and T7L (17 kDa) endolysins belong to the hydrolase and amidase class of peptidoglycan degrading enzymes. Both T4L and T7L exist in partially folded forms and are devoid of lytic activity at low pH conditions. In the current study, we assessed the binding of alginate with T4L and T7L at pH 7 and 3 using variety of biophysical and biochemical techniques. Spectroscopic studies revealed differential structural modulations of partially folded T4L and T7L upon their interaction with alginate. Further, the complex formation of alginate with partially folded T4L/T7L was confirmed by ITC and STEM. Additionally, the formed complexes of alginate with both T4L/T7L PF endolysins were found to be chemically and enzymatically stable. Moreover, such complexes were also marked with differential enhancement in their lytic activities at acidic pH conditions. This implied the potency of alginate as an excellent choice of matrix to preserve the structural and functional integrity of partially folded forms of T4L and T7L at highly acidic conditions.

The impact of sodium periodate oxidation and alginate coating on the capillarity of jute fabric

This study explores the impact of sodium periodate oxidation time (30 min, 60 min, or 120 min) and alginate coating on the capillarity of jute fabrics. Raw jute fabric exhibits a rapid increase in capillary rise height ( h) over time, reaching 72 mm at 1200 s. Fabrics oxidized for 30, 60, and 120 min show significantly lower h values of 19, 18, and 28 mm at 1200 s, respectively, which can be ascribed to the oxidation process itself. Precisely, oxidation-induced conversion of hydroxyl groups of cellulose (the main constituent of jute fiber) to aldehyde groups contributed to the fabric reduced affinity for water, hence negatively affecting its capillarity. Approximately 47% and 55% lower h values of fabrics oxidized for 30 and 60 min compared to that oxidized for 120 min can be explained by their lower weight loss after oxidation as well as less pronounced liberation of elementary fibers. Oxidized fabrics characterized by low capillarity can be used for the manufacturing of packaging materials, outdoor gear, and protective covers for crops. To expand jute fabric applications in biocarpet engineering, that is, to obtain polysaccharide-based supports, raw and oxidized jute fabrics were treated with a 1% sodium alginate solution. At 1200 s, oxidized fabrics coated with alginate exhibited 4.3–5.8 times higher h values compared to solely oxidized counterparts. The diffusion coefficients of oxidized fabrics coated with alginate increase up to 35% with prolonged oxidation, emphasizing the oxidation impact on alginate binding.

Sustained release of chlorogenic acid by co-encapsulation of sodium alginate binding to the Northern pike (Esox Lucius) liver ferritin.

Ferritin has a unique hollow spherical structure, which makes it a promising nanocarrier for food functional substances. In this study, a new ferritin was successfully extracted from the liver of Northern pike, purified, and identified. We used the reversible self-assembly characteristics of ferritin to fabricate chlorogenic acid (CA)-loaded apoferritin (Apo) complex (Apo-CA) and sodium alginate (SA)-apoferritin (Apo) co-encapsulate system. Apo-CA was encapsulated into the SA system to form SA-Apo-CA. The fabricated composites were analyzed using particle size, UV-Vis absorption spectroscopy, fluorescence spectroscopy, flourier transform infrared spectroscopy and transmission electron microscope. Physicochemical property of analysis confirmed th successful preparation of Apo-CA/SA-Apo-CA and improved thermal and UV radiation stability. The effect of sustained-release of CA were tested in vitro of simulated gastrointestinal tract digestion. SA-Apo-CA exhibited greater release ability than unencapsulated CA and Apo-CA. This study provides a new strategy for designing a multilayer delivery system with improved stability and sustained-release property.

1H NMR and EPR Spectroscopies Investigation of Alginate Cross-Linking by Divalent Ions

Alginate is a natural polymer widely applied in materials science, medicine, and biotechnology. Its ability to bind metal ions in order to form insoluble gels has been comprehensively used to create capsules for cell technology, drug delivery, biomedical materials, etc. To modify and predict the properties of cross-linked alginate, knowledge about the mechanism of alginate binding with metal ions and the properties of its gels is necessary. This article presents the results obtained by proton Nuclear Magnetic Resonance Spectroscopy for alginate containing calcium and strontium (alkaline earth metal diamagnetic) ions and by Electron Paramagnetic Resonance Spectroscopy for alginate with copper (Cu) and manganese (Mn) (transition metal paramagnetic) ions. It was found that in the case of calcium (Ca) and Mn ions, their concentration does not affect their distribution in the alginate structure and the cross-linking density. In the case of strontium (Sr) and Cu ions, their number affects the number of binding sites and, accordingly, the cross-linking density. Thus, the cross-linking of alginate depends mainly on the characteristics of specific cations, while the nature of the bond (ionic or coordination type) is less important.

Identification and structural analysis of a carbohydrate-binding module specific to alginate, a representative of a new family, CBM96

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35Å resolution. TM6-N4 exhibited an overall β-sandwich fold architecture with two antiparallel β-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.

Titanium Membranes with Hydroxyapatite/Titania Bioactive Ceramic Coatings: Characterization and In Vivo Biocompatibility Testing.

Titanium membranes and meshes are used for the repair of trauma, tumors, and hernia in dentistry and maxillofacial and abdominal surgery. But such membranes demonstrate the limited effectiveness of integration in recipients due to their bioinertness. In this study, we prepared titania oxide (by microarc oxidation) and/or HAp (by electrophoresis deposition) coatings with alginate soaking. We used annealing at 700 °C for 2.5 h for HAp crystallinity increasing with achievement of an acceptable Ca2+ release rate. The feedstock HAp and prepared coatings were characterized by X-ray diffraction, IR spectroscopy, electron and optical confocal microscopy, and thermal analysis, as well as the in vitro study of solubility in saline and in vivo tests with the animal model of subcutaneous implantation (with Wistar rats). Biocompatible compounds were found for all deposited coatings. We noted that the best biological response was detected for the annealed Ca-P/TiO2 bilayer with alginate binding. In this case, the coating crystallinity was ≈40.5-50.0%. The Ca2+ release rate was 2.042 ± 0.058%/mm2 at 168 h after immersion in saline. Thin and mature tissue capsules with minimal inflammation and vascularization were found in histological sections. We did not detect any unwanted responses around the implants, including inflammation infiltration, suppuration, bacterial infections, tissue lyses, and, finally, implant rejection. This information is expected to be useful for understanding the properties of bioactive ceramic coatings and improving the quality of medical care in dentistry and maxillofacial surgery and other applications of titanium membranes in medicine.

Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.

Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein’s role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

Alginate matrices for protein delivery - a short review.

Growth factors are powerful molecules that regulate cellular growth, proliferation, healing, and cellular differentiation. A delivery matrix that incorporates growth factors with high loading efficiencies, controls their release, and maintains bioactivity would be a powerful tool for regenerative medicine. Alginate has several unique properties that make it an excellent platform for the delivery of proteins. Mild gelling conditions can minimize the risk of protein denaturation; moreover, alginate can serve as protection from degradation until protein release. Various modifications have been proposed to tune alginate binding and release proteins, simultaneously adjusting alginate degradability, mechanical stiffness, swelling, gelation properties and cell affinity. The primary objective of this article is to review the literature related to recent advances in the application of alginate matrices in protein delivery in regenerative medicine. A special emphasis is put on the relevance of delivery of growth factors and chemokine.

Effects of Ground Cardamom on the Functional Properties of Restructured Ground Turkey

ObjectivesThe objectives of this study were to determine the effects of 0, 1.0, 1.5, and 2.0% ground cardamom in a restructured turkey product. The aim was to determine if this ingredient affected the function of an alginate binding system regarding cook yield, pH, texture, water activity, moisture percentage, and water holding capacity.Materials and MethodsEach treatment contained 1 pound (454 g) raw turkey, 0.6% sodium alginate, 0.6% encapsulated lactic acid, 0.3% calcium carbonate, and 5.0% distilled water. Samples were prepared and stuffed into polypropylene centrifuge tubes within 20 min at 23°C and given at least 12 h to set in a 4°C refrigerator. Samples were then cooked to an internal temperature of 72°C via water bath and the experiment was replicated 3 times. The binding strength of each treatment was determined using a Stevens-LFRA Texture Analyzer fitted with a spherical probe to penetrate 1-cm discs of product. One-way ANOVA was used to analyze the data with P < 0.05 as the significance level and a Tukey multiple range test was used to separate means.ResultsThe addition of ground cardamom within the binding system did affect pH, water holding capacity, and cooking yield of each treatment. As ground cardamom increased, the pH decreased from 6.35 to 6.10 and the cook yield increased by 4% (P < 0.05). The 2.0% ground cardamom sample had the highest cooking yield (72.7%) and lowest pH value (6.10). A decreased value in pH across all ground cardamom samples correlated to a decreased value in water holding capacity. This may be explained by competitive interactions between the ground cardamom and the calcium alginate binding system. The cardamom fiber absorbed water adequately to increase cook yield, but did not possess the same water holding capacity as turkey meat bound by calcium alginate. Water activity and moisture percentage appeared to have minimal differences (P > 0.05) of up to 0.003 units and up to 2.8%, respectively, across all 4 treatments. It was observed that 2.0% ground cardamom (166 g) had significantly higher (P < 0.05) binding strength than the 1.0% ground cardamom (146 g) treatment.ConclusionOverall, this study indicated that adding a fiber-rich spice source enhanced the cooking yield of the product while minimally influencing other characteristics. However, no sensory evaluation was performed to determine whether the differences were desirable. Further research can be performed with sensory evaluation to determine if ground cardamom at different levels affects the appearance, flavor, and overall acceptability of a restructured turkey product.

Composite microparticles of halloysite clay nanotubes bound by calcium carbonate

Natural halloysite clay nanotubes with 15nm inner and 75nm outer diameters have been used as vehicles for sustained release of drugs in composite hollow microparticles “glued” with CaCO3. We used a layer-by layer assembly accomplished alginate binding with Ca2+ followed by CO2 bubbling to prepare the composite microspheres of CaCO3 and polyelectrolytes (PE) modified halloysite nanotubes (HNTs–PE2/CaCO3) with the diameter of about 5–10μm. These microparticles have empty spherical structure and abundant pore distributions with maxima at 2.5, 3.9, 6.0 and 13.3nm, and higher surface area of 82.3m2g−1 as characterized by SEM and BET test. We loaded drugs in these micro-nano carriers of tight piles of halloysite nanotube with end clogged with CaCO3. The sustained release of Nifedipine drug from HNTs–PE2/CaCO3 composite microspheres was slower than for pristine halloysite nanotubes.

Structural insights into alginate binding by bacterial cell-surface protein

A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate.

Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.

Structural and Functional Characterization of Pseudomonas aeruginosa AlgX: ROLE OF AlgX IN ALGINATE ACETYLATION

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.

Surfactant Protein A Blocks Recognition of Pseudomonas aeruginosa by CKAP4/P63 on Airway Epithelial Cells

We used isogenic mutant strains that were deficient or over-expressed capsule to study the function of the alginate exopolysaccharide in the interaction of Pseudomonas aeruginosa with the human airway epithelial cells (AEC) in the presence or absence of surfactant protein A (SP-A). SP-A prevented the invasion of AEC by alginate-producing P. aeruginosa strains because of a direct effect on the AEC. Monoclonal antibodies to CKAP4/P63, the principal SP-A-binding receptor on AEC, or inhibition of its expression using specific siRNA reduced the invasion of both highly encapsulated and poorly encapsulated strains, but not the invasion of the acapsular mutant. Treatment of AEC with SP-A, monoclonal antibodies to CKAP4/P63, or CKAP4/P63-specific siRNA decreased the binding of purified alginate exopolysaccharide to AEC. Alginate binding to AEC reduced SP-A release by these cells. Because the alginate exopolysaccharide is surface-exposed, levels of SP-A may be crucial to modulate the interaction of P. aeruginosa with AEC.

Crystal Structure of a Novel Bacterial Cell-Surface Flagellin Binding to a Polysaccharide,

Bacterial flagellins are generally self-assembled into extracellular flagella for cell motility. However, the flagellin homologue p5 is found on the cell surface of Sphingomonas sp. A1 (strain A1) and binds tightly to the alginate polysaccharide. To assimilate alginate, strain A1 forms a mouthlike pit on the cell surface and concentrates the polymer in the pit. p5 is a candidate receptor that recognizes extracellular alginate and controls pit formation. To improve our understanding of the structure and function of p5, we determined the crystal structure of truncated p5 (p5DeltaN53C45) at 2.0 A resolution. This, to our knowledge, is the first structure of flagellin_IN motif-containing flagellin. p5DeltaN53C45 consists of two domains: an alpha-domain rich in alpha-helices that forms the N- and C-terminal regions and a beta-domain rich in beta-strands that constitutes the central region. The alpha-domain is structurally similar to the D1 domain of Salmonella typhimurium flagellin, while the beta-domain is structurally similar to the finger domain of the bacteriophage T4 baseplate protein that is important for intermolecular interactions between baseplate and a long or short tail fiber. Results from the deletion mutant analysis suggest that residues 20-40 and 353-363 are responsible for alginate binding. Truncated N- and C-terminal regions are thought to constitute alpha-helices extending from the alpha-domain. On the basis of the size and surface charge, the cleft in extended alpha-helices is proposed as an alginate binding site of p5. Structural similarity in the beta-domain suggests that the beta-domain is involved in the proper localization and/or orientation of p5 on the cell surface.

Proteomics-Based Identification of Outer-Membrane Proteins Responsible for Import of Macromolecules inSphingomonassp. A1: Alginate-Binding Flagellin on the Cell Surface,

A nonmotile gram-negative bacterium, Sphingomonas sp. A1, directly incorporates macromolecules such as alginate through a "super-channel" consisting of a pit formed on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Here, we demonstrate the proteomics-based identification of cell-surface proteins involved in the formation of the pit and/or import of alginate. Cell-surface proteins were prepared from the outer membrane released as vesicles during the conversion of intact cells to spheroplasts. Seven proteins (p1-p7) with acidic isoelectric points were inducibly expressed in the outer membrane of strain A1 cells grown on alginate and showed significant identity with bacterial cell-surface proteins (p1-p4, TonB-dependent outer-membrane transporter; p5 and p6, flagellin; and p7, lipoprotein). Each mutant with a disruption of the p1-p4 or p6 gene showed significant growth retardation in the alginate medium. Flagellin homologues (p5 and p6) were further analyzed because strain A1 forms no flagellum. p5 was found to be uniformly distributed on the cell surface by immunogold-labeling electron microscopy and to exhibit alginate binding with a nanomolar dissociation constant by a surface plasmon resonance sensor. The cell surface of the p6 gene disruptant differed from that of the wild-type strain A1 in that pit formation was incomplete and cell-surface structures shifted from pleats to networks. These results suggest that, distinct from bacterial flagellins constituting a helical filament of flagella, strain A1 cell-surface flagellin homologues function as receptors for alginate and/or regulators of cell-surface structures.

Effect of comminution method and raw binder system in restructured beef

The effect of particle size, method of particle size reduction (grinding, flaking, slicing) and alginate or Fibrimex ™ binding systems on processing properties of restructured beef were investigated. The size of semimembranosus muscles was reduced with a meat slicer, grinder or flaker (Comitrol) through machine openings that were 2, 4 and 8 mm in size. Bind of meat pieces in raw and cooked beef steakettes were measured. Sensory evaluation to determine the effect of method of size reduction on texture of beef steakettes was done only on steakettes manufactured with alginate. Beef steakettes manufactured with the alginate binding system had significantly ( p < 0.05) higher raw bind values than did the beef steakettes manufactured with Fibrimex. Slicing meat for the manufacture of restructured beef steakettes resulted in a higher raw bind than did either grinding or flaking the meat. Significant ( p < 0.05) interactions were observed for the bind of cooked steakettes between binder and method of size reduction and binder and opening size. When alginates were used to make restructured steakettes the bind of the cooked product was less ( p < 0.05) when meat was sliced than when it was ground or flaked. However, when Fibrimex was used, bind was stronger when sliced meat was used. The alginate system resulted in higher binds for cooked product when particle size was smaller but with the Fibrimex system bind was higher when particle sizes were larger. A consumer panel on alginate restructured steakettes showed no preference for the texture produced by the different methods of particle size reduction.

Investigation of Alginate Binding to Germanium and Polystyrene Substrata Conditioned with Mussel Adhesive Protein

Binding of alginate from Macrocystis pyrifera (kelp) to germanium and polystyrene substrata conditioned with mussel adhesive protein (MAP) from Mytilis edulis , to germanium substrata conditioned with bovine serum albumin (BSA) and polylysine, and to germanium substrata coated with aminopropyltriethoxysilane (APS) was investigated using attenuated total reflection Fourier transform infrared spectrometry. Binding of alginate to MAP appears to be proportional to surface coverage for levels tested. Distinct spectral features appear in the region associated with pyranose ring vibrations upon binding of alginate to MAP, polylysine, and APS, indicating that lysine residues play a prominent role in promoting irreversible adsorption with perturbation of pyranose ring atoms. BSA does not appear to enhance alginate adsorption over that observed on clean germanium and no new spectral features appear as a result of binding. The level of irreversible binding of alginate to germanium and polystyrene substrata conditioned with MAP is similar.

Method for the quantification of alginate in microcapsules.

Alginate is a key reagent in the preparation of microcapsules for cell transplantation. To address the question of the intracapsular alginate concentration, a sensitive assay has been developed to quantify the alginate content of microcapsules. The method is based on the metachromatic change induced by alginate binding to the dye, 1,9-dimethyl methylene blue (DMMB). The assay has a high sensitivity and precision. It covers a wide concentration range enabling the measurement of alginate in dilute supernatants as well as in microcapsules. For the latter, the membrane is initially dissolved by incubating the microcapsules in an alkaline medium. The effect of potentially interfering substances (poly-L-lysine (PLL), citrate, chloride, sodium) and of pH has been studied. Poly-L-lysine interfered with the assay at pH 6.5 but not at pH 13. Interference by sodium augmented with increasing sodium concentration and reached a plateau at 200 mM. This problem was overcome by routinely adjusting all samples to 500 mM sodium. The other substances tested had a negligible effect on the assay. The reliable measurement of alginate with this new assay will allow the optimization of the intracapsular alginate concentration.