Ionic Strength Buffer Research Articles

You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Formation of the calpain-1/calpastatin complex promotes activation of calpain-1 under oxidizing conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165mM or 295mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Retention mechanisms of amitriptyline and its impurities on amide, amino, diol, and silica columns in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has been widely applied to challenging analysis in biomedical and pharmaceutical fields, bridging the gap between normal-phase high-performance liquid chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). This paper comprehensively explores the retention mechanisms of amitriptyline and its impurities A, B, C, D, F, and G on amide, amino, diol, and silica columns. Dual HILIC/RP-HPLC retention mechanisms were developed, and transitional points between HILIC and RP-HPLC mechanisms were calculated on amide, diol, and silica columns. Adsorption and partition contributions to overall retention mechanisms were evaluated using Python software in HILIC and RP-HPLC regions. The cation exchange mechanism dominates overall retention for ionized analytes in the silica column (R2>0.995), whereas the retention of ionized analytes increases with pH. Impacts of acetonitrile content, buffer ionic strength, and pH, along with their interactions on the retention of ionized analytes in the silica column, were determined using the chemometric approach. Acetonitrile content showed the most significant impact on the retention mechanisms. These findings highlight that a detailed investigation into retention mechanisms provides notable insights into factors influencing analyte retention and separation, promising valuable guidance for future analysis.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Critical parameters for highly efficient and reproducible polyelectrolyte multilayer coatings for protein separation by capillary electrophoresis

Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10−2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Potentiometric Determination of Free Fluoride Content in Wines from Dalmatia Region (Croatia)—A Comparative Study of Direct Potentiometry and Standard Addition Method

The aim of this study was to investigate 30 different types of Dalmatian wines as a potential source of fluoride. A fluoride ion selective electrode was used to measure the fluoride concentration in each sample. The direct potentiometric method and the standard addition method were evaluated, the latter being suggested as more accurate and precise. Measurements were performed in two buffers, acetate buffer and total ionic strength adjustment buffer (TISAB), to compare their influence on fluoride determination. The obtained results show that TISAB is a better choice than acetate buffer as a medium for fluoride determination. According to the proposed method, mass concentrations of fluoride of 0.19 and 0.18 mg/L were found in the studied red and white wines, with standard deviations of 0.04 and 0.03 mg/L, respectively. All determined fluorine levels in the tested wines were within the recommended limits and do not pose a risk to human health. No significant difference was found between the fluorine content in white and red wines, but there was a difference depending on the place of origin of the wine. The measured pH values for all the wines studied (except one sample) are very similar and show no significant correlation with the fluoride content.

The melting curves of calf thymus-DNA are buffer specific

The specific effects of salts (strong electrolytes) on biomolecular properties have been investigated for more than a century. By contrast, the specific role of pH buffers (weak electrolytes and their salts) has usually been ignored. Here, specific buffer effects on DNA thermal stability were evaluated by measuring the melting curve of calf thymus DNA through UV–vis spectroscopy. The study was carried out using phosphate, Tris, citrate and cacodylate buffers at fixed pH 7.4 at concentrations varying systematically in the range 1–600 mM. DNA stability increases with buffer concentration and is influenced specifically by buffer type. To interpret empirical data, a theoretical model was applied with parameters quantifying the impact of buffer on the DNA backbone charge. Comparing the buffer effects via buffer ionic strength rather than buffer concentration, we find that the buffers stabilize DNA in the order Tris > cacodylate > phosphate > citrate.

Optimization of the separation of benzoic acid and sulphanilamide by capillary zone electrophoresis (CZE)

Sulfonamide and Benzoic acid play a significant role regarding the treatment and inhibition of bacteria and its side effect on human health. Due to this, different analytical techniques with various adjustments to their parameters have been highlighted as an important challenge that should be tested in order to achieve the most efficient separation process, and thus to enable both qualitative and quantitative measurements of these substances. Therefore, the separation of the above-named compounds had been conducted by capillary zone electrophoresis (CZE) technique. Moreover, the comparison between both compounds in terms of organic, buffer and voltage at 25kV, 20kV had was achieved. Hence, this work was performed to determine the optimum experimental conditions of capillary voltage, with keeping ionic strength of buffer and percentage of organic modifier constant due to experimental time constraints

Design and economic investigation of a Multicolumn Countercurrent Solvent Gradient Purification unit for the separation of an industrially relevant PEGylated protein

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility.

Understanding the Significance of Sample Preparation in Studies of the Nanoparticle Metabolite Corona.

The adsorption of metabolites to the surface of nanomaterials is a growing area of interest in the field of bionanointeractions. Like its more-established protein counterpart, it is thought that the metabolite corona has a key role in the uptake, distribution, and toxicity of nanomaterials in organisms. Previous research has demonstrated that nanomaterials obtain a unique metabolite fingerprint when exposed to biological matrices; however, there have been some concerns raised over the reproducibility of bionanointeraction research due to challenges in dispersion of nanomaterials and their stability. As such, this work investigates a much-overlooked aspect of this field, i.e., sample preparation, which is vital to the accurate, reproducible, and informative analysis of the metabolite corona. The impact of elution buffer pH, volume, and ionic strength on the metabolite corona composition acquired by uncapped and polyvinylpyrrolidone (PVP)-capped TiO2 from mixtures of cationic and anionic metabolites was studied. We demonstrate the temporal evolution of the TiO2 metabolite corona and the recovery of the metabolite corona, which resulted from a complex biological matrix, in this case human plasma. This work also demonstrates that it is vital to optimize sample preparation for each nanomaterial being investigated, as the metabolite recovery from Fe3O4 and Dispex-capped TiO2 nanomaterials is significantly reduced compared to the aforementioned uncapped and PVP-capped TiO2 nanomaterials. These are important findings for future bionanointeraction studies, which is a rapidly emerging area of research in nanoscience.

Novel adsorption approach for the enrichment of R-Phycoerythrin from marine macroalga Gelidium pusillum

R-Phycoerythrin is a fluorescent and commercially valuable pigment protein present in algae. The extraction, purification and concentration of such biomolecules is a challenge for processing industries. In the present work, enrichment of R-Phycoerythrin was attempted by selective adsorption of other contaminant biomolecules from extract of algal biomass. Different adsorbents (zeolite, molecular sieve, ammonium Y zeolite, silver-exchanged zeolite, mesoporous carbon) and nanoparticles (zinc oxide, graphene oxide, gold and silver) were explored for this purpose. Process conditions such as buffer pH (5, 6, 7 and 8), ionic strength (0.1, 0.35, 0.6, 0.85 and 1.1) and adsorbent levels (w/w) were optimized. Optimization resulted in enrichment of pre-treated R-Phycoerythrin by 3.31 fold with 73.36% yield using zeolite and 2.10 fold with 98.91% yield using gold nanoparticles. This work demonstrates the efficacy of adsorbents and nanoparticles as possible alternatives for enrichment and purification of R-Phycoerythrin with appreciable yield.

Phosphoryl guanidine oligonucleotides as primers for RNA-dependent DNA synthesis using murine leukemia virus reverse transcriptase.

Modern approaches to the detection and analysis of low-copy-number RNAs are often based on the use of RNA-dependent DNA polymerases, for example, in reverse-transcription PCR. The accuracy and eff iciency of cDNA synthesis in the reverse-transcription reaction catalyzed by reverse transcriptase (RNA-dependent DNA polymerase) signif icantly affect the correctness of the results of PCR diagnostic assays and/or RNA sequencing. In this regard, many studies are focused on the optimization of the reverse-transcription reaction, including the search for more perfect primers necessary to obtain a full-length DNA copy of RNA under study. The best-known completely uncharged analogs of oligonucleotides – morpholine oligonucleotides and peptide nucleic acids – cannot be substrates for enzymes that process nucleic acids. The aim of this work was to conduct a pilot study of uncharged phosphoryl guanidine oligodeoxyribonucleotides (PGOs) as primers for mouse leukemia virus reverse transcriptase (MMLV H-). Specif ic features of elongation of partially and completely uncharged PGO primers were investigated. It was demonstrated that PGOs can be elongated eff iciently, e. g., in the presence of a fragment of human ribosomal RNA having complex spatial structure. It was shown that the proportion (%) of abortive elongation products of a PGO primer depends on buffer ionic strength, nucleotide sequence of the primer, and the presence and location of phosphoryl guanidine groups in the primer. The results indicate the suitability of PGOs, including completely electroneutral ones, as primers for reverse-transcription PCR, thereby opening up new prospects for the creation of experimental models for the analysis of highly structured RNA.

Electropolymerised pH Insensitive Salicylic Acid Reference Systems: Utilization in a Novel pH Sensor for Food and Environmental Monitoring.

This work summarizes the electrochemical response of a salicylic acid-based carbon electrode for use as a novel solid-state reference electrode in a redox-based pH sensor. This novel reference produces a pH insensitive response over a range of pH 3–10 in solutions with low buffer concentrations, different compositions, conductivities, and ionic strengths is produced. The pH of the local environment is shown to be determined by the chemistry and the electrochemical response of the redox active species on the surface of the electrode; the local pH can be controlled by the electropolymerized salicylic acid moieties due to the acid concentration on the surface, avoiding any perturbation in environmental pH and leading to a stable novel reference system. Sensitivities of −7.1 mV/pH unit, −2.4 mV/pH unit, −0.2 mV/pH unit, and 2.5 mV/pH units were obtained for different food medias, hydroponic solution, seawater, and cell-culture media, respectively, confirming its ability to control the local pH of the electrode. This reference system is paired with a new pH sensing element based on electropolymerized flavanone to provide a calibration free, pH sensitive sensor to effectively and accurately measure the pH of various media with high viscosity, low conductivity, low/high buffer concentration or cell-culture environment, presenting a maximum error of +/−0.03 pH units.

Structural characterization and hepatoprotective activity of an acidic polysaccharide from Ganoderma lucidum

In this study, Ganoderma lucidum crude polysaccharide (GLP) was found to have protective effect on liver damage in mice caused by restraint stress through improving oxidative status. Two polysaccharides, including a neutral β-glucan (GLPB2) and an acidic β-glucan (GLPC2) were purified from GLP through anion-exchange chromatography (AEC) combined with gel permeation. GLPC2, with an average molecular weight of 20.56 kDa, exhibited stronger hepatoprotective effect against H2O2-induced liver injury in HepG2 cells compared to GLPB2. Glycosidic residues and NMR analysis comprehensively revealed that GLPC2 contained d-Glcp-(1→, →3)-d-Glcp-(1→, →4)-d-Glcp-(1→, →6)-d-Glcp-(1→, →3, 6)-d-Glcp-(1 → and → 4)-d-GlcpA-(1 → . AEC can be an effective technique for separating β-glucans into neutral and acidic fractions by different ionic strength buffer. The findings provided a theoretical basis for the potential application of G. lucidum polysaccharides as a hepatoprotective in food and pharmaceutical industry.

Drug-Induced Phase Separation in Polyelectrolyte Microgels.

Polyelectrolyte microgels may undergo volume phase transition upon loading and the release of amphiphilic molecules, a process important in drug delivery. The new phase is “born” in the outermost gel layers, whereby it grows inward as a shell with a sharp boundary to the “mother” phase (core). The swelling and collapse transitions have previously been studied with microgels in large solution volumes, where they go to completion. Our hypothesis is that the boundary between core and shell is stabilized by thermodynamic factors, and thus that collapsed and swollen phases should be able to also coexist at equilibrium. We investigated the interaction between sodium polyacrylate (PA) microgel networks (diameter: 400–850 µm) and the amphiphilic drug amitriptyline hydrochloride (AMT) in the presence of NaCl/phosphate buffer of ionic strength (I) 10 and 155 mM. We used a specially constructed microscopy cell and micromanipulators to study the size and internal morphology of single microgels equilibrated in small liquid volumes of AMT solution. To probe the distribution of AMT micelles we used the fluorescent probe rhodamine B. The amount of AMT in the microgel was determined by a spectrophotometric technique. In separate experiments we studied the binding of AMT and the distribution between different microgels in a suspension. We found that collapsed, AMT-rich, and swollen AMT-lean phases coexisted in equilibrium or as long-lived metastable states at intermediate drug loading levels. In single microgels at I = 10 mM, the collapsed phase formed after loading deviated from the core-shell configuration by forming either discrete domains near the gel boundary or a calotte shaped domain. At I = 155 mM, single microgels, initially fully collapsed, displayed a swollen shell and a collapsed core after partial release of the AMT load. Suspensions displayed a bimodal distribution of swollen and collapsed microgels. The results support the hypothesis that the boundary between collapsed and swollen phases in the same microgel is stabilized by thermodynamic factors.

Dissolution Kinetics of Nifedipine-Ionizable Polymer Amorphous Solid Dispersion: Comparison Between Bicarbonate and Phosphate Buffers.

The intestinal fluid pH is maintained by the bicarbonate buffer system that shows unique properties regarding drug dissolution. Nevertheless, current compendial dissolution tests use phosphate buffers. The purpose of the present study was to investigate the effect of bicarbonate and phosphate buffers on the dissolution profiles of amorphous solid dispersions (ASD) composed of ionizable polymers. Hydroxypropylmethylcellulose acetate succinate (HPMCAS), amino methacrylate copolymer (AMC), and hydroxypropylmethylcellulose (HPMC) were employed as acidic, basic, and neutral polymers, respectively. Nifedipine (NIF) was used as a model drug. Dissolution profiles were measured in pH 6.5 bicarbonate and phosphate buffers by a mini-scale paddle dissolution test. The pH of bicarbonate buffers was maintained by the floating lid method. The pH change of the bicarbonate buffer was suppressed to less than + 0.25 pH for 3h by the floating lid method. In all cases, the NIF concentration was supersaturated against the solubility of crystalline NIF. The dissolution rates of HPMCAS and AMC ASDs were 1.5 to 2.0-fold slower in the bicarbonate buffer than in the phosphate buffer when compared at the same buffer capacity. The dissolution profile of HPMC ASD was not affected by the buffer species. The higher the buffer capacity and ionic strength, the faster the dissolution rate of HPMCAS ASD. The dissolution rate of ASDs with ionizable polymers would be overestimated by using unphysiological phosphate buffer solutions. It is important to use a biorelevant bicarbonate buffer solution for dissolution testing.

Evaluation and application of a positively-charged phenylaminopropyl bonded stationary phase for separation of basic compounds

Silica-based positively-charged stationary phase bonding phenylaminopropyl (named PHN) was found to produce symmetrical peak shape and higher sample loading for basic compounds. In this work, firstly, surface charge property of the PHN was evaluated by ζ-potential and retention of NO3−. A considerable amount of pH-dependent positive charges was confirmed more than that on CSH Phenyl-Hexyl, a commercial positively-charged phenyl stationary phase. Then chromatographic evaluation of standard alkaloids revealed that PHN could offer better peak shape and higher column efficiency at lower pH, and it functioned well under a wide range of buffer ionic strength. The PHN also showed different selectivity for basic compounds compared to the CSH Phenyl-Hexyl. Furthermore, it provided superior peak shape for high sample mass, demonstrating potential applications of this stationary phase in a preparative scale. These results can be explained by the strong charge intensity of the PHN stationary phase. Finally, the PHN was applied to separate a fraction from rhizomes of Corydalis decumbens, and purify dehydrocorybulbine from Corydalis yanhusuo W.T. Wang. Our study indicated the advantages and potential applications of the phenylaminopropyl bonded PHN stationary phase for basic compound separation.

Solution NMR Spectroscopy for Characterizing Protein-Glycosaminoglycan Interactions.

Solution nuclear magnetic resonance (NMR) spectroscopy and, in particular, chemical shift perturbation (CSP) titration experiments are ideally suited for mapping and characterizing the binding interface of macromolecular complexes. 1H-15N-HSQC-based CSP studies have become the method of choice due to their simplicity, short-time requirements, and minimal working knowledge of NMR. CSP studies for characterizing protein-glycosaminoglycan (GAG) interactions can be challenging due to binding-induced aggregation/precipitation and/or poor quality data. In this chapter, we discuss how optimizing experimental conditions such as protein concentration, choice of buffer pH, ionic strength, and GAG size, as well as sensitivity of NMR instrumentation can overcome these roadblocks to obtain meaningful structural insights into protein-GAG interactions.

Role of configurational flexibility on the adsorption kinetics of bivalent bispecific antibodies on porous cation exchange resins

The adsorption kinetics of a monoclonal antibody (mAb) used as a reference and of bivalent bispecific antibodies (BiSAb) on a macroporous cation exchanger is studied experimentally by examining the transient patterns of bound protein within the particles using confocal microscopy for a range of protein concentrations, buffer concentrations and pH, and temperatures. The mAb adsorption kinetics is controlled by pore diffusion and conforms to the classical shrinking core model. While the mAb adsorption rate increases with temperature, the ratio of effective and free solution diffusivity, De /D0, remains constant and has a value of 0.20. The BiSAb's structure is comprised of scFv domains that are genetically fused to a framework IgG through flexible peptide linkers which results in conformational flexibility leading to multiple binding forms with varying affinity for the adsorbent surface. As a result, adsorption of the BiSAbs shows complex patterns of total bound protein within the particles. These BiSAb adsorption patterns are influenced by buffer ionic strength, pH, and temperature in unique ways. Sharper intraparticle profiles are observed for conditions where the binding strength is greater (lower buffer concentration and/or pH) or when the protein is chemically crosslinked to restrict configurational flexibility. Temperature affects the BiSAb pore diffusivity as well as the interconversion kinetics. While the effects of temperature on BiSAb transport are also described by a constant De /D0 = 0.15, the temperature also affects the rate of interconversion between binding forms leading to faster equilibration at higher temperatures. A phenomenological model indicates that the interplay of pore diffusion and adsorption with the kinetically limited interconversion between binding forms is responsible for the experimental trends.