Sedimentation Velocity Analytical Ultracentrifugation Research Articles

Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.

The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.

Characterization of size, anisotropy, and density heterogeneity of nanoparticles by sedimentation velocity.

A critical problem in materials science is the accurate characterization of the size dependent properties of colloidal inorganic nanocrystals. Due to the intrinsic polydispersity present during synthesis, dispersions of such materials exhibit simultaneous heterogeneity in density ρ, molar mass M, and particle diameter d. The density increments ∂ρ/∂d and ∂ρ/∂M of these nanoparticles, if known, can then provide important information about crystal growth and particle size distributions. For most classes of nanocrystals, a mixture of surfactants is added during synthesis to control their shape, size, and optical properties. However, it remains a challenge to accurately determine the amount of passivating ligand bound to the particle surface post synthesis. The presence of the ligand shell hampers an accurate determination of the nanocrystal diameter. Using CdSe and PbS semiconductor nanocrystals, and the ultrastable silver nanoparticle (M4Ag44(p-MBA)30), as model systems, we describe a Custom Grid method implemented in UltraScan-III for the characterization of nanoparticles and macromolecules using sedimentation velocity analytical ultracentrifugation. We show that multiple parametrizations are possible, and that the Custom Grid method can be generalized to provide high resolution composition information for mixtures of solutes that are heterogeneous in two out of three parameters. For such cases, our method can simultaneously resolve arbitrary two-dimensional distributions of hydrodynamic parameters when a third property can be held constant. For example, this method extracts partial specific volume and molar mass from sedimentation velocity data for cases where the anisotropy can be held constant, or provides anisotropy and partial specific volume if the molar mass is known.

In pursuit of mammalian polyamine oxidase crystal structure (774.4)

Mouse Polyamine Oxidase (mPAO) is a mammalian flavoprotein that is necessary for the catabolism of polyamines. mPAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine respectively. It is known that normal levels of polyamines are important for cell growth, yet their actual function is not well understood. The structure of mammalian PAO has yet to be determined; however structures for maize and yeast PAO (Fms1) exist. Although the identity between mPAO, Fms1, and maize PAO is only 20%, it has been shown, through sequence comparison and mutational analysis, that a conserved histidine residue at position 67 of Fms1 and 64 of mPAO helps to properly position the amine substrate for oxidation. Determining the crystal structure of mPAO will help identify other residues that play a role in substrate binding and catalysis.mPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. mPAO was expressed and purified using a Nickel column followed by an anion exchange column. Following the purification process, analytical ultracentrifugation sedimentation velocity experiments were performed to determine the oligomerization state and homogeneity of the protein.The sedimentation velocity experiments revealed a homogeneous, monomeric mPAO in solution, in contrast to the dimeric form of Fms1 in solution. Crystallization trials are underway to determine the structure of mPAO.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies.

Interactions and Stacking in Ordered Mononucleosomes and Folded Chromatin: Effects of Histone Tail Modifications

Folding of genomic DNA into compact but dynamic eukaryotic chromatin is driven by attractive interactions between nucleosome core particles (NCPs) and is inherently related to regulation of DNA replication, transcription and repair.However, little is known about the molecular details of the NCP-NCP contacts as well as of the nature and scale of forces involved. Chromatin folding is sensitive to both the ionic environment and to the sequence and modifications of the positively charged N-terminal histone tails.Combining experimental and computer modelling approaches, we study nucleosome-nucleosome interaction in ordered phases of stacked mono-nucleosomes as well as in folded nucleosome arrays (chromatin). Folding of chromatin fibres and intermolecular association of NCPs have been investigated as a function of ionic conditions and in the presence of posttranslational modifications in the histone tails, prepared by semi-synthetic ligation chemistry methods. Using sedimentation velocity analytical ultracentrifugation (AUC), X-ray diffraction (SAXS) and single molecule force spectroscopy methods, the dependence of NCP stacking and the stability of the folded fibres were investigated.Furthermore, using an advanced coarse-grained (CG) NCP model, computational modelling was used to investigate cation-induced NCP-NCP interactions. The CG model takes into account the detailed shape and charge distribution on the NCP, thus adequately describing the electrostatic interactions. The experimental behaviour is mostly dominated by unspecific electrostatic interactions. However, the close NCP-NCP stacking contacts show great sensitivity to the presence of the H4 histone tail and to modifications within this tail.

The Seca Dimer Interface

As a central component in the general secretion (Sec) pathway of bacteria, the ATPase motor protein, SecA, mediates preprotein translocation through the integral membrane channel, SecYEG. SecA is a potential target for antibacterial therapeutics because it is crucial for protein transport and cell viability. It is highly conserved among species of bacteria, yet it has no close human homologs. In the Sec pathway, SecA interacts with various ligands, including other SecA molecules. The latter interaction, a monomer-dimer equilibrium, is highly sensitive to salt concentration, temperature, and ligand binding. Although the structure of the SecA protomer is well-conserved among bacterial homologs, multiple dimer interfaces have been identified in SecA dimer crystal structures. We employed several biophysical methods to define the SecA dimer interface in solution and map the dimer interface using strategies independent of those used to solve the crystal structures of SecA dimer. By measuring the effects of alanine substitution on dimerization energetics using sedimentation velocity analytical ultracentrifugation, we determined that the substitution of residues at the N-terminus and within the helical scaffold domain significantly decreases dimerization affinity. By monitoring the backbone hydrogen/deuterium exchange rates using mass spectrometry, we found that residues lying within the helical scaffold domain are protected from exchange in the dimer state, consistent with the analytical ultracentrifugation results. These data are consistent with the Bacillus subtilis (1M6N) and Thermus thermophilus (2IPC) SecA dimeric structures.

Investigating High Affinity Protein Self-Association by Fluorescence Optical Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions and the rigorous characterization of binding stoichiometry and affinities in free solution. With a recently introduced commercial fluorescence optical detection system (FDS) it has gained the potential for applications to be extended to high-affinity interactions. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for the study by FDS-SV.

Tubulin Heterodimers Reversibly Dissociate with Moderate Kinetics as Demonstrated using Sedimentation Velocity Analytical Ultracentrifugation

A number of studies have reported reversible αβ-tubulin heterodimer dissociation with Kd =10−8 - 10−7 M, with presumably rapid but undetermined kinetics. However, in a recent study an extremely tight association between subunits (Kd 3 hours). In this work we have revisited the thermodynamics and kinetics of rat brain αβ-tubulin (RBT) dissociation by taking advantage of sedimentation velocity analytical ultracentrifugation methodologies (SV-AUC) developed in recent years. In particular, a combination of optical detection systems was used to cover a wide range of protein concentrations: pseudo-absorbance optics (pABS-AUC) for micro- and submicromolar concentrations, and the newly developed fluorescence optics (FDS-AUC) for nano- and subnanomolar concentrations. SV-AUC data was modeled using SEDFIT software resulting in a sedimentation coefficient continuous distribution, c(s), from which the weight average sedimentation coefficients (sw) were obtained. The isotherms obtained from calculated sw as a function of tubulin concentration were analyzed in SEDPHAT software using the heteroassociation model A+B1 AB to compute the limiting sedimentation coefficients of the dimer and monomer species as well as the equilibrium dissociation constant. In our experimental conditions, RBT heterodimer sediments with sw = 5.1 s, while the monomer species co-sediments with sw = 2.9 s. According to the heteroassociation model, the RBT heterodimer dissociates with Kd = 5.5 x 10−8 M. From the analysis of sedimentation profiles we find the characteristic dissociation time constant to be on the order of hours. In conclusion, the αβ-tubulin heterodimer displays reversible dissociation, moderate Kd and moderate dissociation kinetics.

Analysis of High Affinity Self-Association by Fluorescence Optical Sedimentation Velocity Analytical Ultracentrifugation of Labeled Proteins: Opportunities and Limitations

Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.

Effect of Pegylation on Self-Association of IFN-α2b

Pegylation of therapeutic proteins is an established technology used to enhance the bioavailability of an active pharmaceutical ingredient in the body of patients. While the physiochemical properties of pegylated monomeric proteins have been extensively described, there is still limited information on the characterization of pegylated oligomeric proteins. In this study, we report the characterization of a pegylated interferon alpha2b (PEGIFN-α2b) concentration-dependent oligomerization by a series of orthogonal biochemical and biophysical methods. These methods include sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, matrix-assisted laser desorption ionization, and size exclusion chromatography of bissulfosuccinimidyl suberate cross-linked PEGIFN. We report here that PEGIFN-α2b self-associates in a concentration-dependent manner into mainly monomers, dimers, and trimers. In the presence of the chemical cross-linker, PEGIFN-α2b is primarily monomeric (57%) at concentration lower than 0.3 mg/mL and contains about equal amount of monomers and dimers (47.0% and 37.7%, respectively), about 15% of trimers, and up to 4% of higher molecular weight species at 0.7 mg/mL and above.

The Off-rate of Monomers Dissociating from Amyloid-β Protofibrils

The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.

Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment.

BackgroundSignal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ.ResultsWe obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/− 0.3 μM).ConclusionThe three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Multi-Signal Sedimentation Velocity Analysis with Mass Conservation for Determining the Stoichiometry of Protein Complexes

Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.

Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

Are fluorescence-detected sedimentation velocity data reliable?

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation

AbstractModern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size‐distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self‐ and hetero‐association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective‐particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent‐solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1‐20.12.49. © 2013 by John Wiley & Sons, Inc.

Characterization of the novelTrypanosoma bruceiinosine 5′-monophosphate dehydrogenase

There is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy. Trypanosoma lacks the enzymatic machinery for the de novo synthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5'-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinant Trypanosoma brucei IMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculated K m values of 30 and 1300 μ m for IMP and NAD, respectively. The obtained K m value of TbIMPDH for NAD is approximately 20-200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies show K i values of 3·2 μ m, 21 nM and 3·3 nM for ribavirin 5'-monophosphate, mycophenolic acid and mizoribine 5'-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

Exploring the Interactions between a Coiled-Coil Peptide Model System and Metallated Porphyrins towards the Design of Photoelectronically Active Biomaterials

Porphyrin-peptide structures hold great potential for design of environmentally responsive nanowires, and porphyrin metallation may modulate these structures' electronic properties. To better understand these effects, the binding of metallated meso-tetrakis(4-sulfonatophenyl)porphine (TPPS4) to the peptide Cp3K-N was investigated. Cp3K-N is a non-polymerizing model for the polymerizing peptide Cp3K, allowing for quantitative experiments that would be impossible in an aggregating system. Cu(II)TPPS4, Ni(II)TPPS4, Pd(II)TPPS4, and Zn(II)TPPS4 were studied using circular dichroism (CD) spectroscopy, UV-Visible spectroscopy, and sedimentation velocity analytical ultracentrifugation (SV-AU). CD and UV-Vis titrations of the ratio of Cp3K-N to porphyrin were performed, and binding constants for the complexes were determined. A blue-shifted Soret Band absorbance in the porphyrin, concomitant with induction of Cp3K-N alpha-helicity, was observed for all metals but zinc, which demonstrated a red-shifting of its Soret Band. Such spectroscopic characteristics suggest specific porphyrin-peptide binding. Titration experiments for the copper, nickel, and palladium porphyrins indicated equal peptide:porphyrin stoichiometry, while SV-AU experiments suggest higher order complex formation. These results confirm that metalled TPPS4 can bind Cp3K-N with the specificity and tightness of TPPS4, suggesting that such porphyrins have potential for use in polymerizing systems, towards the goal of designing novel photoelectronically active biomaterials.

Aggregation analysis of pharmaceutical human immunoglobulin preparations using size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity

In the pharmaceutical industry, analysis of soluble aggregates in pharmaceutical formulations is most commonly performed using size-exclusion chromatography (SEC). However, owing to concerns that aggregates can be overlooked by SEC analysis, it has been suggested that its results should be confirmed with orthogonal methods. One of the main alternative methods for SEC is analytical ultracentrifugation sedimentation velocity (AUC-SV), which has been indicated as an important tool for the measurement of protein aggregation. The present study aimed to show that AUC-SV can be effectively applied for the characterization of marketed immunoglobulin pharmaceutical preparations to support the results obtained by SEC. In addition, the present research aimed to assess the appropriateness of two integration approaches for the quantitative analysis of the SEC results. Thus, the aggregates were measured in seven different preparations of human immunoglobulins by AUC-SV and SEC, and the acquired chromatographic data were processed by using either the vertical drop method or the Gaussian skim approach, implemented in the Empower II chromatography data software (Waters, Tokyo, Japan). The results of aggregation measurements performed using AUC-SV were in good agreement with those obtained using SEC. As expected, the Gaussian skim integration approach inherently provided lower estimates of aggregation content than the results of the vertical drop method. The finding of this study confirmed the complementary nature of AUC-SV to SEC for aggregate composition analysis and underscored the important role that the different integration methods can play in the quantitative interpretation of chromatographic results.