Proteins In Aqueous Solution Research Articles

The role of carboxyl groups upon the precipitation of albumin at low pH

Proteins play a major role in the organization of the living matter. Underlying this fact are their unique properties in aqueous solution, for which the available theories provide only a partial explanation. In order to throw some light into the behaviour of proteins in aqueous solution, experiments on the precipitation of electropositive albumin by means of the sodium salts of sulfate, thiocyanate, nitrate, bromide and chloride upon varying the solution pH were undertaken. Results show that the protein undergoes a transition at approximately pH 3, characterized by a change on its precipitation profile. Its shape, of monotonic profile below pH 3, displays a non-monotonic behaviour with separated peaks at higher pH values, which increase in number and decrease in intensity with increasing pH. To elucidate these observations, additional precipitation experiments with sodium thiocyanate in the presence of a secondary salt were undertaken. It is shown that the secondary salt changes significantly the protein's precipitation profile, despite being present in very low concentration. Its influence on the precipitation depends in a great extent on the cation's valence, which induce effects proportional to x, x2, and x3 for monovalent, divalent, and trivalent cations, respectively. The observations reported can be explained under the assumption that i) the protein dissociates into distinct forms, as result of the dissociation of carboxyl groups, and ii) salt cations establish chemical equilibria with the negative charges of the dissociated carboxyl groups present in the emerging protein forms.

Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins

FTIR and Raman spectroscopy are often used to investigate the secondary structure of proteins. Focus is then often laid on the different features that can be distinguished in the Amide I band (1600–1700 cm−1) and, to a lesser extent, the Amide II band (1510–1580 cm−1), signature regions for C=O stretching/N-H bending, and N-H bending/C-N stretching vibrations, respectively. Proper investigation of all hidden and overlapping features/peaks is a necessary step to achieve reliable analysis of FTIR and FT-Raman spectra of proteins. This paper discusses a method to identify, separate, and quantify the hidden peaks in the amide I band region of infrared and Raman spectra of four globular proteins in aqueous solution as well as hydrated zein and gluten proteins. The globular proteins studied, which differ widely in terms of their secondary structures, include immunoglobulin G, concanavalin A, lysozyme, and trypsin. Peak finding was done by analysis of the second derivative of the original spectra. Peak separation and quantification was achieved by curve fitting using the Voigt function. Structural data derived from the FT-Raman and FTIR analyses were compared to literature reports on protein structure. This manuscript proposes an accurate method to analyze protein secondary structure based on the amide I band in vibrational spectra.

Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Structures of the intrinsically disordered Aβ, tau and α-synuclein proteins in aqueous solution from computer simulations

Intrinsically disordered proteins (IDPs) play many biological roles in the human proteome ranging from vesicular transport, signal transduction to neurodegenerative diseases. The Aβ and tau proteins, and the α-synuclein protein, key players in Alzheimer’s and Parkinson’s diseases, respectively are fully disordered at the monomer level. The structural heterogeneity of the monomeric and oligomeric states and the high self-assembly propensity of these three IDPs have precluded experimental structural determination. Simulations have been used to determine the atomic structures of these IDPs. In this article, we review recent computer models to capture the equilibrium ensemble of Aβ, tau and α-synuclein proteins at different association steps in aqueous solution and present new results of the PEP-FOLD framework on α-synuclein monomer.

Effects of atmospheric pressure plasma jet on the physicochemical, functional, and antioxidant properties of flaxseed protein

The aim of this study was to explore the effect of atmospheric pressure plasma jet (APPJ) on the physicochemical, functional, and antioxidant properties of flaxseed protein following APPJ treatment (0 to 240 s). The results showed that the pH value continuously dropped with the minimum value of 3.45±0.15 after 240 s of APPJ treatment (-61.7%, P < 0.05). The relative protein solubility significantly declined after 15 s of APPJ treatment (-43.1%, P < 0.05), which was accompanied by the evident increase in mean particle size of flaxseed protein in aqueous solution (+157%, P < 0.05). Moreover, the surface hydrophobicity and contents of disulfide bonds gradually raised when the APPJ exposure time extended from 30 to 240 s. Notably, the foaming, emulsifying, and in vitro antioxidant properties of flaxseed protein were significantly improved following short time of APPJ treatment (5 to 15 s), which was paralleled with the changes of spatial conformation, mild protein oxidation, as well as the release of phenolic acids and flavonoids from naturally occurring protein-phenolic complex. Our findings elucidated that APPJ may be considered as an effective strategy to improve the functionality and antioxidant activities of flaxseed protein. PRACTICAL APPLICATION: We had evaluated the effect of APPJ treatment on the physicochemical, functional, and antioxidant properties of flaxseed protein, which was conducive to tailor flaxseed protein with the optimal techno-functionality and antioxidant activities as a potential nano-delivery vehicle.

Influence of thermally induced structure changes in diluted β-lactoglobulin solutions on their surface activity and behavior in foam fractionation

Surface activity is an intrinsic protein feature, leading to the capability of aqueous protein solutions to form foam. This feature provides opportunities for downstream processing, such as usage of foam fractionation for purification. In order to investigate the impact of the surface activity on the performance of the foam fractionation process, protein solutions with different surface activity were produced by different thermal denaturation of aqueous β-lactoglobulin solutions. The effectiveness of the denaturation procedure was verified with circular dichroic spectroscopy, and the impact on surface activity was determined via dynamic surface tension measurement. The increased surface activity resulted in higher foamate flow rates. Furthermore, the effects could be correlated with secondary structure changes and with the dynamic surface pressure. The new result of this study is that the effect of the denaturation of a protein on foam fractionation depends on the protein concentration. At the lower feed concentration, effects became visible, which could not be observed at the higher concentration.

Abstract D062: Mechanistic insights into phase 1b/2 study of chitosan for pharmacologic manipulation of AGE (advanced glycation endproducts) levels in prostate cancer patients

Abstract Lifestyle choices have long been known to have a major role in the development or prevention of cancer. Diet, chronic stress, smoking, poverty, physical activity, and socioeconomic variables are recognized as significant factors in prevention of cancer. Increasingly, these behaviors are studied as variables that affect cancer survivorship and response to treatment as well. One general mechanism through which diet could affect cancer growth is through promoting or inhibiting the accumulation of advanced glycation endproducts. Advanced glycation endproducts (AGEs) are reactive metabolites produced as by-products of sugar metabolism and oxidative stress. Furthermore, among PCa patients, AGE accumulation is higher in blacks with African ancestry (AAs) than in European Americans (EAs). As AGEs appear to promote cancer survival and growth, interventions to limit their accumulation may be viewed as potential cancer therapeutic agents. Chitosan, a polysaccharide obtained from shellfish or mushrooms that is thought to be poorly absorbed, directly binds AGE-modified proteins in aqueous solutions. No studies to date have demonstrated that chitosan can reduce either serum or tissue levels of AGEs in humans with AGE-related diseases. The investigators are conducting a Phase 1, assay-guided trial of chitosan in patients with prostate cancer on ADT and propose that chitosan will act as an absorbent of pre-formed AGEs, preventing their absorption from the gut and thus reducing their levels in the body. This intervention produced an 80% reduction in the plasma level of carboxymethyl lysine (CML) AGE adducts and more remarkably there was also a reduction in tissue AGE levels as determined by skin autofluorescence. These data support the idea that chitosan could be an active AGE-reducing agent that is clinically tolerable. Plasma and stool bio-fluid specimens have been collected from the four patients enrolled in the clinical trial and will be assayed. As these mechanistic studies are ongoing, the result will document any systemic effects from the chitosan intervention on inflammation, bowel permeability and microbiome diversity. Citation Format: Shanora G Brown, Brandon Sutton, Taiwo Biotidara, Dave Turner, Robert Wilson, Michael Lilly. Mechanistic insights into phase 1b/2 study of chitosan for pharmacologic manipulation of AGE (advanced glycation endproducts) levels in prostate cancer patients [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D062.

(Invited) Organic Semiconductor, Receptor Material and Circuit Design for Organic Electronic Vapor Sensors and Biosensors

We will present our recent results on the design of semiconducting material, dielectric receptor matrices, and stabilizing and amplifying circuit structures for detection of volatile analytes in the atmosphere and proteins in aqueous solution. Besides our natural interest in designing materials that respond more strongly to analytes of interest than to likely interferents, we also emphasize the use of chemical and electronic amplification methods to increase the ratio of the desired responses to the drift (signal/noise ratio). Printable materials, especially polymers, are emphasized for the responsive layers. The use of multiple sensing elements, typically field-effect transistors (FETs), to create patterns of responses increases the selectivity of the information, either by narrowing the classes of compounds providing the responses, distinguishing time-dependent from dose-dependent responses, and/or increasing the ratio of analyte responses to environmental drifts. As a specific example, using pairs of FETs, in series or in parallel, allows device drifts to be substantially canceled while analyte responses are maintained and even reinforced. This strategy has led to significant improvements in the use of polymer-based FETs to detect pollutant gases such as nitrogen dioxide and ammonia. To increase the stability of systems used to detect analytes in solution, we sometimes separate the sensing surface from the output device in an arrangement known as a remote gate. We show that the output device may be an organic-based or a silicon-based transistor, and can respond to electrochemical potential changes at the sensing surface arising from a variety of chemical interactions. This type of configuration was applied to the understanding of vapor sensors and the effect of receptor polarity on the detection of brain injury biomarker proteins. New details about the mechanisms for sensor responses to analytes can be obtained from remote gate configurations, including connection of interfacial voltages and charge densities in responsive polymer semiconductors.

A Microfluidic Co-Flow Route for Human Serum Albumin-Drug-Nanoparticle Assembly.

Nanoparticles are widely studied as carrier vehicles in biological systems because their size readily allows access through cellular membranes. Moreover, they have the potential to carry cargo molecules and as such, these factors make them especially attractive for intravenous drug delivery purposes. Interest in protein‐based nanoparticles has recently gained attraction due to particle biocompatibility and lack of toxicity. However, the production of homogeneous protein nanoparticles with high encapsulation efficiencies, without the need for additional cross‐linking or further engineering of the molecule, remains challenging. Herein, we present a microfluidic 3D co‐flow device to generate human serum albumin/celastrol nanoparticles by co‐flowing an aqueous protein solution with celastrol in ethanol. This microscale co‐flow method resulted in the formation of nanoparticles with a homogeneous size distribution and an average size, which could be tuned from ≈100 nm to 1 μm by modulating the flow rates used. We show that the high stability of the particles stems from the covalent cross‐linking of the naturally present cysteine residues within the particles formed during the assembly step. By choosing optimal flow rates during synthesis an encapsulation efficiency of 75±24 % was achieved. Finally, we show that this approach achieves significantly enhanced solubility of celastrol in the aqueous phase and, crucially, reduced cellular toxicity.

Validation of specific cation partitioning to molecular surfaces using fluorescent carbon quantum dots

Specific salt effects are significantly involved in the aqueous colloidal solutions of proteins, nucleic acids, surfactants and nanomaterials. The mechanism of such effects has been systematically investigated here with a simple nanomaterial-based model system. Water-soluble fluorescent carbon quantum dots were synthesized for this purpose. This research work highlights the unique advantage offered by fluorescent carbon quantum dots to be utilized as a sensing probe for the detection of selective ion interactions based on their fluorescence turn-on response. Synthesized carbon quantum dots exhibited surface potential dependent strong fluorescence emission. It was further found that the functional group chemistry on the carbon quantum dot surface plays a crucial role in the ion partitioning. A remarkable enhancement of emission intensity observed in the presence of metal cations can be attributed to their relative tendency to form ion-pairs with the nanomaterial surface functionalities. Carbon quantum dots with deprotonated carboxylic groups on their surface showed a stronger affinity for strongly hydrated cations as compared to weakly hydrated cations. Uncharged yet polar nanomaterial surface, however, showed no selectivity towards different ions.

Kinetics of the Light-Oxygen Effect in Aqueous Solutions of Proteins

Molecular mechanisms of the participation of proteins in reactive oxygen species (ROS) generation resulting from the singlet-oxygen effect upon laser exposure in solutions of various proteins are considered. The kinetic equations describing ROS generation are derived, and their approximate general solution is found in the analytical form. By comparison of the found kinetic dependences with experimental data, orders and rate constants of corresponding chemical reactions are determined. An analysis of the results makes it possible to consider that fundamental mechanisms of ROS formation reactions in solutions of various proteins are identical.

The protection of bovine serum albumin against thermal denaturation and gelation by sodium dodecyl sulfate studied by rheology and molecular dynamics simulation

It is well known that when bovine serum albumin (BSA) protein in aqueous solution is heated above the protein denaturation temperature which is typically > 65 °C, the protein unfolds substantially and under continuous heating the unfolded protein aggregates to form a gel. In contrast to conventional wisdom, however, we show that sodium dodecyl sulfate (SDS) can protect BSA against thermal denaturation and gelation at specific concentration range, using 0–1 M SDS and 3, 5 and 7 wt% BSA concentrations. The SDS protective effect which was investigated by time sweep test at 80 °C, increased with the SDS concentration up to 0.85, 0.7 and 0.4 M at 3, 5 and 7 wt% BSA solutions, respectively. Beyond these specified concentrations, the SDS protective effect decreased. The increase or decrease in the protective effect was further confirmed by the frequency sweep test carried out on the pure BSA and BSA/SDS solutions at 80 °C. The frequency sweep test showed that the value of the storage modulus (G′) without the protective effect was more than 3 orders of magnitude larger than its value when the BSA was completely protected from thermal gelation. In order to interpret the SDS effects at the atomic/molecular level, molecular dynamics (MD) simulation was applied to identify the helices of the BSA that were protected in thermal denaturation at 80 °C. The molecular mechanism whereby SDS protects the BSA helices was established as a bridging interaction between the SDS ion and some helical moieties of the BSA.

Ubiquity of complex coacervation of DNA and proteins in aqueous solution.

We report complex coacervation between a primarily hydrophobic protein, elastin, and a strong polyanion DNA (2 kbp) in aqueous and salty solutions at room temperature, 25 °C. The associative interaction at fixed elastin and varying DNA concentration, thereby maintaining a mixing ratio of r = [DNA] : [elastin] = 0.0027 to 0.093, was probed. What distinguishes this study from protein-DNA coacervation reported earlier is that the protein used here was mostly a hydrophobic polyampholyte with low linear charge density, and its complementary polyelectrolyte, DNA, concentration was chosen to be extremely small (1-35 ppm). The interaction profile was found to be strongly hierarchical in the mixing ratio, defined by three distinct regions: (i) Region I (r < 0.02) was defined as the onset of primary binding leading to condensation of DNA; (ii) Region II (0.02 < r < 0.08) indicated secondary binding which led to the formation of fully charge neutralized complexes signaling the onset of coacervation; and (iii) Region III (0.08 < r < 0.12) revealed growth of insoluble complexes of large size facilitating liquid-solid phase separation. The degree of complex coacervation was suppressed in the presence of a monovalent salt implying that screened Coulomb interactions governed the binding. Small angle neutron scattering data attributed an amorphous structure to the coacervates. The elastin-DNA system belongs to a rare class of interacting biopolymers where very weak electrostatic interactions may drive coacervation, thereby implying that coacervation between DNA and proteins may be ubiquitous.

Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins.

Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of "MS-friendly" detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from "MS-friendly" detergents.

Feasibility of electrospraying fully aqueous bovine serum albumin solutions

Electrospraying or electrohydrodynamic atomisation, i.e. the formation of tiny droplets from a jet of conductive liquid under the influence of an electric field, has been gaining in popularity as a particle engineering technique in recent years. In addition to general benefits for particle engineering, e.g. the ability to generate nanometre sized particles with a very narrow size distribution, electrospraying also possesses a number of characteristics, like its applicability at ambient conditions, which could make it especially interesting for formulating therapeutic proteins. However, as fully aqueous solutions of proteins tend to have relatively high electrical conductivities and surface tensions, obtaining a stable Taylor cone-jet mode for these solutions is inherently challenging. This is why in the majority of studies reporting the successful electrospraying of proteins, either emulsions, aqueous suspensions or a mixture of water and one or more organic solvents were used instead of fully aqueous solutions. Therefore, an ab initio electrospraying formulation development study was conducted, using only fully aqueous feed solutions containing protein stabilising excipients commonly used in spray- and freeze-drying of therapeutic proteins. The study included bovine serum albumin (BSA) as a model protein and consisted out of two parts: (1) a one parameter at a time screening study, designed to improve the understanding of how various formulation components influence relevant physicochemical properties and the electrospraying process and (2) two subsequent mixture design of experiments (DoE) studies, designed to aid in the statistical description and prediction of the influence of different protein-excipient combinations on the electrospraying process. Additionally, the influence of physicochemical properties relevant to the electrospraying process, i.e. the volumetric mass density, electrical conductivity, kinematic viscosity and surface tension, was assessed for all feed solutions included in the study.

Proteins in aqueous solution under high frequency electromagnetic field: viscoelastic approximation of a complex system

Proteins in aqueous solution are complex systems that are made of many interacting and non-interacting elements whose behavior can be predictable upon the application of non-linear models. The aim of this study was to show that this complex system behaves like a viscoelastic system under exposure to high frequency electromagnetic (HF-EMF). To this aim, typical proteins in water solution were exposed for 3 h to a high frequency electromagnetic field at the power density of 1 W/m 2 . Proteins α-helices aligned along the direction of the field and the integrated areas of proteins β-sheet content increased linearly as a function of proteins dipole moment. This result can be explained assuming that proteins in aqueous solution under HF-EMF behave like a viscoelastic system.

Effect of aggregation on hydration of HSA protein: Steady-state Terahertz absorption spectroscopic study

Terahertz (THz) absorption behaviour of HSA protein in aqueous buffer solutions has been investigated in the 0.1–2 THz frequency range using a highly intense THz source based on coherent transition radiation (CTR) generated using a femtosecond electron accelerator of 42 MeV energy (for 0.3–2 THz) and a klystron (at 0.1 THz). Like in the cases of other proteins reported earlier, THz absorbance of the protein solutions follow nonlinear behaviour with increasing concentration of HSA protein monitored through the entire frequency range. THz absorbance of the solution initially increases to follow an apparently linear behaviour up to the concentration of ~6 × 10−4 mol dm−3 but decreases gradually with further increase in HSA concentration. The linear behaviour in low concentration regime could be explained considering the increase in concentration of the monomer HSA molecules in solution with a well-defined hydration layer of thickness of about 22 A around it. However, the study of dynamic light scattering measurements suggest the presence of increasing number of protein aggregates in solution with increasing concentration of protein. THz absorbance of each of these samples could be calculated to show that absorbance decreases with increasing number of aggregates in solution and also the relative concentrations of the monomer and aggregated particles existing in solutions could be estimated. This work, for the first time, explains the nonlinear change in THz absorbance of protein solutions with increasing protein concentration considering the protein aggregation effect at very high concentration. At low concentration of Human Serum Albumin protein in aqueous solution, each monomer protein molecule is surrounded by a hydration layer of about 2.2 nm thick.

A Dielectric Barrier Discharge Plasma Degrades Proteins to Peptides by Cleaving the Peptide Bond

Effects of atmospheric pressure plasmas on proteins are studied to assess the quality of plasma decontamination and to gain insights into plasma-triggered molecular events underlying observations made in plasma medicine on the cellular, organ, and systemic level. Atmospheric pressure plasma treatment has been reported to cause protein degradation. Degradation products, however, have not been characterized. Treating different model proteins in aqueous solution with a DBD plasma, we confirmed with different methods (Bradford assay, application of Lambert–Beer’s law on absorption measurements at 280 nm, ninhydrin assay, size exclusion chromatography, SDS-PAGE) that protein degradation takes place. Peptides of different sizes were detected by size exclusion chromatography. The ninhydrin assay indicated that peptide bonds are cleaved. In the presence of hydroxyl radical scavenger d-mannitol, the concentration of amino termini formed during the initial 10 min of plasma treatment was reduced by 96%, while at longer treatment times mannitol did no longer prevent the formation of amino termini, indicating that hydroxyl radicals play an important role in the initial cleaving of peptide bonds in the protein, but other mechanisms are at play in cleaving the peptide bonds in the resulting peptides. The generation of peptides has implications for plasma decontamination and plasma medicine. It is critical to verify that plasma decontamination processes do not result in protein fragments with undesired properties. In plasma medicine, plasma-generated protein fragments may act as molecular triggers in treated cells, tissues, or patients, e.g., regulating signaling cascades in a protease-like fashion.

Following Protein Dynamics in Real Time during Crystallization

The process of protein crystallization from aqueous protein solutions is still insufficiently understood. During macroscopic crystal formation, occurring often on time scales from a few hours to se...

Aggregation selectivity of amyloid β1‐11 peptide in aqueous ionic liquid solutions

AbstractUnderstanding the aggregation selectivity of peptide fragments of full‐length proteins in aqueous solutions with ionic liquids (ILs) could facilitate the elucidation of the relationship between the IL‐protein interactions and structural behavior of intrinsically disordered proteins (IDPs) such as amyloid β protein following the addition of ILs. In the present study, we investigate structural changes in peptide fragment 1‐11 (Aβ1‐11) of amyloid β protein in aqueous solutions with two ILs including 1‐butyl‐3‐methylimidazolium thiocyanate ([bmim][SCN]) and ethylammonium nitrate (EAN) using optical spectroscopy. The addition of [bmim][SCN], which exhibits strong protein denaturant ability, induced the formation of an intermolecular β‐sheet structure (aggregation), while the addition of EAN, which has a weaker denaturant ability compared with [bmim][SCN], did not cause aggregation. Since the role of cations is related to the ability to mask the charged residues of Aβ1‐11, the aggregation selectivity of Aβ1‐11 depends on the anionic species and anions with high denaturation ability enhanced aggregation. Our results demonstrated that the structural change in peptide fragment in aqueous IL solutions could be used to evaluate the relationship between the IL‐protein interactions and aggregation selectivity in IDPs in aqueous IL solutions.