Tuning almond lipase features by the buffer used during immobilization: The apparent biocatalysts stability depends on the immobilization and inactivation buffers and the substrate utilized.

The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.

Interactions of Common Biological Buffers with Iron Oxide Nanoparticles.

Iron oxide nanoparticles (IONPs) have been studied extensively for biomedical applications, which require that they be aqueous-stable at physiological pH. The structures of some of these buffers, however, may also allow for binding to surface iron, thus potentially exchanging with functionally relevant ligands, and altering the desired properties of the nanoparticles. We report here on the interactions of five common biologically relevant buffers (MES, MOPS, phosphate, HEPES, and Tris) with iron oxide nanoparticles through spectroscopic studies. The IONPs in this study are capped with 3,4-dihydroxybenzoic acid (3,4-DHBA) to serve as models for IONP functionalized with catechol ligands. Unlike previous studies, which relied exclusively on dynamic light scattering (DLS) and ζ-potential measurements to characterize buffer interactions with IONPs, we use Fourier transform infrared (FTIR) and ultraviolet-visible (UV-visible) spectroscopic techniques to characterize the IONP surface to demonstrate binding of buffers and etching of the IONP surface. Our findings establish that phosphate and Tris bind to the IONP surface, even in the presence of strongly bound catechol ligands. We further observe significant etching of IONPs in Tris buffer, with the release of surface Fe into solution. Minor etching is noted in HEPES, and to a lesser degree, in MOPS, while no etching is observed in MES. Our findings suggest that, while morpholino buffers, such as MES and MOPS, may be more appropriate for use with IONPs, proper buffer selection should always be considered on a case-by-case basis.

Oxidative damage to ßL-crystallin in vitro by iron compounds formed in physiological buffers.

ßL-crystallin aggregation due to oxidative damage in the presence of H2O2 and ferric chloride was studied in-vitro under conditions close to physiological. It was shown that the protein aggregation characterized by the nucleation time and the aggregation rate significantly depended on the composition of the isoosmotic buffers used, and decreased in the series HEPES buffer > Tris buffer > PBS. Ferric chloride at neutral pH was converted into water-insoluble iron hydroxide III (≡FeIIIOH). According to the data of scanning electron microscopy the ≡FeIIIOH particles formed in HEPES buffer, Tris buffer, and PBS practically did not differ in structure. However, the sizes of ≡FeIIIOH floating particles measured by dynamic light scattering differed significantly and were 44 ± 28 nm, 93 ± 66 nm, 433 ± 316 nm (Zaver ± SD) for HEPES buffer, Tris buffer, and PBS, respectively. It was found by the spin trap method that the ability of ≡FeIIIOH to decompose H2O2 with the formation of a •OH decreases in the series HEPES buffer, Tris buffer, and PBS. The authors suggest that the ability to generate •OH during the decomposition of H2O2 is determined by the total surface area of ≡FeIIIOH particles, which significantly depends on the composition of the buffer in which these particles are formed.

The Influence of Small Biomolecules, Salts and Buffers on the Molecular Recognition of Amide Naphthotube in Aqueous Solutions.

We found the binding affinities of amide naphthotube to neutral organic molecules in water are not influenced by most of small biomolecules, inorganic salts, and PBS and Tris buffers but are reduced in HEPES buffer through competitive binding. Nevertheless, salts do change the binding affinities of amide naphthotube to charged molecules through a screening effect.

Investigating Reaction Intermediates during the Seedless Growth of Gold Nanostars Using Electron Tomography.

Good's buffers can act both as nucleating and shape-directing agents during the synthesis of anisotropic gold nanostars (AuNS). Although different Good's buffers can produce AuNS shapes with branches that are oriented along specific crystallographic directions, the mechanism is not fully understood. This paper reports how an analysis of the intermediate structures during AuNS synthesis from HEPES, EPPS, and MOPS Good's buffers can provide insight into the formation of seedless AuNS. Electron tomography of AuNS structures quenched at early times (minutes) was used to characterize the morphology of the incipient seeds, and later times were used to construct the growth maps. Through this approach, we identified how the crystallinity and shape of the first structures synthesized with different Good's buffers determine the final AuNS morphologies.

Formation of pH-Responsive Supramolecular Hydrogels in Basic Buffers: Self-assembly of Amphiphilic Tris-Urea.

An amphiphilic tris-urea compound (1) containing hydrophilic resorcinol units was designed and synthesized. Compound 1 formed supramolecular hydrogels in basic buffers, such as glycine-NaOH, phosphate-NaOH, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH, and borate-NaOH. The optimum pH range of the buffer solution for gelation was 10-11 and insoluble suspensions or solutions were formed when the pH was outside this range. When the borate-NaOH buffer was used, supramolecular hydrogels were formed over a wide pH range (7.5-11.0). The thermal stabilities and viscoelastic properties of the supramolecular hydrogels were determined from the gel-to-sol phase transition temperatures and rheological properties, respectively. The supramolecular hydrogel formed from compound 1 and the borate-NaOH buffer exhibited a pH-responsive reversible gel-to-sol phase transition property. Gel-to-sol phase transition could be achieved by adding NaOH and regelation of the sol was realized by adding an appropriate amount of boric acid. Increasing the amount of the acid resulted in a gel-to-sol phase transition.

A quantitative method for the assessment of homocysteine thiolactonase enzyme activity.

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

Effect of a Good buffer on the fate of metastable protein-rich droplets near physiological composition.

Metastable protein-rich microdroplets are produced from liquid-liquid phase separation (LLPS) of protein aqueous solutions. These globules can be intermediates for the formation of other protein-rich phases. Lysozyme aqueous solutions undergo LLPS around 0 °C in the presence of NaCl near physiological conditions. Here, it is shown that insertion of small amounts of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.1 M) as a second additive to lysozyme-NaCl-water solutions near physiological ionic strength (0.2 M) is an essential step for triggering conversion of protein-rich droplets into another phase. Specifically, LLPS induced by cooling reproducibly leads to a rapid and high-yield formation of compact tetragonal crystalline microparticles only in the presence of HEPES. These microcrystals exhibit small size (1-3 µm), narrow size distribution and guest-binding properties. The temperature-concentration phase diagram shows a characteristic topology with LLPS boundary metastable with respect to tetragonal microcrystals, which in turn become less stable than rod-shaped orthorhombic crystals above 40 °C. Interestingly, dynamic light scattering, hydrogen-ion titrations and isothermal titration calorimetry reveal that lysozyme-HEPES interactions were found to be weakly attractive and exothermic. Our findings indicate that additives of salting-in type can represent an important factor controlling the fate of metastable protein-rich microdroplets relevant to drug formulations, femtosecond crystallography, and potential implications in protein-driven cytoplasmic compartmentalization.

Development of an innovative salt-mediated pH gradient cation exchange chromatography method for the characterization of therapeutic antibodies.

The successful application of monoclonal antibodies (mAb) in oncology and autoimmune diseases paved the way for the development of therapeutic antibodies with a wider range of structural and physico-chemical properties. A pH-gradient combining 2-(N-morpholino)ethanesulfonic (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers and mediated with potassium chloride was developed to sufficiently retain acidic mAbs (pI < 7) in cation exchange chromatography (CEX), while keeping suitable separation performance for basic mAbs (pI > 7). Firstly, the MES and HEPES buffers were individually evaluated in their useful pH range by applying a salt gradient. The performance of a salt-mediated pH gradient combining the MES and HEPES buffers was then compared to a commercial pH gradient kit. The developed conditions were found superior to the salt-gradient approaches and provided a useful alternative to commercial pH gradient kits. In this study, the developed conditions were applied to separate a bispecific antibody (BsAb) from its two parental mAbs.

Gold nanostars as a colloidal substrate for in-solution SERS measurements using a handheld Raman spectrometer.

The evolution of Raman spectroscopy into a useful analytical technique has been due, in part, to the development of inexpensive, compact instrumentation and advancements in methodologies that enhance Raman intensities. Surface enhanced Raman scattering (SERS) is a primary methodology for quantitative and low detection limit measurements. While a broad array of applications using solid SERS substrates have been demonstrated, in-solution SERS measurements are not as widely pursued. This work seeks to optimize the synthesis of gold nanostars (AuNS) as a colloidal SERS substrate for in-solution measurements using handheld instrumentation. The types and concentrations of two buffers typically used for AuNS synthesis are examined to optimize the SERS intensity of a chemisorbed Raman probe. The observed SERS intensity primarily depends on conditions that allow higher surface coverage of the probe. Conditions that result in AuNS aggregates are found to be most optimal for SERS, similar to other nanoparticle shapes. A method to quantitate methimazole, an anti-hormone pharmaceutical, in urine is developed and reported. The primary impact of this work is the demonstration of the combination of water dispersible substrates and handheld instrumentation for rapid and sensitive analytical measurements.

A high-sensitive sensor with HEPES-enhanced electrochemiluminescence of benzo[3]uril for Fe3+ and its application in human serum.

An electrochemiluminescence (ECL) sensor based on a benzo[3]uril-modified glassy carbon electrode with sensitized luminescence, with the coexistence of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as the coreactant, was successfully constructed. The sensitization mechanism was proposed by analyzing the results of the control experiments for establishing the relationship of the luminescence effect with the concentration of HEPES. Under the optimized conditions, the fabricated sensor system was applied for the detection of Fe3+ in an aqueous solution with good sensitivity and selectivity. A low detection limit of 0.41 nM was achieved, indicating superior sensor performance over the previous analytical methods. The ECL sensor system was employed for the detection of Fe3+ in human serum samples to produce excellent recoveries ranging from 96.17% to 101.81%.

Early-stage corrosion, ion release, and the antibacterial effect of copper and cuprous oxide in physiological buffers: Phosphate-buffered saline vs Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Copper surfaces are well known for their antibacterial effects due to the release of copper ions. This benefit has been shown in many antibacterial efficiency tests, however, without considering the corrosion behaviors of copper in the physiological solutions, which could play an indispensable role in ion release from the metallic surface. This study compared the ground copper surface and sputtered cuprous oxide (Cu2O) coating in two common physiological buffers: phosphate-buffered saline (PBS) and Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Na-HEPES). The growth of the cuprous oxide (Cu2O) layer was found on copper in pure PBS, inhibiting further copper ion release. In contrast, a continuous release of copper ions was recorded in Na-HEPES for 3 h, where no oxide formation was observed. The antibacterial efficiency of copper (against E. coli) was measured and discussed with the ion release kinetics in the presence of E. coli. Similar results were obtained from Cu2O coating, ruling out its assisting role in showing the antibacterial property from copper surfaces, but they did indicate the importance of taking environmental parameters into consideration in interpreting the antibacterial efficiency of copper surfaces.

Evidence-based guidelines for controlling pH in mammalian live-cell culture systems.

A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3- and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase.

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

Measurement of Specific Heat and Crystallization in VS55, DP6, and M22 Cryoprotectant Systems With and Without Sucrose.

Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).

Ultrarapid Inductive Rewarming of Vitrified Biomaterials with Thin Metal Forms.

Arteries with 1-mm thick walls can be successfully vitrified by loading cryoprotective agents (CPAs) such as VS55 (8.4 M) or less concentrated DP6 (6 M) and cooling at or beyond their critical cooling rates of 2.5 and 40 °C/min, respectively. Successful warming from this vitrified state, however, can be challenging. For example, convective warming by simple warm-bath immersion achieves 70 °C/min, which is faster than VS55's critical warming rate of 55 °C/min, but remains far below that of DP6 (185 °C/min). Here we present a new method that can dramatically increase the warming rates within either a solution or tissue by inductively warming commercially available metal components placed within solutions or in proximity to tissues with non-invasive radiofrequency fields (360 kHz, 20 kA/m). Directly measured warming rates within solutions exceeded 1000 °C/min with specific absorption rates (W/g) of 100, 450 and 1000 for copper foam, aluminum foil, and nitinol mesh, respectively. As proof of principle, a carotid artery diffusively loaded with VS55 and DP6 CPA was successfully warmed with high viability using aluminum foil, while standard convection failed for the DP6 loaded tissue. Modeling suggests this approach can improve warming in tissues up to 4-mm thick where diffusive loading of CPA may be incomplete. Finally, this technology is not dependent on the size of the system and should therefore scale up where convection cannot.

Vitrification tendency and stability of DP6-based vitrification solutions for complex tissue cryopreservation.

Vitrification tendency and stability of the amorphous state were analyzed by means of differential scanning calorimetry (DSC) for the vitrification solution DP6, with and without additional solutes to enhance ice suppression. This study is a part of an ongoing research effort to characterize the thermophysical and mechanical properties of DP6 and its derivatives, and their qualities as cryoprotective solutions. DP6 was determined to have a critical cooling rate necessary to ensure vitrification of 2.7 °C/min. The following additional solutions were tested: DP6 + 6% (2R, 3R) 2,3-butanediol, DP6 + 6% 1,3-cyclohexanediol, DP6 + 6% (0.175M) sucrose, DP6 + 12% PEG 400, and DP6 + 17.1% (0.5 M) sucrose. The additives decreased the critical cooling rate of the DP6 solution to rates below 1 °C/min that were not quantifiable by the DSC techniques used. The following critical warming rates necessary to avoid devitrification were identified for DP6 and the modified solutions, respectively: 189 °C/min, 5 °C/min, ≈ 1 °C/min, 15 °C/min, <1 °C/min, and <1 °C/min. Glass transition temperatures and melting temperatures were also measured. Sucrose was the least effective additive on a per mass basis, with 1,3-cyclohexanediol appearing to be the most effective additive for suppressing ice formation in DP6.

A "turn-off" red-emitting fluorophore for nanomolar detection of heparin.

A simple fluorophore bearing a diethylaminocoumarin donor and a pyridinium acceptor was synthesized and utilized for the ultra-sensitive detection of heparin. The synthesized dicationic push-pull coumarin derivative emits strongly in the red-region (665 nm) and detects nanomolar concentrations (14.8 nM to 148 nM) of heparin in HEPES buffer and FBS serum solutions. The dication exhibits excellent fluorescence selectivity and sensitivity towards heparin over its analogues such as chondroitin 4-sulfate (CS), hyaluronic acid (HA) and dextran. This fluorescence assay is a convenient, sensitive method for monitoring heparin levels in biological samples. These findings were confirmed using coarse-grained Monte Carlo simulations, which provide us with a rationale for the selective binding of heparin.

Effect of different buffer systems on the xanthine oxidase inhibitory activity of tuna (Katsuwonus pelamis) protein hydrolysate.

This study systematically explored the effect of HEPES, Tris and sodium phosphate (PB) buffers on the xanthine oxidase (XO) inhibitory activity of tuna protein hydrolysate (TPH, containing over 90% of constituents with molecular weight below 5kDa). The greatest XO inhibition by TPH was observed in HEPES buffer. The optimal HEPES concentration was 100mmol/L. Tryptophan fluorescence and circular dichroism measurements revealed the comparable stability of XO and TPH in the three buffers. The buffers did not alter the majority of XO or TPH structure but induced slight modifications to specific domains (e.g. Trp residues on α-helices) and certain rearrangements (e.g. XO unfolding or refolding). HEPES buffer exerted stronger interactions with XO or TPH, causing a lower α-helical content in XO and consequently a lower XO catalytic activity but greater XO inhibition, compared to Tris and PB buffers.

Influence of buffer solutions in the adsorption of human serum proteins onto layered double hydroxide.

The adsorption of human immunoglobulin G (IgG) and human serum albumin (HSA) on a non-calcined Mg-Al layered double hydroxide (3:1 Mg-Al LDH) was studied in batch and fixed bed experiments, focusing on the effect of buffer solution and pH over sorbent uptake. Mg-Al LDH was synthesized and characterized by X-ray diffraction (XRD), N2 adsorption-desorption isotherms at -196°C, X-ray photoelectron spectroscopy (XPS), Zero point charge (pHzpc), particle size distribution and Fourier transform infra-red (FTIR). Batch adsorption experiments were performed in order to investigate the effects of pH on IgG and HSA adsorption with different buffers: sodium acetate (ACETATE), sodium phosphate (PHOSPHATE), 3-(N-morpholino) propanesulfonic acid (MOPS), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and trizma-hydrochloric acid (TRIS-HCl). Maximum adsorption capacities estimated by the Langmuir model were 239mgg-1 for IgG and 105mgg-1 for HSA in TRIS-HCl buffer. On the other hand, the highest selectivity for IgG adsorption over HSA was obtained with buffer PHOSPHATE (pH 6.5). The maximum IgG and HSA adsorption uptake in this case were 165 and 36mgg-1, respectively. Fixed bed experiments were carried out with both proteins using PHOSPHATE buffer (pH 6.5), which confirmed that IgG was more selectively adsorbed than HSA on Mg-Al LDH and both could be fully recovered by elution with sodium chloride (NaCl).