Polyamines (PAs) but not small peptides with closely spaced positively charged groups interact with DNA and RNA, but they do not represent a relevant buffer system at physiological pH values.

Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity.

Nobody's Perfect: Choice of the Buffer and the Rate of Cu2+ Ion-Peptide Interaction.

The choice of correct pH buffer is crucial in chemical studies modeling biological processes involving Cu2+ ions. Popular buffers for physiological pH are known to form Cu(II) complexes, but their impact on kinetics of Cu(II) complexation has not been considered. We performed a stopped-flow kinetic study of Cu2+ ion interactions with four popular buffers (phosphate, Tris, HEPES, and MOPS) and two buffers considered as nonbinding (MES and PIPPS). Next, we studied their effects on the rate of Cu2+ reaction with Gly-Gly-His (GGH), a tripeptide modeling physiological Cu(II) sites, which we studied previously at conditions presumably excluding the buffer interference [Kotuniak, R.; Angew. Chem., Int. Ed. 2020, 59, 11234-11239]. We observed that (i) all tested pH 7.4 buffers formed Cu(II) complexes within the stopped-flow instrument dead time; (ii) Cu(II)-peptide complexes were formed via ternary complexes with the buffers; (iii) nevertheless, Good buffers affected the observed rate of Cu(II)-GGH complex formation only slightly; (iv) Tris was a competitive inhibitor of Cu(II)-GGH complexation; while (v) phosphate was a reaction catalyst. This is particularly important as phosphate is a biological buffer.

Cross Talking Calcium, IP3 and Buffer Dynamics Alters ATP and NADH Level in Obese and Normal Hepatocyte Cell.

The cross talk between calcium (Ca2+), IP3 and buffer dynamics regulate various mechanisms in hepatocyte cells. The study of independent systems of calcium, IP3, and buffer signaling provides limited information about cell dynamics. In the current study, coupled reaction-diffusion equations are used to design a cross-talk model for IP3, buffer, and calcium dynamics in a hepatocyte cell. The one-way feedback of calcium, buffer, and IP3 in ATP production, ATP degradation, and NADH production rate is incorporated into the model. Numerical simulation has been done using the Finite Element Method (FEM) along the spatial direction and the Crank-Nicolson (C-N) method along the temporal direction. The numerical results are analysed to determine the effects of alterations in processes of cross-talking dynamics of IP3, buffer, and calcium on ATP and NADH production and degradation rate of ATP in a hepatocyte cell under normal and obesity conditions. The comparative analysis of these findings unveils notable distinctions induced by obesity in calcium dynamics, ATP and NADH synthesis, and ATP degradation kinetics.

Assessing the Efficacy of Buffered Versus Nonbuffered Lidocaine in Dental Extractions: A Double-Blinded Randomized Controlled Trial.

BACKGROUND: Injections using buffered lidocaine may decrease discomfort, have a quicker onset, and be a more efficacious local anesthetic. Previous studies have been inconclusive in the oral context. PURPOSE: To address if bicarbonate buffered 2% lidocaine can decrease pain from the use of local anesthesia, has a quicker onset time, and is more efficacious. STUDY DESIGN: The design was a single-center double-blinded randomized control trial, set in an outpatient oral and maxillofacial clinic housed in the University of Cincinnati Medical Center. Inclusion criteria for the study were patients requiring a single tooth extraction due either to caries or periodontal disease. PREDICTOR VARIABLE: The predictor variable was the local anesthetic used either nonbuffered 2% lidocaine with 1:100,000 epinephrine (control) or bicarbonate buffered 2% lidocaine with 1:100,000 epinephrine (study) was randomly assigned. MAIN OUTCOME VARIABLES: Primary outcome variables were injection pain score, and postoperative pain, time to anesthetic onset, and the number of rounds of injections required to achieve adequate anesthesia. COVARIATES: The covariates were jaw involved, age, sex, and race, American Society of Anesthesiologists score, body mass index, current tobacco use, history of psychiatric illness, chronic pain, and preoperative pain score. ANALYSES: Test statistics were calculated using Wilcoxon rank-sum test, Kruskal-Wallis test, Spearman rank correlation test, χ2 test for bivariate analyses, and Fisher's exact test. P values ≤ .05 were considered statistically significant. RESULTS: The final sample was 114 subjects. The mean age of the sample was 42.97 years, standard deviation ±13.43 years. The sample was 39.47% male. The racial demographics were Caucasian (62.28%) and African American (33.33%). Buffered lidocaine did not have a statistically significant relationship with any of the outcomes. The jaw involved had a statistically significant association to the injection pain score (P value = .006), and the number of rounds of anesthetic required (P value = .047). Age showed a statistically significant association to injection pain score (P value = .032), and the number of rounds of anesthetic required (P value = .027). Finally, preoperative pain had a statistically significant relationship with injection pain score (P value = < .001). CONCLUSION AND RELEVANCE: In this study, bicarbonate buffered lidocaine did not exhibit any discernible advantages over nonbuffered lidocaine for any study outcomes.

Optimized Refolding Buffers Oriented Humoral Immune Responses Versus PfGCS1 Self-Assembled Peptide Nanoparticle.

Developing a novel class of vaccine is pivotal for eliminating and eradicating malaria. Preceding investigations demonstrated partial blocking activity in malaria transmission against recombinant vaccine PfHAP2-GCS1 and conserved region of the cd loop. The effectiveness of immune response varies with the size and shape of the self-assembly of peptide nanoparticles (SAPNs) displaying antigen, affected by different components in refolding buffers. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising malaria transmission-blocking vaccine (TBV) candidate, was expressed, purified, and followed by a four-step refolding process to form nanoparticles (PfGCS1-SAPNs). The influence of buffer components on the size and shape of SAPNs was investigated by DLS and FESEM. Furthermore, the immunogenicity of nanostructures was assessed in different mouse groups. The results showed that PfGCS1-SAPN was immunogenic and its administration with Poly (I:C), stimulated humoral and cellular responses in the mouse model. In the immunized mice groups, the level of IgG antibodies against PfGCS1-SAPN was significantly increased in different time points (second and third boost) and heterogeneous boosters. The various IgG-subclasses profile shifted to Th1, Th2, or Th1/Th2 mix responses in mice immunized with PfGCS1-SAPN refolded in different buffers, indicating a prerequisite for further investigations to optimize vaccine formulation to enhance and modulate Th1/cellular responses. Such studies pave the way to improve biophysical features related to the nanoparticles' size, shape, and conformational epitopes of candidate antigens and T- and B-cells presented on the superficial structure to elicit robust immune responses.

Evaluation of the efficacy of buffered local anesthetic in extraction of infected teeth-randomized double-blind study.

PURPOSE: In the presence of infection, acidic pH of a lignocaine local anesthetic causes undesirable effects such as burning on injection, relatively slow onset, and lack of numbness. Buffered lignocaine will increase the pH of the solution and may resolve above problems. Thus, the objective of this study is to compare the efficacy of buffered lignocaine with that of commercial lignocaine. METHOD: Seventy patients with infected teeth were randomly divided into two equal groups. The study group received buffered lignocaine (8.4% sodium bicarbonate added to 2% lignocaine mixture) while the control received commercial lignocaine preparation (2% lignocaine with 1:80,000 adrenaline). Burning while injection, pain using VAS scale and onset of action with EPT (electric pulp tester) were recorded. RESULTS: In the study group, the VAS score after injection was 1.20 ± 0.68 and the control group was 2.57 ± 0.92 (p = 0.001). There was a statistically significant reduction in pain reduction in the study group. The time of onset was 3.97 ± 0.71 and 5.67 ± 1.15 min, respectively, and the difference was statically significant. Only one-third of the study group experienced burning on injection as compared to two-thirds in the control group. CONCLUSION: Buffered lignocaine is more effective as compared to commercial lignocaine in the extraction of infected teeth. CTRI NUMBER: CTRI/2022/01/039476.

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.

Luminescence Sensing of Biomacromolecules Heparin and Protamine in 100% Human Serum and Plasma by Supramolecular Polymeric Assemblies.

Heparin, an anionic biomacromolecule, is routinely used as an anticoagulant during medical surgery to prevent blood clot formation and in the treatment of several heart, lung, and circulatory disorders having a higher risk of blood clotting. We herein report supramolecular polymeric nanoassemblies of cationic pyrene-tagged bis-imidazolium amphiphiles for heparin detection with high sensitivity and selectivity in aqueous buffer, plasma, and serum media. The nano-assemblies exhibited cyan-green excimeric emission in aqueous media, and their multivalent array of positive surface charges allowed them to form co-assemblies with heparin, resulting in significantly enhanced emission. This provided a convenient method for heparin detection in buffer at nanomolar concentrations, and most notably, a ratiometric fluorescence response was obtained even in highly competitive 100% human serum and 100% human plasma in a clinically relevant concentration range. Moreover, using the heparin-based luminescent co-assemblies, protamine sulfate, a clinically administered antidote to heparin, was also detected in 100% human serum and 100% human plasma at sub-micromolar concentrations.

Native Mass Spectrometry for Peptide-Metal Interaction in Picoliter Cell Lysate.

Native mass spectrometry, which takes a high concentration of ammonium acetate (NH4OAc) for ionization, coupled with tedious and solvent-consuming purification, which separates proteins from complicated environments, has shown great potential for proteins and their complexes. A high level of nonvolatile salts in the endogenous intracellular environment results in serious ion suppression and has been one of the bottlenecks for native mass spectrometry, especially for protein complexes. Herein, an integrated protocol utilizing the inner surface of a micropipette for rapid purification, desorption, and ionization of peptide-metal interaction at subfemtomole level in cell lysate was demonstrated for native mass spectrometry. The methods showed robust and reproducibility in protein measurement within 1 min from various buffers. The E. coli cells expressing with various proteins were lysed and used to test our method. The specific interaction between the peptide-metal complex in cell lysates could be reserved and distinguished by mass spectrometry.

Improving packed red blood cell storage with a high-viscosity buffered storage solution.

BACKGROUND: Massive transfusion with older packed red blood cells is associated with increased morbidity and mortality. As packed red blood cells age, they undergo biochemical and structural changes known as the storage lesion. We developed a novel solution to increase viscosity in stored packed red blood cells. We hypothesized that packed red blood cell storage in this solution would blunt storage lesion formation and mitigate the inflammatory response after resuscitation. METHODS: Blood was obtained from 8- to 10-week-old C57BL/6 male donor mice or human volunteers and stored as packed red blood cell units for 14 days for mice or 42 days for humans in either standard AS-3 storage solution or EAS-1587, the novel packed red blood cell storage solution. Packed red blood cells were analyzed for microvesicles, cell-free hemoglobin, phosphatidylserine, band-3 protein, glucose utilization, and osmotic fragility. Additional mice underwent hemorrhage and resuscitation with packed red blood cells stored in either AS-3 or EAS-1587. Serum was analyzed for inflammatory markers. RESULTS: Murine packed red blood cells stored in EAS-1587 demonstrated reductions in microvesicle and cell-free hemoglobin accumulation as well as preserved band-3 expression, increase glucose utilization, reductions in phosphatidylserine expression, and susceptibility to osmotic stress. Serum from mice resuscitated with packed red blood cells stored in EAS-1587 demonstrated reduced proinflammatory cytokines. Human packed red blood cells demonstrated a reduction in microvesicle and cell-free hemoglobin as well as an increase in glucose utilization. CONCLUSION: Storage of packed red blood cells in a novel storage solution mitigated many aspects of the red blood cell storage lesion as well as the inflammatory response to resuscitation after hemorrhage. This modified storage solution may lead to improvement of packed red blood cell storage and reduce harm after massive transfusion.

Stabilization of VEGF i-motif structure by CpG methylation.

The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemi-protonated cytosine base pairs (C-C+) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motif-forming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C+ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tm) by 5.1 °C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tm by 0.5, 1.7, and 2.0 °C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure.

1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1.

2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), α/ß hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.

Evaluation of the dentinal tubule penetration of an endodontic bioceramic sealer after three final irrigation protocols

Introduction: This study aimed to evaluate the dentinal tubule penetration of an endodontic bioceramic sealer, Sealer Plus BC, after three final irrigation protocols. Methods: Thirty distobuccal roots of maxillary molars were selected. Root canal preparation was performed up to an #40.06 instrument (X1 Blue) under 2.5% sodium hypochlorite irrigation. Specimens were randomly divided into three groups (n=10), according to the final irrigation protocol: G-NaOCl (2.5% sodium hypochlorite + PUI), G-SS (0.9% saline solution + PUI) and G-H20 (Deionized water + PUI). After final irrigation protocols, all specimens were irrigated with phosphate buffer solution. Root canal obturation was performed using the single cone technique and Sealer Plus BC, stained with a specific fluorophore. Specimens were transversely sectioned and each root third was evaluated in a confocal scanning laser microscopy. Images obtained were analyzed for sealer penetration in the dentinal tubules. Results: Dentinal tubule penetration of Sealer Plus BC was not observed in any root third, regardless of the final irrigation protocol investigated. Conclusions: Sealer Plus BC dentinal tubule penetration was not observed after none of the protocols tested. Dentinal tubule penetrability of Sealer Plus BC may be related to other factors rather than the final irrigation protocol.

A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium.

Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.

Automated multi-attribute method sample preparation using high-throughput buffer exchange tips.

RATIONALE: The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability. METHODS: In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system. RESULTS: No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates. CONCLUSIONS: This automated protocol enables the preparation of up to 96 samples with less "at-bench" time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

Simultaneous determination of paracetamol, dextromethorphan, chlorpheniramine, pseudoephedrine and major impurities of paracetamol and pseudoephedrine by using capillary electrophoresis

Abstract A capillary electrophoresis method was developed for the first time and optimized for the determination of paracetamol, pseudoephedrine, dextromethorphan, chlorpheniramine, 4-aminophenol and ephedrine in tablet formulation. Optimum electrophoretic conditions were achieved by using a background electrolyte of 75 mmol L-1 sodium borate buffer at pH 8.0, a capillary temperature of 30°C, a separation voltage of 30 kV and a pressure injection of the sample at 50 mbar for 10 s. Calibration graphs showed a good linearity with a coefficient of determination (R2) of at least 0.999 for all compounds. Intraday and interday precision (expressed as relative standard deviation (RSD) %) were lower than 1.39% for capillary electrophoresis method. The developed method was demonstrated to be simple and rapid for the determination of paracetamol, pseudoephedrine, dextromethorphan, chlorpheniramine, 4-aminophenol and ephedrine in tablet formulation providing recoveries in the range between 99.62 and 100.57% for all analytes.

Optimization of Reaction Conditions for γ-Glutamylcysteine Production from Glutathione Using a Phytochelatin Synthase-Like Enzyme from Nostoc sp. Pasteur Culture Collection 7120.

γ-Glutamylcysteine (γ-EC) has antioxidant properties similar to those of glutathione (GSH) and acts as its precursor in mammals. There are a few procedures for the production of γ-EC, such as chemical synthesis or enzymatic synthesis from glutamate and cysteine; however, they are very costly and not suitable for industrial production. A phytochelatin synthase-like enzyme derived from Nostoc sp. Pasteur Culture Collection 7120 (NsPCS) catalyzes the hydrolysis of GSH to γ-EC and glycine in the absence of ATP or other additives. Our research aims to establish an alternative γ-EC production procedure with low cost and high productivity. To this end, we optimized the reaction conditions of NsPCS and characterized its properties in this study. We found that 200 mM potassium phosphate buffer, pH 8.0, at 37 °C, had the highest NsPCS activity among the conditions we tested. Under these conditions, NsPCS had a Km of 385 µM and a Vmax of 26 mol/min/mg-protein. In addition, NsPCS converted 100 mM GSH into γ-EC with high yields. These results suggest that the NsPCS reaction has great potential for the low-cost, industrial-scale production of γ-EC.

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Scaling Down Large-Scale Thawing of Monoclonal Antibody Solutions: 3D Temperature Profiles, Changes in Concentration, and Density Gradients.

PURPOSE: Scale-down devices (SDD) are designed to simulate large-scale thawing of protein drug substance, but require only a fraction of the material. To evaluate the performance of a new SDD that aims to predict thawing in large-scale 2 L bottles, we characterised 3D temperature profiles and changes in concentration and density in comparison to 125 mL and 2 L bottles. Differences in diffusion between a monoclonal antibody (mAb) and histidine buffer after thawing were examined. METHODS: Temperature profiles at six distinct positions were recorded with type T thermocouples. Size-exclusion chromatography allowed quantification of mAb and histidine. Polysorbate 80 was quantified using a fluorescent dye assay. In addition, the solution's density at different locations in bottles and the SDD was identified. RESULTS: The temperature profiles in the SDD and the large-scale 2 L bottle during thawing were similar. Significant concentration gradients were detected in the 2 L bottle leading to marked density gradients. The SDD slightly overestimated the dilution in the top region and the maximum concentrations at the bottom. Fast diffusion resulted in rapid equilibration of histidine. CONCLUSION: The innovative SDD allows a realistic characterisation and helps to understand thawing processes of mAb solutions in large-scale 2 L bottles. Only a fraction of material is needed to gain insights into the thawing behaviour that is associated with several possible detrimental limitations.