Mechanism of Protein Aggregation Inhibition by Arginine: Blockage of Anionic Side Chains Favors Unproductive Encounter Complexes.

Aggregation refers to the assembly of proteins into nonphysiological higher order structures. While amyloid has been studied extensively, much less is known about amorphous aggregation, a process that interferes with protein expression and storage. Free arginine (Arg+) is a widely used aggregation inhibitor, but its mechanism remains elusive. Focusing on myoglobin (Mb), we recently applied atomistic molecular dynamics (MD) simulations for gaining detailed insights into amorphous aggregation (Ng J. Phys. Chem. B 2021, 125, 13099). Building on that approach, the current work for the first time demonstrates that MD simulations can directly elucidate aggregation inhibition mechanisms. Comparative simulations with and without Arg+ reproduced the experimental finding that Arg+ significantly decreased the Mb aggregation propensity. Our data reveal that, without Arg+, protein-protein encounter complexes readily form salt bridges and hydrophobic contacts, culminating in firmly linked dimeric aggregation nuclei. Arg+ promotes the dissociation of encounter complexes. These "unproductive" encounter complexes are favored because Arg+ binding to D- and E- lowers the tendency of these anionic residues to form interprotein salt bridges. Side chain blockage is mediated largely by the guanidinium group of Arg+, which binds carboxylates through H-bond-reinforced ionic contacts. Our MD data revealed Arg+ self-association into a dynamic quasi-infinite network, but we found no evidence that this self-association is important for protein aggregation inhibition. Instead, aggregation inhibition by Arg+ is similar to that mediated by free guanidinium ions. The computational strategy used here should be suitable for the rational design of aggregation inhibitors with enhanced potency.

Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action.

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Genetically encoded fluorescent sensor to monitor intracellular arginine methylation.

Arginine methylation (ArgMet), as a post-translational modification, plays crucial roles in RNA processing, transcriptional regulation, signal transduction, DNA repair, apoptosis and liquid-liquid phase separation (LLPS). Since arginine methylation is associated with cancer pathogenesis and progression, protein arginine methyltransferases have gained interest as targets for anti-cancer therapy. Despite considerable process made to elucidate (patho)physiological mechanisms regulated by arginine methylation, there remains a lack of tools to visualize arginine methylation with high spatiotemporal resolution in live cells. To address this unmet need, we generated an ArgMet-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based biosensor, called GEMS, capable of quantitative real-time monitoring of ArgMet dynamics. We optimized these biosensors by using different ArgMet-binding domains, arginine-glycine-rich regions and adjusting the linkers within the biosensors to improve their performance. Using a set of mammalian cell lines and modulators, we demonstrated the applicability of GEMS for monitoring changes in arginine methylation with single-cell and temporal resolution. The GEMS can facilitate the in vitro screening to find potential protein arginine methyltransferase inhibitors and will contribute to a better understanding of the regulation of ArgMet related to differentiation, development and disease.

Combinations of arginine and pullulan reveal the selective effect of stabilization mechanisms on different lyophilized proteins.

The stability of lactate dehydrogenase (LDH) and ß-galactosidase (ß-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while ß-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, ß-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and ß-gal may be dependent on the interaction with arginine.

l-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation.

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

Submillisecond Atomistic Molecular Dynamics Simulations Reveal Hydrogen Bond-Driven Diffusion of a Guest Peptide in Protein-RNA Condensate.

Liquid-liquid phase separation mediated by proteins and/or nucleic acids is believed to underlie the formation of many distinct condensed phases, or membraneless organelles, within living cells. These condensates have been proposed to orchestrate a variety of important processes. Despite recent advances, the interactions that regulate the dynamics of molecules within a condensate remain poorly understood. We performed accumulated 564.7 µs all-atom molecular dynamics (MD) simulations (system size ∼200k atoms) of model condensates formed by a scaffold RNA oligomer and a scaffold peptide rich in arginine (Arg). These model condensates contained one of three possible guest peptides: the scaffold peptide itself or a variant in which six Arg residues were replaced by lysine (Lys) or asymmetric dimethyl arginine (ADMA). We found that the Arg-rich peptide can form the largest number of hydrogen bonds and bind the strongest to the scaffold RNA in the condensate, relative to the Lys- and ADMA-rich peptides. Our MD simulations also showed that the Arg-rich peptide diffused more slowly in the condensate relative to the other two guest peptides, which is consistent with a recent fluorescence microscopy study. There was no significant increase in the number of cation-π interactions between the Arg-rich peptide and the scaffold RNA compared to the Lys-rich and ADMA-rich peptides. Our results indicate that hydrogen bonds between the peptides and the RNA backbone, rather than cation-π interactions, play a major role in regulating peptide diffusion in the condensate.

Synthesis of Amino Acid-Based Cationic Lipids and Study of the Role of the Cationic Head Group for Enhanced Drug and Nucleic Acid Delivery.

Leveraging liposomes for drug and nucleic acid delivery, though promising due to reduced toxicity and ease of preparation, faces challenges in stability and efficiency. To address this, we synthesized cationic amphiphiles from amino acids (arginine, lysine, and histidine). Histidine emerged as the superior candidate, leading to the development of three histidine-rich cationic amphiphiles for liposomes. Using the hydration method, we have prepared the liposomes and determined the optimal N/P ratios for lipoplex formation via gel electrophoresis. In vitro transfection assays compared the efficacy of our lipids to Fugene, while MTT assays gauged biocompatibility across cancer cell lines (MDA-MB 231 and MCF-7). The histidine-based lipid demonstrated marked potential in enhancing drug and nucleic acid delivery. This improvement stemmed from increased zeta potential, enhancing electrostatic interactions with nucleic acids and cellular uptake. Our findings underscore histidine's crucial role over lysine and arginine for effective delivery, revealing a significant correlation between histidine abundance and optimal performance. This study paves the way for histidine-enriched lipids as promising candidates for efficient drug and nucleic acid delivery, addressing key challenges in the field.

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Amorphization of Low Soluble Drug with Amino Acids to Improve Its Therapeutic Efficacy: a State-of-Art-Review.

Low aqueous solubility of drug candidates is an ongoing challenge and pharmaceutical manufacturers pay close attention to amorphization (AMORP) technology to improve the solubility of drugs that dissolve poorly. Amorphous drug typically exhibits much higher apparent solubility than their crystalline form due to high energy state that enable them to produce a supersaturated state in the gastrointestinal tract and thereby improve bioavailability. The stability and augmented solubility in co-amorphous (COA) formulations is influenced by molecular interactions. COA are excellent carriers-based drug delivery systems for biopharmaceutical classification system (BCS) class II and class IV drugs. The three important critical quality attributes, such as co-formability, physical stability, and dissolution performance, are necessary to illustrate the COA systems. New amorphous-stabilized carriers-based fabrication techniques that improve drug loading and degree of AMORP have been the focus of emerging AMORP technology. Numerous low-molecular-weight compounds, particularly amino acids such as glutamic acid, arginine, isoleucine, leucine, valine, alanine, glycine, etc., have been employed as potential co-formers. The review focus on the prevailing drug AMORP strategies used in pharmaceutical research, including in situ AMORP, COA systems, and mesoporous particle-based methods. Moreover, brief characterization techniques and the application of the different amino acids in stabilization and solubility improvements have been related.

Synthesis of Novel Arginine Building Blocks with Increased Lipophilicity Compatible with Solid-Phase Peptide Synthesis.

Arginine, due to the guanidine moiety, increases peptides' hydrophilicity and enables interactions with charged molecules, but at the same time, its presence in a peptide chain might reduce its permeability through biological membranes. This might be resolved by temporary coverage of the peptide charge by lipophilic, enzyme-sensitive alkoxycarbonyl groups. Unfortunately, such a modification of a guanidine moiety has not been reported to date and turned out to be challenging. Here, we present a new, optimized strategy to obtain arginine building blocks with increased lipophilicity that were successfully utilized in the solid-phase peptide synthesis of novel arginine vasopressin prodrugs.

LC-MS/MS Analysis of Reaction Products of Arginine/Methylarginines with Methylglyoxal/Glyoxal.

Methylglyoxal (MGO) and glyoxal (GO) are toxic α-dicarbonyl compounds that undergo reactions with amine containing molecules such as proteins and amino acids and result in the formation of advanced glycation end products (AGEs). This study aimed at investigating the reactivity of arginine (Arg) or dimethylarginine (SDMA or ADMA) with MGO or GO. The solutions of arginine and MGO or GO were prepared in PBS buffer (pH 7.4) and incubated at 37 °C. Direct electrospray ionization-high-resolution mass spectrometry (ESI-HRMS) analysis of the reaction mixture of Arg and MGO revealed the formation of Arg-MGO (1:1) and Arg-2MGO (1:2) products and their corresponding dehydrated products. Further liquid chromatography (LC)-MS analyses revealed the presence of isomeric products in each 1:1 and 1:2 product. The [M + H]+ of each isomeric product was subjected to MS/MS experiments for structural elucidation. The MS/MS spectra of some of the products showed a distinct structure indicative fragment ions, while others showed similar data. The types of products formed by the arginines with GO were also found to be similar to that of MGO. The importance of the guanidine group in the formation of the AGEs was reflected in similar incubation experiments with ADMA and SDMA. The structures of the products were proposed based on the comparison of the retention times and HRMS and MS/MS data interpretation, and some of them were confirmed by drawing analogy to the data reported in the literature.

A comprehensive evaluation of arginine and its derivatives as protein formulation stabilizers.

Arginine and its derivatives (such as arginine ethyl ester and acetyl arginine) have varying degrees of protein aggregation suppressor effect across different protein solutions. To understand this performance ambiguity, we evaluated the activity of arginine, acetyl arginine, and arginine ethyl ester for aggregation suppressor effect against human intravenous immunoglobulin G (IgG) solution at pH 4.8. Both arginine and its cationic derivative arginine ethyl ester in their hydrochloride salt forms significantly reduced the colloidal and conformational stability (reduced kd and Tm) of IgG. Consequently, the monomer content was decreased with an increase in subvisible particulates after agitation or thermal stress. Furthermore, compared to arginine, arginine ethyl ester with one more cationic charge and hydrochloride salt form readily precipitated IgG at temperatures higher than 25 °C. On the contrary, acetyl arginine, which mostly exists in a neutral state at pH 4.8, efficiently suppressed the formation of subvisible particles retaining a high amount of monomer owing to its higher colloidal and conformational stability. Concisely, the charged state of additives significantly impacts protein stability. This study demonstrated that contrary to popular belief, arginine and its derivatives may either enhance or suppress protein aggregation depending on their net charge and concentration.

PRMxAI: protein arginine methylation sites prediction based on amino acid spatial distribution using explainable artificial intelligence.

BACKGROUND: Protein methylation, a post-translational modification, is crucial in regulating various cellular functions. Arginine methylation is required to understand crucial biochemical activities and biological functions, like gene regulation, signal transduction, etc. However, some experimental methods, including Chip-Chip, mass spectrometry, and methylation-specific antibodies, exist for the prediction of methylated proteins. These experimental methods are expensive and tedious. As a result, computational methods based on machine learning play an efficient role in predicting arginine methylation sites. RESULTS: In this research, a novel method called PRMxAI has been proposed to predict arginine methylation sites. The proposed PRMxAI extract sequence-based features, such as dipeptide composition, physicochemical properties, amino acid composition, and information theory-based features (Arimoto, Havrda-Charvat, Renyi, and Shannon entropy), to represent the protein sequences into numerical format. Various machine learning algorithms are implemented to select the better classifier, such as Decision trees, Naive Bayes, Random Forest, Support vector machines, and K-nearest neighbors. The random forest algorithm is selected as the underlying classifier for the PRMxAI model. The performance of PRMxAI is evaluated by employing 10-fold cross-validation, and it yields 87.17% and 90.40% accuracy on mono-methylarginine and di-methylarginine data sets, respectively. This research also examines the impact of various features on both data sets using explainable artificial intelligence. CONCLUSIONS: The proposed PRMxAI shows the effectiveness of the features for predicting arginine methylation sites. Additionally, the SHapley Additive exPlanation method is used to interpret the predictive mechanism of the proposed model. The results indicate that the proposed PRMxAI model outperforms other state-of-the-art predictors.

Cellular uptake and trafficking of peptide-based drug delivery systems for miRNA.

Today, macromolecular compounds such as microRNAs (miRNAs) are becoming more and more widespread as leading therapeutics. However, their application is limited mostly due to their poor stability, limited cellular uptake, and poor target specificity. Cell-penetrating peptides (CPPs), a group of positively charged peptides, represent a breakthrough as delivery systems for macromolecules. In the present study, we used two types of nanoparticles which differ in the type of CPP used for their manufacturing. The first type is composed of protamine, an arginine rich CPP, which is highly positively charged. The arginine residues are able to form electrostatic interactions with miRNAs, stabilize them, and deliver them to cells. The second type is composed of the N-Ter peptide (also known as MPG), an amphipathic peptide rich in lysine. The positively charged parts of the N-Ter peptide electrostatically stabilize miRNAs, whereas its amphipathic character allows it to successfully traverse cell membranes. We used miRNA-27a, a negative regulator of adipogenesis, to form nanoparticles with the peptides and traced their uptake in 3T3-L1 preadipocytes. Motivated by the lengthy discourse regarding the uptake mechanism of CPPs, the focus of our study was to analyse and understand the internalization of proticles (protamine nanoparticles) and N-Ter complexes. The nanoparticles were characterized regarding size, size distribution, and zeta potential, and their cytotoxicity was tested in 3T3-L1 cells. The uptake studies were performed by varying the experimental conditions such as time, concentration, and temperature, as well as by applying different inhibitors of endocytosis. Furthermore, we assessed the biological effect of miRNA-27a on the pro-adipogenic machinery. The obtained data have shown that protamine and the N-Ter peptide form positively charged nanoparticles through non-covalent complexation. The uptake of proticles and N-Ter complexes was found to be dependent on time, concentration, and temperature, and different uptake pathways were discovered to be involved in the internalization of the different nanoparticles. Furthermore, both types of nanoparticles induced the anti-adipogenic effect of miRNA-27a, demonstrating that this approach can be used as a novel miRNA replacement therapy in the treatment of obesity and obesity-related disorders.

Identification of two short peptide motifs from serine/arginine-rich protein ribonucleic acid recognition motif-1 domain acting as splicing regulators.

Background: Serine/arginine-rich (SR) proteins regulate pre-mRNA splicing. However, structurally similar proteins often behave differently in splicing regulation and the underlying mechanisms are largely unknown. Here, using SMN1/2 minigenes we extensively analyzed four SR proteins, SRSF1/5/6/9. Methods: In this study, the effects of these proteins on SMN1/2 exon 7 splicing when tethered at either intron 6 or 7 were evaluated using an MS2-tethering assay. Deletion analysis in four SR proteins and co-overexpression analysis were performed. Results: Splicing outcomes varied among all four SR proteins, SRSF1 and SRSF5 function the same at the two sites, acting as repressor and stimulator, respectively; while SRSF6 and SRSF9 promote exon 7 inclusion at only one site. Further, the key domains of each SR proteins were investigated, which identified a potent inhibitory nonapeptide in the C-terminus of SRSF1/9 ribonucleic acid recognition motif-1 (RRM1) and a potent stimulatory heptapeptide at the N-terminus of SRSF5/6 RRM1. Conclusion: The insight of the four SR proteins and their domains in affecting SMN gene splicing brings a new perspective on the modes of action of SR proteins; and the functional peptides obtained here offers new ideas for developing splice switching-related therapies.

An investigation of excipients for a stable Orf viral vector formulation.

The Orf virus (ORFV) is a promising candidate for vector vaccines as well as for immunomodulatory and oncolytic therapies. However, few publications are available on its infectivity degradation or on suitable additives for prolonging its viral stability. In this study, the non-supplemented ORFV itself showed a very high stability at storage temperatures up to 28 °C, with a linear titer loss of 0.10 log infectious particles per day at 4 °C over a period of five weeks. To prolong this inherent stability, thirty additives, i.e., detergents, sugars, proteins, salts, and buffers as well as amino acids, were tested for their time- and temperature-dependent influence on the ORFV infectivity. A stabilizing effect on the infectivity was identified for the addition of all tested proteins, i.e., gelatine, bovine serum albumin, and recombinant human serum albumin (rHSA), of several sugars, i.e., mannitol, galactose, sucrose, and trehalose, of amino acids, i.e., arginine and proline, of the detergent Pluronic F68, and of the salt Na2SO4. The infectivity preservation was especially pronounced for proteins in liquid and frozen formulations, sugars in frozen state, and arginine und Pluronic in liquid formulations at high storage temperatures (37 °C). The addition of 1% rHSA with and without 5% sucrose was evaluated as a very stable formulation with a high safety profile and economic validity at storage temperatures up to 28 °C. At increased temperatures, the supplementation with 200 mM arginine performed better than with rHSA. In summary, this comprehensive data provides different options for a stable ORFV formulation, considering temperature, storage time, economic aspects, and downstream processing integrity.

C-Terminal Arginine-Selective Cleavage of Peptides as a Method for Mimicking Carboxypeptidase B.

C-Terminal residues play a pivotal role in dictating the structure and functions of proteins. Herein, we report a mild, efficient, chemoselective, and site-selective chemical method that allows for precise chemical proteolysis at C-terminal arginine dictated by 9,10-phenanthrenequinone independent of the remaining sequence. This biomimetic approach also exhibits the potential to synthesize C-terminal methyl ester (-CO2Me) peptides.

Computational Insights on the Hydride and Proton Transfer Mechanisms of D-Arginine Dehydrogenase.

D-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) is an amine oxidase which catalyzes the conversion of D-arginine into iminoarginine. It contains a non-covalent FAD cofactor that is involved in the oxidation mechanism. Based on substrate, solvent, and multiple kinetic isotope effects studies, a stepwise hydride transfer mechanism is proposed. It was shown that D-arginine binds to the active site of enzyme as α-amino group protonated, and it is deprotonated before a hydride ion is transferred from its α-C to FAD. Based on a mutagenesis study, it was concluded that a water molecule is the most likely catalytic base responsible from the deprotonation of α-amino group. In this study, we formulated computational models based on ONIOM method to elucidate the oxidation mechanism of D-arginine into iminoarginine using the crystal structure of enzyme complexed with iminoarginine. The calculations showed that Arg222, Arg305, Tyr249, Glu87, His 48, and two active site water molecules play key roles in binding and catalysis. Model systems showed that the deprotonation step occurs prior to hydride transfer step, and active site water molecule(s) may have participated in the deprotonation process.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.