Salting-out effect of quaternary alkylammonium cations on protein solubility derived from the excluded-volume and hydrophobicity of cations

Salting-out effect of quaternary alkylammonium cations on protein solubility derived from the excluded-volume and hydrophobicity of cations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Salting-out effect of quaternary alkylammonium cations on protein solubility derived from the excluded-volume and hydrophobicity of cations Tatsushi Nakayama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5728577/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The solubility of several proteins, including hen egg-white lysozyme, bovine serum albumin, cytochrome C, and canavalia ensiformis urease (from Jack bean), was examined in the presence of quaternary alkylammonium salts, tetramethylammonium perchlorate, tetraethylammonium perchlorate, tetrapropylammonium perchlorate, and tetrabutylammonium perchlorate. The quaternary alkylammonium cations markedly reduced protein solubility, and were more potent than Hofmeister cations, such as sodium, potassium, and cesium. The quaternary alkylammonium cations reduced protein solubility mainly through the excluded-volume effect rather than electrostatic shielding or hydrophobic effect. This result highlights the importance of the excluded-volume derived from salts themselves in the salting-out effect and provides insights into the mechanism of the Hofmeister series. protein solubility Hofmeister series quaternary alkylammonium cations salt effect excluded-volume effect Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The solubility of proteins is a significant factor in structural biology and protein therapeutics, in which scientists work with proteins at a high concentrations [ 1 , 2 ]. A low protein solubility is also implicated in diseases caused by protein aggregates, such as Alzheimer’s disease, Parkinson’s disease, and Creutzfeldt-Jakob diseases [ 3 – 6 ]. The protein solubility is determined by a number of extrinsic factors (pH, temperature, ionic strength, hydrostatic pressure, organic solvents, and polymers) and intrinsic factors encoded in the amino acid sequence of protein (hydrophobicity/ hydrophilicity, and net charge, and charge distribution on the molecular surface) [ 1 , 2 , 7 – 9 ]. Salt and ionic strengths are among the most potent extrinsic factors affecting the protein solubility [ 2 , 10 ] and have been a subject of intensive research for many years [ 11 ]. Previous studies have established that the addition of salt at a low concentration increases the protein solubility (“salting-in” effect) due to the electrostatic shielding of Coulomb interactions at the protein surface by counterions (described in the Debye–Hückel theory). At a higher salt concentration, Coulomb interactions are further shielded to induce attractive van der Waals interactions between proteins, leading to a decrease in the protein solubility (“salting-out” effect). The Hofmeister series is a classification of ions based on their ability to salt in/out proteins [ 12 – 14 ]. The Hofmeister series is equivalent for most proteins, indicating that these ions are authentic extrinsic factors that determine protein solubility. In general, ions with a high salting-out effect increase the surface tension of the solvent and decrease the solubility of nonpolar solutes. These ions can also lower the entropy of water to enhance the hydrophobic effect, or can modulate protein-protein interaction by the electrostatic shielding effect. However, the mechanism underlying the salting-out effect remains unclear. In this study, I examined the effects of four kinds of quaternary alkylammonium cations that differ in length of alkyl chains, tetramethylammonium perchlorate (TMAP), tetraethylammonium perchlorate (TEAP), tetrapropylammonium perchlorate (TPAP), and tetrabutylammonium perchlorate (TBAP), on protein solubility to obtain a quantitative correlation between the salting-out effect and physiochemical factors of the salts. The quaternary alkylammonium cations are popular electrolytes because of their stability and nonfunctionality without acidic protons, especially in electrochemistry and electroanalytical chemistry as the supporting electrolyte [ 15 – 19 ]. Any of the quaternary alkylammonium are monovalent cations [ 20 ] and have some rotation symmetries (Figure S1 ), which enable multiple comparable factors for the salts. This is the greatest advantage for verifying other factors that determine the residual salting-out effect. The methylammonium cation was previously shown to have a high salting-out effect in the Hofmeister series [ 14 ], but other ammonium cations have not been examined. Goals in this study were 1) to estimate the salting-out effect of quaternary alkylammonium cations and 2) to understand the mechanism by which quaternary alkylammonium cations would affect protein solubility. 2. Materials and Methods Materials Proteins, hen egg-white lysozyme (LYZ, > 95.0%), bovine serum albumin (BSA, > 96.0%), cytochrome C (CYT, > 95.0%), and canavalia ensiformis urease (from Jack bean, URE, > 95.0%), were purchased from Sigma-Aldrich Inc. (Tokyo, Japan) and were used as received. Additionally, Acetonitrile (ACN, > 99.5%), N , N -dimethylformamide (DMF, > 99.5%), dimethyl Sulfoxide (DMSO, > 99.0%), methanol (> 99.8%), and ethanol (> 99.8%), employed as organic solvents, were also procured from Sigma-Aldrich Inc. Quaternary alkylammonium salts, TMAP, TEAP, TPAP, and TBAP, were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The quaternary alkylammonium salts were recrystallized several times and dried overnight under conditions of heating and reduced pressure before use. Other salts, lithium perchlorate (LiClO 4 , > 98.0%), sodium perchlorate (NaClO 4 > 98.0%, monohydrate), magnesium perchlorate (Mg(ClO 4 ) 2 >95.0%, tetrahydrate), potassium perchlorate (KClO 4 > 98.0%), calcium perchlorate (Ca(ClO 4 ) 2 >95.0%, tetrahydrate), and cesium perchlorate (CsClO 4 > 99.9%), were purchased from Tokyo Chemical Industry Co., Ltd. and were used without further purification or drying. The counter anion of all salts employed in this study is perchloric ion (ClO 4 − ), since it is available commercially. Methods ACN–water-mixtured solutions containing each of the salts without a buffer were prepared before adding proteins. Proteins in dry powder form were added until saturation; saturation was confirmed visually based on the remaining solid protein residue after extensive vortex mixing. The solution samples were then centrifuged at 9,000 g (at 20°C for 10 minutes) immediately before measurement. The supernatant was collected and diluted as necessary, and the protein concentration was determined via ultraviolet-visible (UV-vis) spectrometry for absorbance at 280 nm (LYZ, BSA, URE) or 410 nm (CYT) using UV-3600 Plus spectrometer (Shimadzu Co., Ltd.) in the room temperature. Solution-phase Density functional theory (DFT) calculations were performed with the Becke three-parameter Lee–Yang–Parr functional (B3LYP), implemented in the Gaussian 16 Program package [ 21 ]. The B3LYP functional was chosen because it give good geometries and electronic states of ions [ 22 , 23 ]. In the calculations, I applied the standard split-valence double ζ basis sets augmented by the polarization d,p and diffusion orbitals 6-311 + + G(d,p). The polarized continuum model (PCM) was employed for solvent contributions to the standard Gibbs free energies under the default settings of Gaussian 16 that is widely employed. The internal energies were converted to standard Gibbs energies at 298.15 K using the zero-point energies, thermal correction, and entropy. The geometry of each ionic structure was optimized. The frequency analyses were performed to obtain the electrostatic potentials (ESP) of ions. 3. Results 3.1. An organic solvent for evaluating the effects of quaternary alkylammonium salts on protein solubility The aim of this study is to examine how quaternary alkylammonium salts affect the protein solubility. However, these hydrophobic salts were less soluble in water; in particular, quaternary alkylammonium salts with longer alkyl chains (TEAP, TPAP, and TBAP) were completely insoluble even at the lowest concentration employed (0.1 mol dm − 3 ). To overcome the low solubility in aqueous solvents, I examined the solubility of quaternary alkylammonium salts in five kinds of organic solvent (ACN, DMF, DMSO, methanol, and ethanol) as well as in a 50/50% (v/v) organic-aqueous solvent mixture. Each quaternary alkylammonium salt was dissolved at concentrations of 0.1, 0.5, and 1.0 mol dm − 3 using ClO 4 − as a counter anion. As shown in Table 1 , at 0.1 mol dm − 3 concentration, all four quaternary alkylammonium salts were soluble in pure ACN, pure DMF, 50/50% ACN–water mixture, and 50/50% DMF-water mixture. In contrast, quaternary alkylammonium salts were less soluble in polar aprotic (DMSO) or protic solvents (methanol, ethanol), suggesting the lipophilic nature of the quaternary alkylammonium salts. Notably, the addition of the quaternary alkylammonium salts into the 50/50% organic-aqueous mixture often resulted in the gelation of water (“G” in Table 1 ). This effect was prominent in quaternary alkylammonium salts with longer alkyl chains (TPAP and TBAP) and in aprotic solvent (ACN, DMF, and DMSO), but the detailed mechanism was not pursued in this study. Collectively, I selected ACN as a co-solvent to examine the effects of quaternary alkylammonium salts on protein solubility. Table 1 States of an organic solution and an organic-aqueous mixture (50% v/v) containing quaternary alkylammonium salt at concentrations of 0.1, 0.5, and 1.0 mol dm − 3 . Properties States of solution (50% organic-aqueous solvent, 100% organic solvent) Molecular weight 2 ACN DMF DMSO Methanol Ethanol Density (g/ml) Log P o/w mol dm − 3 50% 100% 50% 100% 50% 100% 50% 100% 50% 100% 1 TMAP 173.59 0.1 3 ○ N ○ ○ ○ ○ N N N N 1.3739 0.5 N N N N N N N N N N 0.30 1.0 N N N N N N N N N N TEAP 229.70 0.1 ○ ○ ○ ○ ○ ○ ○ N ○ N 1.2711 0.5 ○ ○ ○ ○ ○ ○ N N N N 1.70 1.0 ○ ○ N ○ N ○ N N N N TPAP 285.81 0.1 ○ ○ ○ ○ ○ ○ N N N N 1.1057 0.5 G ○ N ○ N ○ N N N N 3.90 1.0 G ○ N N N N N N N N TBAP 341.92 0.1 ○ ○ ○ ○ N ○ N N N N 1.0387 0.5 G ○ G ○ G ○ N N N N 5.30 1.0 G ○ G ○ G ○ N N N N 1 Tetramethylammonium perchlorate (TMAP), tetraethylammonium perchlorate (TEAP), tetrapropylammonium perchlorate (TPAP), tetrabutylammonium perchlorate (TBAP), 2 acetonitrile (ACN), N , N -dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 3 ○: solution / homogeneous clear mixture, N: insoluble, G: forming gel 3.2. Measurement of protein solubility using the midpoint of the solubility curves Next, I examined the effect of ammonium salts on protein solubility in a ACN–water mixture. DMF was excluded from further analysis, because of water gelation at high protein concentrations (data not shown), which hampered the precise measurement of protein solubility. Figure 1 (a) shows the solubility of model proteins (LYZ, BSA, CYT and URE) in the absence of ammonium salts. Protein solubility was determined by measuring the UV-Vis absorbance of the solution after the removal of insoluble proteins, as described in the Methods. The horizontal axis shows the mixing ratio of ACN–water in terms of the composition of ACN (% v/v), and the vertical axis shows the ratio of solubility. All four proteins were completely insoluble in 100% ACN; however, as the proportion of water increased, the solubility significantly increased, showing a sigmoidal transition in the solubility curve for LYZ, BSA, and CYT. Accordingly, the midpoint of the sigmoidal transition was thought to provide a qualitative measurement for the protein solubility, where URE was excluded from further analysis because of its broaden sigmoidal curve. Figure 1 (b) shows the solubility curves of LYZ in ACN–water solution in the presence of Hofmeister cations at a concentration of 0.01 mol dm − 3 . The midpoint of the sigmoidal transition was shifted to the left side (water-rich side) in the order of Hofmeister series, cesium (Cs + ) > potassium (K + ) > sodium (Na + ) > lithium (Li + ) > magnesium (Mg 2+ ) > calcium (Ca 2+ ). In Fig. 1 (b), cations with greater salting-out effects are plotted lower at each solvent ratio, and the order remains constant, where Cs + have the strongest salting-out effect. This result confirmed that the midpoint of the sigmoidal transition can be used as a qualitative measure of protein solubility. 3.3. Quaternary alkylammonium cations markedly reduced protein solubility. Figure 1 (b) shows the solubility curves of LYZ in the presence of quaternary alkylammonium cations at a concentration of 0.01 mol dm − 3 . Notably, all the quaternary alkylammonium cations reduced the protein solubility of LYZ more strongly than any of Hofmeister cations (the midpoint of the sigmoidal transition was shifted to left side). This salting-out effect was more prominent in quaternary alkylammonium cations with longer alkyl chains, such as TPA + and TBA + . Protein solubility of (a) LYZ, (b) BSA, and (c) CYT were examined in the presence of quaternary alkylammonium salts at the concentration of 0.1 mol dm − 3 (Fig. 2 ). Consistent with the above observations, the quaternary alkylammonium cations significantly reduced the protein solubility in the order of TBAP > TPAP > TEAP > TMAP. Thus, quaternary alkylammonium cations with longer alkyl chains effectively reduced the solubility of various proteins. Figure 3 shows the protein solubility in the presence of various concentrations of TBAP (0.01–0.10 mol dm − 3 ) and TMAP (0.1-1.0 mol dm − 3 ). Protein solubility data at low ACN compositions were sometimes incomplete because of the gelation and low solubility of quaternary alkylammonium cations (see Figs. 3 d, 3 e, and 3 f), as described above (Table 1 ). Nonetheless, as the TBAP and TEAP concentrations increased, the solubility curves were gradually shifted to the left water-rich side. Thus, the quaternary alkylammonium cations reduced protein solubility in a concentration-dependent manner. 3.4. Quaternary alkylammonium cations reduced protein solubility mainly through the excluded-volume effect. Next, I examined how quaternary alkylammonium cations reduce protein solubility. I thought that the salting-out ability of ions could be determined by (1) electrostatic effects to shield the surface charge of protein which are divided into the bulk property derived from ionic strength and the surface property from electrostatic potentials, (2) excluded-volume of ions, and (3) other mechanisms involving hydrophobicity of ions or effects on solvent. Here, I examine how (1) and (2) would contribute to the salting-out ability of quaternary alkylammonium cations. As described above, quaternary alkylammonium cations with longer alkyl chains more effectively reduced protein solubility, despite of the same molar concentrations (same ionic strengths) at 0.1 mol dm − 3 (Fig. 2 ). Therefore, the ESP of the ions was calculated using the DFT-B3LYP/6-311 + + G(d,p) method. As shown in Fig. 2 (d), the ESPs of the quaternary alkylammonium cations were increased by shortening the alkyl chains (0.180e for TMA + and 0.144e for TBA + ), opposite to the trend in the observed salting-out effect (Fig. 3 ). In general, high ESPs (and high shielding effects) are linked to strong salting-out effects. Even in organic solvents such as ACN, DMF, DMSO, and water, each alkylammonium salt has a symmetric and stable chemical structure, and ESPs also show a similar tendency (Table S1 ). Thus, the reverse correlation between ESPs and salting-out effects indicates that the electrostatic effect contributes negatively to the observed phenomenon. To estimate the excluded-volume effect of the quaternary alkylammonium salts, I re-examined protein solubility in the presence of the same volume of salts (12.63 or 25.26 ml/L, equivalent to 0.100 or 0.200 mol dm − 3 of TMAP, respectively) (Fig. 4 ). The salt volumes were estimated from the weights and densities in solid states of the salts (Table 1 ). Compared with the solubility curves in the presence of the same molar concentration of salts (Fig. 2 ), the difference between the quaternary alkylammonium salts was significantly diminished. Thus, the excluded-volume effect effectively reduce protein solubility. Slight differences in the solubility curves between the different quaternary alkylammonium salts were present even after matching the excluded-volume of the salts (Fig. 4 ). Moreover, the same order of the salting-out effect was preserved, as shown in Figs. 2 (a–c). This indicates that other mechanisms, which overcome the opposite effect of ESP (Fig. 2 d), must exist to explain the residual salting-out effect. This may include (a) the hydrophobic effect of longer quaternary alkylammonium salts and (b) a diminished salting-in effect of the counter anion (ClO 4 − ), whose concentration was difficult to match in this experiment (i.e., longer alkylammonium salts include less ClO 4 − and may have somewhat lower salting-in effect of ClO 4 − ). The partition coefficient (P o/w ) of the quaternary alkylammonium cations showed as logP o/w (Table 1 ) markedly increases by lengthening the alkyl chains (0.30 for TMA + and 5.30 for TBA + ), corresponding to the order of the residual salting-out effect. Therefore, it is reasonable that the hydrophobic effect (a) mainly gives slight differences, changing the solvation (water and ACN) and ion coordination of protein surfaces as to be preferential hydrophobic structure. 4. Discussion In this study, I observed that quaternary alkylammonium cations markedly decrease protein solubility, and they were more potent than any of Hofmeister cations (Fig. 2 b), and proved that the excluded-volume effect mainly contributes to the salting-out effect (Fig. 4 ). By contrast, other mechanisms such as electrostatic shielding or hydrophobic effect, contributes negatively or slightly to the salting-out effect of quaternary alkylammonium cations. Middaugh et al. previously examined protein solubility in the presence of polyethylene glycol (PEG) and revealed that PEG reduced protein solubility through the excluded-volume effect [ 24 ]. In addition, the results of this study highlight the importance of the excluded-volume effect of salt, which provides insights into understanding the mechanism of salting-out effect induced by Hofmeister cations. This excluded-volume effect needed to be verified using similar salts with different volumes, such as the tetraalkyl groups used in this study, and therefore required the use of an organic solvent capable of dissolving bulky salts with high solubility. The results allowed us to gain insight into the Hofmeister series that would not have been noticed in studies using aqueous solvents. Declarations Acknowledgements The author would like to thank Takuma Fujii for his experimental assistance. Funding This work was supported by the Foundation of Public Interest of Tatematsu, Amano Institute of Technology, the Koshiyama Science and Technology Foundation, and the OGAWA Science and Technology Foundation. Data Availability: Data are available at the following link: doi: 10.17632/pfkbjvjvsc.1 Statements and Declarations Competing Interests: The author declares no competing interests. Ethical Approval: This is not applicable. 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J Biol Chem 254:367–370 Additional Declarations No competing interests reported. Supplementary Files Supplementary.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5728577","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":397004504,"identity":"d07bc1f4-eaad-4cd6-9c5e-bdc2dd431a64","order_by":0,"name":"Tatsushi Nakayama","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYHACZgjF3sBwoAIuyINbPQ9cC88BhgNnSNMikcDAcAa3QgSwl25+bPCzzS6af+bziwcOtjHkGRxgfviBQeYObltkjhkn9rYl5864nVMA0lJscIDNWIKB5xluLRIJxgd425hzG27nJBz+2PY/ccMBBjOg+GE8WtI/H/zbVp87/+aZBJAtQC3s3whoyTFO5m07nLvhBvsBqBYeArbcyCk2ljl3PHfjmRyGAwfOMRRLHuYplkjA4xf2GembJd+UVefOO3788YcDZQx5fMfbN3742IM7xMCAkQ1soQGITADHU2LPAfxaGP6ALXwA0QIGPwhpGQWjYBSMghEEAMTcXzxHZGruAAAAAElFTkSuQmCC","orcid":"","institution":"Gifu Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Tatsushi","middleName":"","lastName":"Nakayama","suffix":""}],"badges":[],"createdAt":"2024-12-29 06:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5728577/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5728577/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73138889,"identity":"baab12e0-13f7-4eab-ab50-423bac5b6661","added_by":"auto","created_at":"2025-01-07 06:34:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12751134,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Change in solubility of model proteins in a mixed solution of acetonitrile (ACN) and water obtained by measuring the absorbance spectral of wavelength at 280 nm (A\u003csub\u003e280nm\u003c/sub\u003e) for hen egg-white lysozyme (LYZ), bovine serum albumin (BSA), and canavalia ensiformis urease (URE), then, at 410 nm (A\u003csub\u003e410nm\u003c/sub\u003e) for cytochrome C (CYT). The composition [% v/v] shows the percentage of ACN in a mixed solution. The solubility ratio [C\u003csub\u003ein mixture\u003c/sub\u003e/C\u003csub\u003ein water\u003c/sub\u003e] indicates the ratio of the saturated concentration of proteins in a mixed solution relative to that in pure water (= 1.0). (b) Effects of cation, calcium (Ca\u003csup\u003e2+\u003c/sup\u003e), magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e), lithium (Li\u003csup\u003e+\u003c/sup\u003e), sodium (Na\u003csup\u003e+\u003c/sup\u003e), potassium (K\u003csup\u003e+\u003c/sup\u003e), cesium (Cs\u003csup\u003e+\u003c/sup\u003e), tetramethylammonium (TMA\u003csup\u003e+\u003c/sup\u003e), tetraethylammonium (TEA\u003csup\u003e+\u003c/sup\u003e), tetrapropylammonium (TPA\u003csup\u003e+\u003c/sup\u003e), and tetrabutylammonium (TBA\u003csup\u003e+\u003c/sup\u003e), on change of the solubility of LYZ; each cation is added as a perchlorate salt at a concentration of 0.01 mol dm\u003csup\u003e−3\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5728577/v1/47a4566f427c50032830005a.png"},{"id":73138493,"identity":"1f303f14-148a-4f5e-a8ee-89747d274836","added_by":"auto","created_at":"2025-01-07 06:26:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93667801,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the solubility of (a) hen egg-white lysozyme (LYZ), (b) bovine serum albumin (BSA), and (c) cytochrome C (CYT) in the absence (blank) and presence of 0.1 mol dm\u003csup\u003e−3\u003c/sup\u003e quaternary alkylammonium electrolyte. The vertical axis shows the concentration of the protein [mg/ml] obtained by A280nm (a, b) and A410nm (c) spectral measurements. (d) Electrostatic potential surfaces for quaternary alkylammonium salts in water calculated using the density functional theory-B3LYP/PCM/6-311++G(d,p) method.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5728577/v1/98203c724536c6e2ff1314ec.png"},{"id":73138495,"identity":"2b21ef6a-8c4a-488e-a123-4297202b7c67","added_by":"auto","created_at":"2025-01-07 06:26:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130054035,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of the solubility of protein on the concentration of quaternary alkylammonium salts in the mixed solution of acetonitrile–water. (a, d) Hen egg-white lysozyme (LYZ), (b, e) bovine serum albumin (BSA), and (c, f) cytochrome C (CYT). The concentrations of tetrabutylammonium perchlorate (TBAP) are 0, 0.01, 0.05, and 0.10 mol dm\u003csup\u003e−3\u003c/sup\u003e (a–c), and those of tetraethylammonium perchlorate (TEAP) are 0, 0.1, 0.5, and 1.0 mol dm\u003csup\u003e−3\u003c/sup\u003e (d–f).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5728577/v1/19d93eaca968111a1d0ebc5f.png"},{"id":73138491,"identity":"ba90bfd9-b177-48b1-9872-3343494efac7","added_by":"auto","created_at":"2025-01-07 06:26:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65176905,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the solubility of (a) hen egg-white lysozyme (LYZ), (b) bovine serum albumin (BSA), and (c) cytochrome C (CYT) in the presence of the same volume of quaternary alkylammonium salts; tetramethylammonium perchlorate (TMAP), tetraethylammonium perchlorate (TEAP), tetrapropylammonium perchlorate (TPAP), and tetrabutylammonium perchlorate (TBAP). The concentrations of each salt are adjusted to 0.10/0.20 mol dm\u003csup\u003e−3\u003c/sup\u003e TMAP as the volumes are the same at 12.63/25.26 ml/L.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5728577/v1/84e4d48079a70a48eb018719.png"},{"id":73138473,"identity":"75c977df-c5be-4bc9-a7c5-7d3f985accbd","added_by":"auto","created_at":"2025-01-07 06:26:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":433501,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5728577/v1/100b1daf6e45a6b76c288cdc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Salting-out effect of quaternary alkylammonium cations on protein solubility derived from the excluded-volume and hydrophobicity of cations","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe solubility of proteins is a significant factor in structural biology and protein therapeutics, in which scientists work with proteins at a high concentrations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A low protein solubility is also implicated in diseases caused by protein aggregates, such as Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, and Creutzfeldt-Jakob diseases [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The protein solubility is determined by a number of extrinsic factors (pH, temperature, ionic strength, hydrostatic pressure, organic solvents, and polymers) and intrinsic factors encoded in the amino acid sequence of protein (hydrophobicity/ hydrophilicity, and net charge, and charge distribution on the molecular surface) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Salt and ionic strengths are among the most potent extrinsic factors affecting the protein solubility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and have been a subject of intensive research for many years [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous studies have established that the addition of salt at a low concentration increases the protein solubility (\u0026ldquo;salting-in\u0026rdquo; effect) due to the electrostatic shielding of Coulomb interactions at the protein surface by counterions (described in the Debye\u0026ndash;H\u0026uuml;ckel theory). At a higher salt concentration, Coulomb interactions are further shielded to induce attractive van der Waals interactions between proteins, leading to a decrease in the protein solubility (\u0026ldquo;salting-out\u0026rdquo; effect).\u003c/p\u003e \u003cp\u003eThe Hofmeister series is a classification of ions based on their ability to salt in/out proteins [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The Hofmeister series is equivalent for most proteins, indicating that these ions are authentic extrinsic factors that determine protein solubility. In general, ions with a high salting-out effect increase the surface tension of the solvent and decrease the solubility of nonpolar solutes. These ions can also lower the entropy of water to enhance the hydrophobic effect, or can modulate protein-protein interaction by the electrostatic shielding effect. However, the mechanism underlying the salting-out effect remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, I examined the effects of four kinds of quaternary alkylammonium cations that differ in length of alkyl chains, tetramethylammonium perchlorate (TMAP), tetraethylammonium perchlorate (TEAP), tetrapropylammonium perchlorate (TPAP), and tetrabutylammonium perchlorate (TBAP), on protein solubility to obtain a quantitative correlation between the salting-out effect and physiochemical factors of the salts. The quaternary alkylammonium cations are popular electrolytes because of their stability and nonfunctionality without acidic protons, especially in electrochemistry and electroanalytical chemistry as the supporting electrolyte [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Any of the quaternary alkylammonium are monovalent cations [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and have some rotation symmetries (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which enable multiple comparable factors for the salts. This is the greatest advantage for verifying other factors that determine the residual salting-out effect. The methylammonium cation was previously shown to have a high salting-out effect in the Hofmeister series [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but other ammonium cations have not been examined. Goals in this study were 1) to estimate the salting-out effect of quaternary alkylammonium cations and 2) to understand the mechanism by which quaternary alkylammonium cations would affect protein solubility.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eMaterials\u003c/p\u003e \u003cp\u003eProteins, hen egg-white lysozyme (LYZ, \u0026gt;\u0026thinsp;95.0%), bovine serum albumin (BSA, \u0026gt;\u0026thinsp;96.0%), cytochrome C (CYT, \u0026gt;\u0026thinsp;95.0%), and canavalia ensiformis urease (from Jack bean, URE, \u0026gt;\u0026thinsp;95.0%), were purchased from Sigma-Aldrich Inc. (Tokyo, Japan) and were used as received. Additionally, Acetonitrile (ACN, \u0026gt;\u0026thinsp;99.5%), \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylformamide (DMF, \u0026gt;\u0026thinsp;99.5%), dimethyl Sulfoxide (DMSO, \u0026gt;\u0026thinsp;99.0%), methanol (\u0026gt;\u0026thinsp;99.8%), and ethanol (\u0026gt;\u0026thinsp;99.8%), employed as organic solvents, were also procured from Sigma-Aldrich Inc. Quaternary alkylammonium salts, TMAP, TEAP, TPAP, and TBAP, were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The quaternary alkylammonium salts were recrystallized several times and dried overnight under conditions of heating and reduced pressure before use. Other salts, lithium perchlorate (LiClO\u003csub\u003e4\u003c/sub\u003e, \u0026gt;\u0026thinsp;98.0%), sodium perchlorate (NaClO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;98.0%, monohydrate), magnesium perchlorate (Mg(ClO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e \u0026gt;95.0%, tetrahydrate), potassium perchlorate (KClO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;98.0%), calcium perchlorate (Ca(ClO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e \u0026gt;95.0%, tetrahydrate), and cesium perchlorate (CsClO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;99.9%), were purchased from Tokyo Chemical Industry Co., Ltd. and were used without further purification or drying. The counter anion of all salts employed in this study is perchloric ion (ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), since it is available commercially.\u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eACN\u0026ndash;water-mixtured solutions containing each of the salts without a buffer were prepared before adding proteins. Proteins in dry powder form were added until saturation; saturation was confirmed visually based on the remaining solid protein residue after extensive vortex mixing. The solution samples were then centrifuged at 9,000 g (at 20\u0026deg;C for 10 minutes) immediately before measurement. The supernatant was collected and diluted as necessary, and the protein concentration was determined via ultraviolet-visible (UV-vis) spectrometry for absorbance at 280 nm (LYZ, BSA, URE) or 410 nm (CYT) using UV-3600 Plus spectrometer (Shimadzu Co., Ltd.) in the room temperature.\u003c/p\u003e \u003cp\u003eSolution-phase Density functional theory (DFT) calculations were performed with the Becke three-parameter Lee\u0026ndash;Yang\u0026ndash;Parr functional (B3LYP), implemented in the Gaussian 16 Program package [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The B3LYP functional was chosen because it give good geometries and electronic states of ions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the calculations, I applied the standard split-valence double ζ basis sets augmented by the polarization d,p and diffusion orbitals 6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p). The polarized continuum model (PCM) was employed for solvent contributions to the standard Gibbs free energies under the default settings of Gaussian 16 that is widely employed. The internal energies were converted to standard Gibbs energies at 298.15 K using the zero-point energies, thermal correction, and entropy. The geometry of each ionic structure was optimized. The frequency analyses were performed to obtain the electrostatic potentials (ESP) of ions.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1. An organic solvent for evaluating the effects of quaternary alkylammonium salts on protein solubility\u003c/h2\u003e \u003cp\u003eThe aim of this study is to examine how quaternary alkylammonium salts affect the protein solubility. However, these hydrophobic salts were less soluble in water; in particular, quaternary alkylammonium salts with longer alkyl chains (TEAP, TPAP, and TBAP) were completely insoluble even at the lowest concentration employed (0.1 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). To overcome the low solubility in aqueous solvents, I examined the solubility of quaternary alkylammonium salts in five kinds of organic solvent (ACN, DMF, DMSO, methanol, and ethanol) as well as in a 50/50% (v/v) organic-aqueous solvent mixture. Each quaternary alkylammonium salt was dissolved at concentrations of 0.1, 0.5, and 1.0 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e using ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e as a counter anion. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, at 0.1 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e concentration, all four quaternary alkylammonium salts were soluble in pure ACN, pure DMF, 50/50% ACN\u0026ndash;water mixture, and 50/50% DMF-water mixture. In contrast, quaternary alkylammonium salts were less soluble in polar aprotic (DMSO) or protic solvents (methanol, ethanol), suggesting the lipophilic nature of the quaternary alkylammonium salts. Notably, the addition of the quaternary alkylammonium salts into the 50/50% organic-aqueous mixture often resulted in the gelation of water (\u0026ldquo;G\u0026rdquo; in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This effect was prominent in quaternary alkylammonium salts with longer alkyl chains (TPAP and TBAP) and in aprotic solvent (ACN, DMF, and DMSO), but the detailed mechanism was not pursued in this study. Collectively, I selected ACN as a co-solvent to examine the effects of quaternary alkylammonium salts on protein solubility.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStates of an organic solution and an organic-aqueous mixture (50% v/v) containing quaternary alkylammonium salt at concentrations of 0.1, 0.5, and 1.0 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eProperties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c13\" namest=\"c4\"\u003e \u003cp\u003eStates of solution (50% organic-aqueous solvent, 100% organic solvent)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c5\" namest=\"c4\" rowspan=\"2\"\u003e \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eACN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c7\" namest=\"c6\" rowspan=\"2\"\u003e \u003cp\u003eDMF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c9\" namest=\"c8\" rowspan=\"2\"\u003e \u003cp\u003eDMSO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c11\" namest=\"c10\" rowspan=\"2\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c13\" namest=\"c12\" rowspan=\"2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDensity (g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eTMAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e173.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e3\u003c/sup\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e 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align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e 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colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e 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align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e○\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e1\u003c/sup\u003eTetramethylammonium perchlorate (TMAP), tetraethylammonium perchlorate (TEAP), tetrapropylammonium perchlorate (TPAP), tetrabutylammonium perchlorate (TBAP), \u003csup\u003e2\u003c/sup\u003eacetonitrile (ACN), \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), \u003csup\u003e3\u003c/sup\u003e○: solution / homogeneous clear mixture, N: insoluble, G: forming gel\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Measurement of protein solubility using the midpoint of the solubility curves\u003c/h2\u003e \u003cp\u003eNext, I examined the effect of ammonium salts on protein solubility in a ACN\u0026ndash;water mixture. DMF was excluded from further analysis, because of water gelation at high protein concentrations (data not shown), which hampered the precise measurement of protein solubility. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a) shows the solubility of model proteins (LYZ, BSA, CYT and URE) in the absence of ammonium salts. Protein solubility was determined by measuring the UV-Vis absorbance of the solution after the removal of insoluble proteins, as described in the Methods. The horizontal axis shows the mixing ratio of ACN\u0026ndash;water in terms of the composition of ACN (% v/v), and the vertical axis shows the ratio of solubility. All four proteins were completely insoluble in 100% ACN; however, as the proportion of water increased, the solubility significantly increased, showing a sigmoidal transition in the solubility curve for LYZ, BSA, and CYT. Accordingly, the midpoint of the sigmoidal transition was thought to provide a qualitative measurement for the protein solubility, where URE was excluded from further analysis because of its broaden sigmoidal curve. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b) shows the solubility curves of LYZ in ACN\u0026ndash;water solution in the presence of Hofmeister cations at a concentration of 0.01 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. The midpoint of the sigmoidal transition was shifted to the left side (water-rich side) in the order of Hofmeister series, cesium (Cs\u003csup\u003e+\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;potassium (K\u003csup\u003e+\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;sodium (Na\u003csup\u003e+\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;lithium (Li\u003csup\u003e+\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e)\u0026thinsp;\u0026gt;\u0026thinsp;calcium (Ca\u003csup\u003e2+\u003c/sup\u003e). In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b), cations with greater salting-out effects are plotted lower at each solvent ratio, and the order remains constant, where Cs\u003csup\u003e+\u003c/sup\u003e have the strongest salting-out effect. This result confirmed that the midpoint of the sigmoidal transition can be used as a qualitative measure of protein solubility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Quaternary alkylammonium cations markedly reduced protein solubility.\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b) shows the solubility curves of LYZ in the presence of quaternary alkylammonium cations at a concentration of 0.01 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. Notably, all the quaternary alkylammonium cations reduced the protein solubility of LYZ more strongly than any of Hofmeister cations (the midpoint of the sigmoidal transition was shifted to left side). This salting-out effect was more prominent in quaternary alkylammonium cations with longer alkyl chains, such as TPA\u003csup\u003e+\u003c/sup\u003e and TBA\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProtein solubility of (a) LYZ, (b) BSA, and (c) CYT were examined in the presence of quaternary alkylammonium salts at the concentration of 0.1 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consistent with the above observations, the quaternary alkylammonium cations significantly reduced the protein solubility in the order of TBAP\u0026thinsp;\u0026gt;\u0026thinsp;TPAP\u0026thinsp;\u0026gt;\u0026thinsp;TEAP\u0026thinsp;\u0026gt;\u0026thinsp;TMAP. Thus, quaternary alkylammonium cations with longer alkyl chains effectively reduced the solubility of various proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the protein solubility in the presence of various concentrations of TBAP (0.01\u0026ndash;0.10 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) and TMAP (0.1-1.0 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e). Protein solubility data at low ACN compositions were sometimes incomplete because of the gelation and low solubility of quaternary alkylammonium cations (see Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), as described above (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nonetheless, as the TBAP and TEAP concentrations increased, the solubility curves were gradually shifted to the left water-rich side. Thus, the quaternary alkylammonium cations reduced protein solubility in a concentration-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Quaternary alkylammonium cations reduced protein solubility mainly through the excluded-volume effect.\u003c/h2\u003e \u003cp\u003eNext, I examined how quaternary alkylammonium cations reduce protein solubility. I thought that the salting-out ability of ions could be determined by (1) electrostatic effects to shield the surface charge of protein which are divided into the bulk property derived from ionic strength and the surface property from electrostatic potentials, (2) excluded-volume of ions, and (3) other mechanisms involving hydrophobicity of ions or effects on solvent. Here, I examine how (1) and (2) would contribute to the salting-out ability of quaternary alkylammonium cations.\u003c/p\u003e \u003cp\u003eAs described above, quaternary alkylammonium cations with longer alkyl chains more effectively reduced protein solubility, despite of the same molar concentrations (same ionic strengths) at 0.1 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the ESP of the ions was calculated using the DFT-B3LYP/6-311\u0026thinsp;+\u0026thinsp;+\u0026thinsp;G(d,p) method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d), the ESPs of the quaternary alkylammonium cations were increased by shortening the alkyl chains (0.180e for TMA\u003csup\u003e+\u003c/sup\u003e and 0.144e for TBA\u003csup\u003e+\u003c/sup\u003e), opposite to the trend in the observed salting-out effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In general, high ESPs (and high shielding effects) are linked to strong salting-out effects. Even in organic solvents such as ACN, DMF, DMSO, and water, each alkylammonium salt has a symmetric and stable chemical structure, and ESPs also show a similar tendency (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, the reverse correlation between ESPs and salting-out effects indicates that the electrostatic effect contributes negatively to the observed phenomenon.\u003c/p\u003e \u003cp\u003eTo estimate the excluded-volume effect of the quaternary alkylammonium salts, I re-examined protein solubility in the presence of the same volume of salts (12.63 or 25.26 ml/L, equivalent to 0.100 or 0.200 mol dm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e of TMAP, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The salt volumes were estimated from the weights and densities in solid states of the salts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared with the solubility curves in the presence of the same molar concentration of salts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the difference between the quaternary alkylammonium salts was significantly diminished. Thus, the excluded-volume effect effectively reduce protein solubility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSlight differences in the solubility curves between the different quaternary alkylammonium salts were present even after matching the excluded-volume of the salts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, the same order of the salting-out effect was preserved, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a\u0026ndash;c). This indicates that other mechanisms, which overcome the opposite effect of ESP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), must exist to explain the residual salting-out effect. This may include (a) the hydrophobic effect of longer quaternary alkylammonium salts and (b) a diminished salting-in effect of the counter anion (ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), whose concentration was difficult to match in this experiment (i.e., longer alkylammonium salts include less ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and may have somewhat lower salting-in effect of ClO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). The partition coefficient (P\u003csub\u003eo/w\u003c/sub\u003e) of the quaternary alkylammonium cations showed as logP\u003csub\u003eo/w\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) markedly increases by lengthening the alkyl chains (0.30 for TMA\u003csup\u003e+\u003c/sup\u003e and 5.30 for TBA\u003csup\u003e+\u003c/sup\u003e), corresponding to the order of the residual salting-out effect. Therefore, it is reasonable that the hydrophobic effect (a) mainly gives slight differences, changing the solvation (water and ACN) and ion coordination of protein surfaces as to be preferential hydrophobic structure.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, I observed that quaternary alkylammonium cations markedly decrease protein solubility, and they were more potent than any of Hofmeister cations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and proved that the excluded-volume effect mainly contributes to the salting-out effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). By contrast, other mechanisms such as electrostatic shielding or hydrophobic effect, contributes negatively or slightly to the salting-out effect of quaternary alkylammonium cations. Middaugh et al. previously examined protein solubility in the presence of polyethylene glycol (PEG) and revealed that PEG reduced protein solubility through the excluded-volume effect [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, the results of this study highlight the importance of the excluded-volume effect of salt, which provides insights into understanding the mechanism of salting-out effect induced by Hofmeister cations. This excluded-volume effect needed to be verified using similar salts with different volumes, such as the tetraalkyl groups used in this study, and therefore required the use of an organic solvent capable of dissolving bulky salts with high solubility. The results allowed us to gain insight into the Hofmeister series that would not have been noticed in studies using aqueous solvents.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe author would like to thank Takuma Fujii for his experimental assistance.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Foundation of Public Interest of Tatematsu, Amano Institute of Technology, the Koshiyama Science and Technology Foundation, and the OGAWA Science and Technology Foundation.\u003c/p\u003e\n\u003cp\u003eData Availability: Data are available at the following link: doi: 10.17632/pfkbjvjvsc.1\u003c/p\u003e\n\u003cp\u003eStatements and Declarations\u003c/p\u003e\n\u003cp\u003eCompeting Interests: The author declares no competing interests.\u003c/p\u003e\n\u003cp\u003eEthical Approval: This is not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKramer RM, Shende VR, Motl N et al (2012) Toward a molecular understanding of protein solubility: Increased negative surface charge correlates with increased solubility. 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J Biol Chem 254:367\u0026ndash;370\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"protein solubility, Hofmeister series, quaternary alkylammonium cations, salt effect, excluded-volume effect","lastPublishedDoi":"10.21203/rs.3.rs-5728577/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5728577/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe solubility of several proteins, including hen egg-white lysozyme, bovine serum albumin, cytochrome C, and canavalia ensiformis urease (from Jack bean), was examined in the presence of quaternary alkylammonium salts, tetramethylammonium perchlorate, tetraethylammonium perchlorate, tetrapropylammonium perchlorate, and tetrabutylammonium perchlorate. The quaternary alkylammonium cations markedly reduced protein solubility, and were more potent than Hofmeister cations, such as sodium, potassium, and cesium. The quaternary alkylammonium cations reduced protein solubility mainly through the excluded-volume effect rather than electrostatic shielding or hydrophobic effect. This result highlights the importance of the excluded-volume derived from salts themselves in the salting-out effect and provides insights into the mechanism of the Hofmeister series.\u003c/p\u003e","manuscriptTitle":"Salting-out effect of quaternary alkylammonium cations on protein solubility derived from the excluded-volume and hydrophobicity of cations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-07 06:25:56","doi":"10.21203/rs.3.rs-5728577/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f02d1b9-a904-430f-8bfe-0ea05b3721a8","owner":[],"postedDate":"January 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-07T06:25:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-07 06:25:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5728577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5728577","identity":"rs-5728577","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}