Extreme enthalpy‒entropy compensation in the dimerization of small solutes in aqueous solution.

This communication summarizes findings from the earliest encounters with extreme enthalpy‒entropy compensation, a phenomenon first detected in the 1950s by a reappraisal of isopiestic and calorimetric measurements on aqueous urea solutions in terms of solute self-association. Because concurrent studies of carboxylic acid association were confined to measurement of the equilibrium constant by conductance, IR spectrophotometry or potentiometric titration measurements, temperature-independence of the dimerization constant was mistakenly taken to signify a value of zero for Δ H o instead of (Δ H o  â€’ TΔ S o ). In those studies of small-solute self-association the extreme enthalpy‒entropy compensation was reflecting the action of water as a reactant whose hydroxyl groups were competing for the solute carbonyl involved in self-association. Such action gives rise to a positive temperature dependence of Δ H o that could well be operating in concert with that responsible for the commonly observed negative dependence for protein‒ligand interactions exhibiting extreme enthalpy‒entropy compensation, where the solvent contribution to the energetics reflects changes in the extent of ordered water structure in hydrophobic environments.

Is there reversible dimerization of albumin in blood plasma? And does it matter?

Most albumin in blood plasma is thought to be monomeric with some 5% covalently dimerized. However, many reports in the recent biophysics literature find that albumin is reversibly dimerized or even oligomerized. We review data on this from X-ray crystallography and diverse biophysical techniques. The number-average molecular weight of albumin would be increased by dimerization, affecting size-dependent filtration processes of albumin such as at the glycocalyx of the capillary endothelium and the podocyte slit-diaphragm of the renal glomerulus. If correct, and depending on characteristics of the process, such as Kd, reversible dimerization of albumin in plasma would have major implications for normal physiology and medicine. We present quantitative models of the impact of dimerization on albumin molecular forms, on the number-average molecular weight of albumin, and estimate the effect on the colloid osmotic pressure of albumin. Dimerization reduces colloid osmotic pressure as total albumin concentration increases below that expected in the absence of dimerization. Current models of albumin filtration by the renal glomerulus would need revision to account for the dynamic size of albumin molecules filtered. More robust biophysical data are needed to give a definitive answer to the questions posed and we suggest possible approaches to this.

Defining the mechanism of galectin-3-mediated TGF-ß1 activation and its role in lung fibrosis.

Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.

Assessment of internet-based information on statin therapy.

AIMS: The use of statin therapy is deemed to be controversial by mainstream media. Patients increasingly source medical information from the internet, and the use of statins is no exception. This study aims to determine the quality and educational content of statin-focused information on the internet and YouTube. METHODS AND RESULTS: 'Statin' was searched on Google, Yahoo!, Bing, and YouTube. The first 50 results obtained from each search engine and the first 20 YouTube videos were screened by two assessors. Websites were assessed using the Flesch Reading Ease (FRE) score, University of Michigan Consumer Health Website Evaluation Checklist, and a customized scoring system evaluating statin-focused content for quality. Videos were scored using the Journal of the American Medical Association (JAMA) benchmark criteria, Global Quality Score (GQS), and the customized scoring system. Websites scored a median FRE score of 57.5 [interquartile range (IQR) 52.1-62.3], median Michigan score of 36 (IQR 32-41.5), and median content score of 5 (IQR 3.75-7). Good interobserver agreement was demonstrated [Michigan score interobserver coefficient correlation (ICC) = 0.968; content score ICC = 0.944]. Videos scored a median JAMA score of 2, median GQS score of 2.5, and median content score of 2.5. Good interobserver agreement was demonstrated (JAMA ICC = 0.746; GQS ICC = 0.874; content score ICC = 0.946). CONCLUSION: Quality and readability of statin-focused online information are poor. Healthcare professionals should be aware of the limitations of the current available sources and design online resources that are accurate and patient-friendly.

The adaptability of the ion-binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram-negative bacteria. Sil proteins also bind Cu(I) but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry, we show that binding of both ions is not only tighter than previously thought but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram-negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.

Retrospective rationalization of disparities between the concentration dependence of diffusion coefficients obtained by boundary spreading and dynamic light scattering.

This study establishes the existence of substantial agreement between published results from traditional boundary spreading measurements (including synthetic boundary measurements in the analytical ultracenrifuge) on two globular proteins (bovine serum albumin, ovalbumin) and the concentration dependence of diffusion coefficient predicted for experiments conducted under the operative thermodynamic constraints of constant temperature and solvent chemical potential. Although slight negative concentration dependence of the translational diffusion coefficient is the experimentally observed as well as theoretically predicted, the extent of the concentration dependence is within the limits of experimental uncertainty inherent in diffusion coefficient measurement. Attention is then directed toward the ionic strength dependence of the concentration dependence coefficient ([Formula: see text]) describing diffusion coefficients obtained by dynamic light scattering, where, in principle, the operative thermodynamic constraints of constant temperature and pressure preclude consideration of results in terms of single-solute theory. Nevertheless, good agreement between predicted and published experimental ionic strength dependencies of [Formula: see text] for lysozyme and an immunoglobulin is observed by a minor adaptation of the theoretical treatment to accommodate the fact that thermodynamic activity is monitored on the molal concentration scale because of the constraint of constant pressure that pertains in dynamic light scattering experiments.

Experimental support for reclassification of the light scattering second virial coefficient from macromolecular solutions as a hydrodynamic parameter.

This investigation examines the source of the disparity between experimental values of the light scattering second virial coefficient [Formula: see text] (mL.mol/g2) for proteins and those predicted on the statistical mechanical basis of excluded volume. A much better theoretical description of published results for lysozyme is obtained by considering the experimental parameters to monitor the difference between the thermodynamic excluded volume term and its hydrodynamic counterpart. This involves a combination of parameters quantifying concentration dependence of the translational diffusion coefficient obtained from dynamic light scattering measurements. That finding is shown to account for observations of a strong correlation between [Formula: see text] (mL/g), where M2 is the molar mass (molecular weight) of the macromolecule and the diffusion concentration parameter [Formula: see text] (mL/g). On the grounds that [Formula: see text] is regarded as a hydrodynamic parameter, the same status should be accorded the light scattering second virial coefficient rather than its current incorrect thermodynamic designation as [Formula: see text] (mL.mol/g2), or just B, the osmotic second virial coefficient for protein self-interaction.

The Effect of Complex Training on Physical Performance in Rugby League Players.

PURPOSE: To compare the effects of variable-resistance complex training (VRCT) versus traditional complex training (TCT) on strength, power, speed, and leg stiffness (Kleg) in rugby league players during a 6-week mesocycle. METHODS: Twenty-four rugby league players competing in the British University and Colleges Sport Premier North Division were randomized to VRCT (n = 8), TCT (n = 8), or control (CON; n = 8). Experimental groups completed a 6-week lower-body complex training intervention (2×/wk) that involved alternating high-load resistance exercise with plyometric exercise within the same session. The VRCT group performed resistance exercises at 70% of 1-repetition maximum (1RM) + 0% to 23% of 1RM from band resistance with a 90-second intracontrast rest interval, whereas the TCT group performed resistance exercise at 93% of 1RM with a 4-minute intracontrast rest interval. Back-squat 1RM, countermovement jump peak power, reactive strength index, sprint times, and Kleg were assessed pretraining and posttraining. RESULTS: VRCT and TCT significantly improved 1RM back squat, countermovement jump peak power, and 5-m sprint time (all P < .05). VRCT also improved Kleg, whereas TCT improved 10- and 20-m sprint times (all P < .05). Between groups, both VRCT and TCT improved 1RM back squat compared with CON (both P < .001). Additionally, VRCT improved Kleg compared with CON (right leg: P = .016) and TCT improved 20-m sprint time compared with CON (P = .042). CONCLUSIONS: VRCT and TCT can be implemented during the competitive season to improve strength, power, and 5-m sprint time. VRCT may lead to greater improvements in reactive strength index and Kleg, whereas TCT may enhance 10- and 20-m sprint times.

TBX3 Promotes Cervical Cancer Proliferation and Migration via HPV E6 and E7 Signaling.

Cervical cancer is a leading cause of cancer-related deaths in women globally and 99% of cases are caused by persistent infection with high-risk strains of the human papillomavirus (HPV). The HPV oncoproteins E6 and E7 establish the cancer phenotype by cooperating with host proteins and identifying them may have important therapeutic benefits. T-box transcription factor 3 (TBX3) is a critical developmental regulator, and when it is overexpressed postnatally, it contributes to several cancers, but little is known about its expression and role in cervical cancer. The current study shows that TBX3 is upregulated in cervical cancer cell lines as well as precancerous and cervical cancer patient tissue and is associated with larger and more invasive tumors. Knockdown and overexpression cell culture models show that TBX3 promotes HPV-positive cell proliferation, migration, and spheroid growth; however, TBX3 inhibits these processes in HPV-negative cells. Importantly, we show that the tumor promoting activity of TBX3 in cervical cancer is dependent on E6/E7. IMPLICATIONS: In summary, our study highlights the importance of TBX3 as a cooperating partner of E6/E7 in HPV-positive cervical cancer and identifies TBX3 as a potential therapeutic target to treat this neoplasm.

The Effect of Complex Training on Muscle Architecture in Rugby League Players.

PURPOSE: To compare the effects of variable-resistance complex training (VRCT) versus traditional complex training (TCT) on muscle architecture in rugby league players during a 6-week mesocycle. METHODS: Twenty-four rugby league players competing in the British University & Colleges Sport (BUCS) Premier North Division were randomized to VRCT (n = 8), TCT (n = 8), or control (n = 8). Experimental groups completed a 6-week lower-body complex training intervention (2×/wk), which involved alternating high-load resistance exercise with plyometric exercise in the same session. The VRCT group performed resistance exercises at 70% of 1-repetition maximum (1RM) + 0% to 23% of 1RM from band resistance with a 90-second intracontrast rest interval, whereas the TCT group performed resistance exercise at 93% of 1RM with a 4-minute intracontrast rest interval. Muscle thickness (MT), pennation angle, and fascicle length (Lf) were assessed for the vastus lateralis (VL) and gastrocnemius medialis using ultrasound imaging. RESULTS: Both TCT and VRCT groups significantly improved VL MT and VL Lf compared with control (all P < .05). Standardized within-group changes in MT and Lf (Cohen dav ± 95% CI) were moderate for TCT (dav = 0.91 ± 1.0; dav = 1.1 ± 1.1) and unclear for VRCT (dav = 0.44 ± 0.99; dav = 0.47 ± 0.99), respectively. Differences in change scores between TCT and VRCT were unclear. CONCLUSIONS: VRCT and TCT can be utilized during the competitive season to induce favorable MT and Lf muscle architecture adaptations for the VL. TCT may induce greater muscle architecture adaptations of the VL, whereas VRCT may be of more practical value given the shorter intracontrast rest interval between resistance and plyometric exercises.

Flavour compounds affect protein structure: The effect of methyl anthranilate on bovine serum albumin conformation.

This study aims to understand possible effects of flavour compounds on the structure and conformation of endogenous proteins. Using methyl anthranilate (a grape flavour compound added to drinks, confectionery, and vape-liquids) and bovine serum albumin (BSA, a model serum protein) we designed experimental investigations using analytical ultracentrifugation, size exclusion chromatography small angle X-ray scattering, and fluorescence spectroscopy to reveal that methyl anthranilate spontaneously binds to BSA (ΔG°, ca. -21 KJ mol-1) which induces a conformational compactness (ca. 10 %) in the monomer structure. Complementary molecular modelling and dynamics simulations suggested the binding occurs at Sudlow II of BSA via establishment of hydrogen bonds with arginine409, lysine413 and serine488 leading to an increased conformational order in domains IA, IIB and IIIB. This work aims to set the foundation for future research on flavour-protein interactions and offer new sets of opportunities for understanding the effects of small compounds on protein structure.

High-Frequency Three-Dimensional Lumen Volume Ultrasound Is a Sensitive Method to Detect Early Aneurysmal Change in Elastase-Induced Murine Abdominal Aortic Aneurysm.

OBJECTIVE: The aim of this study was to investigate the reproducibility of anterior-posterior diameter (APdmax) and three-dimensional lumen volume (3DLV) measurements of abdominal aortic aneurysms (AAA) in a classical murine AAA model. We also compared the magnitude of change in the aortic size detected with each method of assessment. METHODS: Periadventitial application of porcine pancreatic elastase (PPE AAA) or sham surgery was performed in two cohorts of mice. Cohort 1 was used to assess for observer variability with the APdmax and 3DLV measurements. Cohort 2 highlighted the relationship between APdmax and 3DLV and changes in AAA detected. RESULTS: There was no significant observer variability detected with APdmax measurement. Similarly, no significant intraobserver variability was evident with 3DLV; however, a small but significant interobserver difference was present. APdmax and 3DLV measurements of PPE AAA significantly correlated. However, changes in the AAA morphology were detected earlier with 3DLV. CONCLUSION: APdmax and 3DLV are both reliable methods for measuring an AAA. Both these methods correlate with each other. However, changes in AAA morphology were detected earlier with 3DLV, which is important to detect subtle but important changes to aortic geometry in a laboratory setting. 3DLV measurement of AAA is a simple, reproducible, and comprehensive method for assessing changes in disease morphology.

Thermodynamics of semi-specific ligand recognition: the binding of dipeptides to the E.coli dipeptide binding protein DppA.

This investigation of the temperature dependence of DppA interactions with a subset of three dipeptides (AA. AF and FA) by isothermal titration calorimetry has revealed the negative heat capacity ([Formula: see text]) that is a characteristic of hydrophobic interactions. The observation of enthalpy-entropy compensation is interpreted in terms of the increased structuring of water molecules trapped in a hydrophobic environment, the enthalpic energy gain from which is automatically countered by the entropy decrease associated with consequent loss of water structure flexibility. Specificity for dipeptides stems from appropriate spacing of designated DppA aspartate and arginine residues for electrostatic interaction with the terminal amino and carboxyl groups of a dipeptide, after which the binding pocket closes to become completely isolated from the aqueous environment. Any differences in chemical reactivity of the dipeptide sidechains are thereby modulated by their occurrence in a hydrophobic environment where changes in the structural state of entrapped water molecules give rise to the phenomenon of enthalpy-entropy compensation. The consequent minimization of differences in the value of ΔG0 for all DppA-dipeptide interactions thus provides thermodynamic insight into the biological role of DppA as a transporter of all dipeptides across the periplasmic membrane.

Quantifying the concentration dependence of sedimentation coefficients for globular macromolecules: a continuing age-old problem.

This retrospective investigation has established that the early theoretical attempts to directly incorporate the consequences of radial dilution into expressions for variation of the sedimentation coefficient as a function of the loading concentration in sedimentation velocity experiments require concentration distributions exhibiting far greater precision than that achieved by the optical systems of past and current analytical ultracentrifuges. In terms of current methods of sedimentation coefficient measurement, until such improvement is made, the simplest procedure for quantifying linear s-c dependence (or linear concentration dependence of 1/s) for dilute systems therefore entails consideration of the sedimentation coefficient obtained by standard c(s), g*(s) or G(s) analysis) as an average parameter ( s ¯ ) that pertains to the corresponding mean plateau concentration (following radial dilution) ( c ¯ ) over the range of sedimentation velocity distributions used for the determination of s ¯ . The relation of this with current descriptions of the concentration dependence of the sedimentation and translational diffusion coefficients is considered, together with a suggestion for the necessary improvement in the optical system.

The abdominal waist circumference and 4-year outcomes following peripheral bypass grafting.

BACKGROUND: Current literature evaluating the relationship between obesity, utilizing measures other than the Body Mass Index (BMI), and postoperative outcomes following vascular surgery are sparse. This study aimed to investigate any association between abdominal waist circumference (AWC) and waist-hip ratio (WHR) in relation to postoperative morbidity and mortality following peripheral artery bypass graft (PABG) surgery. METHODS: AWC and hip circumference (HC) were measured from pre-intervention magnetic resonance (MR) and computed tomography (CT) scans of patients undergoing elective and nonelective PABG. The AWC and WHR were assessed in relation to: the need for higher level care (i.e. level 2/3), the duration of higher level care, postoperative limb ischemia, postoperative hospital stay, graft patency on discharge and 30 day readmission, using logistic and linear regression analysis. Mortality was assessed using cox-regression analysis with calculation of hazard ratios at 30 days and 4 years. RESULTS: In total, 177 patient images performed between January 2014 to January 2017 were analyzed. There were no significant intra-observer and interobserver differences in measurements of AWC and HC. Pre-intervention AWC was predictive of the need for higher level care following non-elective PABG (adjusted OR 1.1 [95% CI: 1.0-1.1, P=0.026]). An inverse relationship between AWC and mortality at 4 years was also observed (adjusted HR=0.9, 95% CI: 0.9-1.0, P=0.028). However, pre-intervention WHR failed to predict mortality and morbidity. CONCLUSIONS: AWC may potentially be a suitable risk stratification tool in patients undergoing non-elective PABG. The association of AWC with long-term mortality outcomes require further investigation so that suitable cut-off values may be determined.

Morphometric and traditional frailty assessment in transcatheter aortic valve implantation.

OBJECTIVES: Frailty is common amongst patients undergoing transcatheter aortic valve implantation (TAVI). The aim of this study was to determine the prognostic relevance of newer objective and traditional measures of frailty after TAVI. METHODS: Consecutive patients were identified from the Leeds Teaching Hospitals Trust TAVI database. Frailty was quantified objectively by measuring the total psoas muscle area (TPMA) on routine computer tomography scans and compared against Canadian Study of Health and Aging Clinical Frailty Score, Katz Index of independence in activities of daily living and Clinician Estimated Poor Mobility. Postintervention morbidity and mortality were examined between these scoring systems. RESULTS: The current study included 420 patients who had undergone TAVI between January 2013 and December 2015. Median clinical follow-up was 4.0 years (interquartile range 2.9-5.0). Standardized measurements of the TPMA were not associated with either postintervention morbidity or mortality. Only the Canadian Study of Health and Aging Clinical Frailty Score was associated with hospital stay (adjusted regression coefficient 0.70, 95% confidence interval 0.04-1.36, P = 0.038) and overall all-cause mortality (adjusted regression coefficient 1.26, 95% confidence interval 1.05-1.50, P = 0.013). There were no significant correlations between TPMA and any of the traditional frailty tools. CONCLUSION: We demonstrate TPMA to be a poor measure of patient frailty when compared with traditional methods of assessment which failed to predict postintervention outcomes. Furthermore, morphometric sarcopaenia correlated poorly with established measures of frailty.

Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended ß-sheet conformation.

BACKGROUND: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3'-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. METHODS: Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. RESULTS: AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed ß-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14-3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. CONCLUSIONS: This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection. SIGNIFICANCE: The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14-3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14-3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy.

Experimental determination of second virial coefficients by small-angle X-ray scattering: a problem revisited.

This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions-the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20-100 g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein-cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory [Formula: see text] still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source-protein self-interactions and/or protein-cosolute contributions.

Structure-Guided Enhancement of Selectivity of Chemical Probe Inhibitors Targeting Bacterial Seryl-tRNA Synthetase.

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.

Use of molecular crowding for the detection of protein self-association by size-exclusion chromatography.

The feasibility of employing molecular crowding cosolutes to facilitate the detection of protein self-association by zonal size exclusion chromatography is investigated. Theoretical considerations have established that although the cosolute-induced displacement of a self-association equilibrium towards the oligomeric state invariably occurs in the mobile phase of the column, that displacement is only manifested as a decreased protein elution volume for cosolutes sufficiently small to partition between the mobile and stationary phases. Indeed, the use of a crowding agent sufficiently large to be confined to the mobile phase gives rise to an increased elution volume that could be misconstrued as evidence of cosolute-induced protein dissociation. Those theoretical considerations are reinforced by experimental studies of α-chymotrypsin (a reversibly dimerizing enzyme) on Superdex 200. The use of cosolutes such as sucrose and small polyethylene glycol fractions such as PEG-2000 is therefore recommended for the detection of protein self-association by molecular crowding effects in size exclusion chromatography.