Interactions of endogenous ligands with human serum albumin

Location of High and Low Affinity Fatty Acid Binding Sites on Human Serum Albumin Revealed by NMR Drug-competition Analysis

Molecular Docking (Mol.dock) of resorcinol based acridinedione dyes (ADR1 and ADR2) with a globular protein, Human Serum Albumin (HSA) were carried out. Docking studies reveal that ADR2 dye binding with HSA is energetically more stable and feasible than ADR1 dye. ADR1 dye predominantly resides in site I and III of HSA rather than binding site II wherein, ADR1 dye acts as hydrogen bonding (HB) acceptor through its carbonyl oxygen. On the contrary, ADR2 dye resides in all the binding sites of HSA such that the dye acts as the HB donor through the NH hydrogen atom and the carbonyl oxygen of the amino acid acts as the HB acceptor. The stability of dye-protein complex in the presence of several non-steroidal anti-inflammatory drugs (NSAIDs) was carried out by employing specific site selective drugs (Sudlow binding site drugs). The energetics and the bimolecular interactions of various drugs with ADR1-HSA and ADR2-HSA were generated to ascertain the influence of drug and its governance on the binding affinity of dye-protein complex. Sudlow site I binding drugs were effective in decreasing the energetics of ADR1 dye-HSA complex whereas site II binding drugs predominantly decreases the affinity of ADR2 dye with HSA. However, the dyes efficiently displaces the site specific drugs from their specific binding sites of HSA which was not observed in the case of drugs on the displacing ability over dyes situated in different domains of protein. Mol.dock studies are employed as an authentic, reliable and most effective tool to ascertain the binding stability of host–guest complex as well as to ascertain the most probable location of several competing ligands in various domains of HSA.

Protein G is a streptococcal cell wall protein with separate and repetitively arranged binding domains for immunoglobulin G (IgG) and human serum albumin (HSA). In this work, the binding of protein G to HSA was studied. The results suggest that a single binding site is present on HSA: the apparent size of the HSA-protein G complex (230 kDa) corresponded to two or three HSA molecules bound to one protein G molecule, and Ouchterlony immunodiffusion did not yield any precipitate between protein G and HSA. HSA was cleaved by pepsin and CNBr into several fragments which were identified by SDS-PAGE and N-terminal amino acid sequencing, and the binding of protein G to the fragments was studied in Western blot experiments. The results indicated that the binding area was located in disulfide loops 6-8, involving both the second (loop 6) and the third (loops 7 and 8) domain of HSA. One of the protein G binding pepsin fragments, with an apparent molecular mass of 5.5 kDa, located in loops 7 and 8, was isolated and found to completely inhibit the binding between protein G and the intact HSA, again suggesting a single protein G binding site on serum albumin. Reducing the disulfide bonds of HSA, and subsequent alkylation of the half-cystine residues, significantly decreased the affinity for protein G. Protein G bound to albumin from baboon, cat, guinea pig, hamster, hen, horse, man, mouse, and rat, but not to albumin from cow, dog, goat, pig, rabbit, sheep, snake, or turkey.

A wide variety of drugs bind to human serum albumin (HSA) at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand) and ibuprofen (site II ligand) at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole) to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a ‘competitive-like’ allosteric interaction in the case of the defatted HSA conformer to a ‘nearly independent’ binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a ‘competitive-like’ allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to ‘nearly independent’ binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

Identifying Steroid Hormone Binding Sites on Human Serum Albumin by 2D NMR

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[(13)C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-β-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-β-cyclodextrin extracted OA from individual sites in the reverse order of filling. FA bound in three low-affinity sites were displaced by drugs shown to bind in crystalline HSA to FA sites 7 and 3 (Sudlow's drug sites I and II, respectively) and FA site 6. With this strategy, 2D NMR spectral analysis permits site-specific characterization of the binding of drugs and FA and provides a sensitive probe of the mutual effects of FA and ligand binding.

Electron paramagnetic resonance spectroscopy quantitatively correlates structural damages of serum albumin with COVID-19 severity and mortality thus suggesting albumin replacement therapy as a strategy to rescue patients at risk of mortality.

Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors’ HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors' HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress. Editor's evaluation This submission is novel since it provides information on the structure changes of albumin in COVID-19. https://doi.org/10.7554/eLife.69417.sa0 Decision letter Reviews on Sciety eLife's review process Introduction COVID-19 pandemic continues as a global health crisis while the underlying SARS-CoV-2 virus defies all attempted treatment strategies. While writing this report, there have been more than 135 million confirmed cases including around 3 million deaths worldwide according to the World Health Organization Coronavirus Disease Dashboard (https://covid19.who.int/). Although 50% of cases are reported to be in the 25–64 age group, the percentage of deaths increases dramatically with age, and approximately 75% of deaths are in those aged 65 years and above (COVID-19 Hospitalization and Death by Age | CDC). People in the age groups 30–39 years, 40–49 years, and 50–64 years are 4, 10, and 30 times more likely to die from COVID-19 complications compared to the 18–29 years age group. Nevertheless, molecular and cellular factors contributing to mortality outcome in a homogeneous cohort of patients are not yet clear. Lack of diagnostic markers that predict mortality in COVID-19 patients impedes current efforts to siege the pandemic. It is thus critical to identify prognostic tests that can assess the risk of death in critically ill patients to guide clinical protocols and prioritize interventions. Furthermore, mechanistic clues for determining the underlying molecular factors contributing to the hypercoagulability, inflammation, and cytokine storm have been so far illusive. It is therefore imperative to intensify efforts focusing on understanding the molecular pathophysiology of COVID-19 infection and to identify prognostic markers to guide and prioritize clinical decisions. Human serum albumin (HSA) is the most abundant constituent of soluble proteins in the circulatory system. HSA has been suggested and used as a diagnostic and prognostic marker of numerous diseases and conditions including ischemia, rheumatoid arthritis, cancer, septic shock, among many others. In addition to its numerous physiological and pharmacological functions including the maintenance of blood/tissue osmotic balance (Singh-Zocchi et al., 1999), blood pH, metal cation transport and homeostasis (Bal et al., 2013; Stewart et al., 2003), nutrients and drug shuttling (Fujiwara and Amisaki, 2013; Wishart et al., 2018), and toxin neutralization (Ascenzi et al., 2006; Vorum and Honoré, 1996), HSA is suggested to be a major circulating antioxidant (Cha and Kim, 1996; Loban et al., 1997). HSA can remarkably bind with a diverse array of drugs and toxins thus controlling their bioavailability and pharmacologic effects (Fasano et al., 2005). It has been previously shown that more than 70% of the free radical-trapping capacity of serum was due to HSA (reviewed in Roche et al., 2008). Importantly, several reports indicated that inflammation enhances vascular permeability of various tissues to HSA apparently to confer antioxidant beneficial effects against reactive species released by activated neutrophils (Cross et al., 1994; Halliwell, 1988; Sitar et al., 2013). Although currently without direct experimental evidence, neutrophilia-mediated oxidative stress was implicated in the COVID-19 pathology and speculated to exacerbate the inflammatory immune response eventually causing multi-organ failure and death (Laforge et al., 2020). We hypothesized that COVID-19-mediated oxidative stress may be differentially reflected in HSA's structure and functions and employed electron paramagnetic resonance (EPR) spin labeling spectroscopy to explore HSA's structural changes in correlation with severity and mortality of critically ill COVID-19 patients. Spin-labeled fatty acids (SLFAs) are established probes to explore structural and functional changes in albumin by EPR spectroscopy (Ge et al., 1990; Haeri et al., 2019). This approach relies on the well-studied ability of albumin to strongly and exclusively bind with fatty acids in blood. Albumin has at least seven different specific binding sites for long-chain fatty acids located in different domains within the protein (Bhattacharya et al., 2000; Curry et al., 1999; Simard et al., 2006). Effectively, structural and functional changes in HSA may be assessed through the detection of parallel changes in mobility and binding affinity of SLFAs, in addition to the distribution of the spin labels on the albumin molecule (Haeri et al., 2019). EPR spectra of spin labels bound to different domains of the protein provide information on the local fatty acids/protein interactions, which may probe changes in the overall structure of the protein under unfolding or damaging conditions (Figure 1A; Bhattacharya et al., 2000). Here, we compare changes that occur to the mobility, binding affinity, and distribution of the HSA-bound SLFA in whole blood and plasma from COVID-19 patients in critical care unit relative to those observed in normal healthy individuals. Figure 1 Download asset Open asset Probing structual changes of serum albumin through spin labeling EPR spectroscopy. (A) HSA crystal structure containing seven copies of stearic acid. (B) Representative EPR spectra of free and HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood from the same COVID-19 recovered patient. Chemical structures of the two spin-labeled fatty acids are given on the right side of the figure. EPR, electron paramagnetic resonance; HSA, human serum albumin. Results Demographic, clinical, and laboratory hematologic characteristics of COVID-19 patients Table 1 lists demographic data, comorbidities, ongoing medications, and administered anti-COVID-19 medications applied to treat current study participants that were divided into survivors (Sev-R) and deceased (Sev-D). No clinical or demographic characteristic showed statistically significant difference between Sev-R and Sev-D groups when analyzed by Pearson's Chi-square test. In Table 2, we show and statistically compare laboratory results of survivors versus non-survivor COVID-19 groups. Although when comparing all parameters in the two COVID-19 groups, we observed changes following the same reported trends in the literature, means' comparisons by Tukey test reported non-significant changes in all parameters except for a significant decrease in albumin level (p<0.05) and a strong trend observed for C-reactive protein (CRP) (greater levels in Sev-D group, p=0.06). Nevertheless, non-survivors' blood carried the frequently observed hallmarks of increased CRP, D-dimer, IL-6, ferritin, and the liver enzymes ALT and AST (reviewed in: Singh et al., 2021; Velavan and Meyer, 2020). However, it is conceivable that the clinical severe category and the same ICU status of patients in the two groups in addition to relatively small sample sizes underlie the observed lack of robust statistical differences between these parameters. Table 1 Demographic and clinical characteristics of the studied subjects. Sev-RSev-DTukey 95% CIpn1623Age (mean ± SD)60.7±9.567.8±13.24.1–17.80.09Male56.25%63.16%0.677†sO2 (mean ± SD)82.1±18.776.1±18.6–20.5 to 8.40.40Hypertension12.5%42.1%0.053†Diabetes25%75%0.08†Cardiovascular disease0%15.8%0.10†Cancer0%10.5%0.18†Bronchial asthma6.25%10.5%0.65†ACE inhibitors0%7.14%0.47†ARBs9.09%7.14%0.85†calcium channel blocker14.28%7.14%0.60†Beta blockers0%7.14%0.47†Diuretics0%7.14%0.47†Sulphonylurea14.28%21.43%0.69†Other oral hypoglycemic0%21.43%0.18†Insulin42.85%21.43%0.30†Anticoagulant57.14%50.0%0.76†Steroids71.42%64.28%0.74†Hydroxychloro-quine14.28%7.14%0.60†IL-6 receptor antibody28.57%21.42%0.72†Proton-pump inhibitor28.57%42.85%0.52†Azithromycin14.28%28.57%0.47†Cephalosporin42.85%21.43%0.30†Carbapenem42.85%42.85%1.0†Oxazolidinone42.85%28.57%0.51†Fluoro-quinolone42.85%21.43%0.30†Nitrofuran14.28%0%0.15†Remdesivir14.28%28.57%0.47†Ivermectin0%28.57%0.11† sO2, blood oxygen saturation level; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blocker; IL-6, interleukin-6. † p values obtained through Pearson's χ2 test. Table 2 Laboratory parameters of the current study patients. WBC, white blood cell; INR, international normalized ratio; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; ALT, alanine transaminase; AST, aspartate transaminase. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Sev-R (mean ± SD)Sev-D (mean ± SD)Tukey 95% CIpWBCs (×103 /ml)10.6±4.013.9±8.0–1.47 to 8.060.17Platelets (×106 /ml)260±75.5213.7±115.8–116.7 to 24.20.19INR1.29±0.561.23±0.23–0.38 to 0.260.69CRP (mg/L)51.19±54.4103.77±86.3–2.35 to 107.50.06D-dimer (mg/ml)1.47±1.93.17±3.56–0.55 to 3.960.13IL-6 (pg/ml)314.1±527325.3±619–591 to 6140.97Ferritin922.6±5651078±578–281 to 5940.47Albumin (g/ml)31.47±7.9526.97±5.1–8.7 to –0.260.038Hemoglobin (g/dl)12.26±2.012.16±2.0–1.53 to –1.330.97ALT (U/L)33.64±24.1546.5±37.2–10.37 to 36.13 ALT, alanine transaminase; AST, aspartate transaminase; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; INR, international normalized ratio; WBC, white blood cell. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Neutrophils are a major source of reactive oxygen species It has been recently proposed that the high neutrophil-to-lymphocyte ratio (NLR) observed in critically ill COVID-19 patients may tip the redox homeostasis due to increased reactive oxygen species (ROS) production (Laforge et al., 2020). Our hypothesis implicates elevated oxidative stress as a major cause of HSA damage in severe COVID-19 patients. As a result, we started by following the dependence of clinical outcomes and mortality on ROS levels in blood cells. First, we used flow cytometry to assess percentages of neutrophils, lymphocytes, and platelets in all patients as described in Materials and methods (Figure 2A). Furthermore, we used the ROS-sensitive DCF dye to probe intracellular ROS levels in various cell populations in whole blood from all groups. Figure 2 shows that while lymphocyte counts decrease, a parallel dramatic increase in neutrophil counts (% total) was observable when going from Control (40.78±14.0, n=9) to Sev-R (64.0±20.0, n=10) to Sev-D (76.4± 6.8, n=11) groups (overall ANOVA p=3.9×10–5). Similar trend was clearly seen in the heat map depicting parameters for all patients analyzed by flow cytometry (Figure 2B). It is also clear from Figure 2B&C that changes in DCF-positive neutrophils follow similar trend observed for neutrophil counts. To confirm this relation, we compared neutrophil counts with DCF-positive neutrophil counts and found that the two parameters were strongly correlated (Pearson's r=0.8, p=3×10–7; Figure 2C). Moreover, both parameters individually showed statistically significant increases in both of the studied COVID-19 groups when compared with the control group (Figure 2D&E). These results suggest that neutrophils are major sources of elevated oxidative stress in critically ill patients. Note that the observed trends in platelets, lymphocyte, neutrophils, and NLR are similar to reported values (Sun et al., 2020; Yang et al., 2020). Figure 2 Download asset Open asset Hematologic cellular counts and neutrophil-ROS levels reflect severity and mortality in COVID-19 patients. (A) Representative flow cytometric diagrams comparing morphologic, hematologic, and ROS levels in control (representative of n=9; upper row), Sev-R (representative of n=10; middle row), and Sev-D (representative of n=11; lower row) groups. (B) Heat diagram comparing lymphocyte, neutrophils, platelets, and DCF-positive neutrophil counts as the percentage of total cell counts in all of the studied subjects. Yellow areas are either group separators or missing data due to insufficient sample size or processing errors. (C) A diagram showing statistically positive correlation between neutrophil count and count of neutrophils stained positive for DCF dye in all groups (black dots denote controls; blue are Sev-R; and red represent Sev-D patients). (D) When neutrophil counts were compared for all groups, both Sev-R and Sev-D groups showed statistically significant neutrophilia relative to control groups. However, only a weak trend has been observed when comparing the two groups with COVID-19. (E) DCF staining revealed increased levels of ROS in Sev-R and Sev-D groups relative to control neutrophils. Sev-D showed a trend of increased ROS level relative to Sev-R group. Multiple comparisons were carried out using ANOVA followed by Tukey test and p values are given. ROS, reactive oxygen species. Figure 2—source data 1 Raw source data for Figure 2B-E. https://cdn.elifesciences.org/articles/69417/elife-69417-fig2-data1-v2.xlsx Download elife-69417-fig2-data1-v2.xlsx Hydrogen peroxide levels in plasma correlate with mortality Next, we reasoned that elevated oxidative stress in both groups with critical COVID-19 infection would be echoed in plasma levels of hydrogen peroxide. Hydrogen peroxide is the most stable ROS and is highly stable under prolonged storage at low temperatures. We used a highly specific catalase-based assay that we developed and verified in our laboratory to quantify [H2O2] in plasma samples of all groups. The assay relies on high-resolution detection and quantification of released oxygen due to hydrogen peroxide decompostion by catalase (Figure 3A&B). We constructed a calibration curve to confirm the catalase-mediated H2O2 to O2 stoichiometric conversion (Figure 3B). A linear relation was obtained with zero intercept and slope of 0.47±0.03 which closely matches the theoretically expected value of 0.5 (95% confidence interval [CI]: 0.37‒0.56, p=5.6×10–4, Pearson's r=0.994). Indeed, we detected striking differences between groups even with relatively small sample sizes (Mean ± SD, Control, n=11: 2.95±0.77, Sev-R, n=16: 7.21±2.4, Sev-D, n=23: 9.67±2.0; overall ANOVA p=2.6×10–11; Figure 3C). The differences between groups have reached statistical significance (Sev-R vs. Control, 95% CI: 2.37‒6.13, p=4.9×10–6; Sev-D vs. Control, 95% CI: 4.95‒8.48, p=0.0; Sev-D vs. Sev-R, 95% CI: 0.90‒4.03, p=0.001). It appears from these results that a measure of oxidative stress, that is, [H2O2] in plasma, is doubled in survivors and tripled in deceased COVID-19 patients relative to controls' plasma average levels. Figure 3 Download asset Open asset Hydrogen peroxide levels in plasma and neutrophils reflect mortality in COVID-19 patients. Catalase was used to specifically and quantitatively determine levels of hydrogen peroxide in identical plasma volumes collected from control (n=11), Sev-R (n=16), and Sev-D (n=23) groups. (A) Oxygen levels are monitored and recorded while 50 μl batches of plasma from control, Sev-R, and Sev-D subjects are sequentially infused into tightly air-controlled O2k chamber containing catalase (315 units/ml) in deoxygenated buffer. In addition to the initial rise due to residual oxygen in the added plasma samples, the decomposition of hydrogen peroxide in these samples produces oxygen quantitatively. (B) To verify the assay we measured the released oxygen upon adding an increasing volume of standard hydrogen peroxide solution in PBS buffer with 0.2, 0.8, 1.2, and 1.6 μM final concentrations; inset. Linear fitting of the plotted [O2] versus [H2O2] relation yielded a slope=0.47±0.03 (Pearson's r=0.994, p=5.6×10–4), which is very close to the theoretically expected value of 0.5 as the catalase-mediated decomposition of one mole of H2O2 produces ½-mole O2. (C) Plasma contents of H2O2 in plasma significantly increased in the order Sev-D>Sev-R>Cont using ANOVA followed by Tukey test applied on n=11, 16, and 23 for control, survivors, and non-survivors, respectively. (D) Fluorescence imaging was used to assess levels of ROS in freshly isolated neutrophils using DCF (2,7-Dichlorodihydrofluorescein diacetate, green) staining in all groups. Hoechst binds strongly to adenine–thymine-rich regions in DNA thus mapping nuclei through emitting blue fluorescence. Merged DCF and Hoechst images are shown in the third column. Images were acquired using Cytation 5 Cell Imaging Multi-Mode Reader (Agilent) and analyzed using Gen5 Software package 3.08. Scale bar: 100 µm. Figure 3—source data 1 Raw polarographic data for released oxygen (A), calibration curve (B), and calculated plasma hydrogen peroxide levels in all groups (C). https://cdn.elifesciences.org/articles/69417/elife-69417-fig3-data1-v2.xlsx Download elife-69417-fig3-data1-v2.xlsx To confirm this finding, we performed DCF fluorescence imaging on freshly isolated neutrophils of representative group of individuals from each group (Figure 3D). We simultaneously stained neutrophils' nuclei with Hoechst 33342 (blue stain) to follow nuclear morphologic changes and DNA diffusion in all groups. Although requiring more detailed studies, a closer look at the acquired images of Hoechst-stained neutrophils from a survivor patient showed significantly reduced average neutrophil size DNA vs. Sev-R DNA two samples with more more nuclei and However, neutrophils from a non-survivor and in a DNA than normal and times that of Sev-R neutrophils vs. both control and Sev-R of the DCF fluorescence images indicated that the neutrophils populations of appears to be and that are highly of mean DCF fluorescence cell confirmed results obtained by flow cytometry and catalase assay increased levels of ROS in the order ± Control Sev-R Sev-D two samples for all parameters reflecting albumin changes are of COVID-19 mortality It has been previously shown that non-survivor COVID-19 patients relative to survivors et al., 2020). We started by albumin levels in the studied cohort of subjects to confirm follow similar We found that in plasma in the order (Mean ± SD, Control, Sev-R, n=16: Sev-D, n=23: overall ANOVA Figure We detected statistically significant decrease in in plasma of Sev-R and Sev-D groups (Sev-R vs. Control, 95% CI: to Sev-D vs. Control, 95% CI: to Sev-D vs. Sev-D, 95% CI: to The of HSA in serum is approximately we and groups found that COVID-19 mortality with et al., 2020). However, high of in numerous and the of this protein its diagnostic and As a result, we parameters to HSA protein in whole blood and plasma of all groups as of this critical protein functions. Figure Download asset Open asset EPR of binding strong dependence of binding on mortality in COVID-19 patients. (A) Albumin level in plasma of control Sev-R (n=16), and Sev-D groups showed that both survivors and COVID-19 patients statistically significant between representative spectra showing changes in that are to mobility and of HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood of a control (black a Sev-R (blue and a Sev-D patients. parameters including order rotational correlation and the ratio between strongly bound to bound spin labels as in Figure 1 and described in Materials and methods comparisons by ANOVA followed by Tukey tests were used for means' comparisons and revealed decrease in the binding and packing of the local the spin calculated parameters with p values are given in the into acids binding are followed by with which into the EPR Representative EPR of 16-DSA in whole blood of control and Sev-D are showing the of 16-DSA and 5-DSA bound to HSA by in whole blood. are shown as the percentage of the of the middle samples spin and 3 and measured at and of the EPR the of the spin EPR, electron paramagnetic resonance; HSA, human serum albumin. Figure data 1 and EPR parameters used for statistical Download reported that changes in HSA may be to reflect critical functional changes in albumin and diagnostic and prognostic values of these changes in (Haeri et al., et al., 2006). It has also been found that long-chain fatty acids binding the binding of in the two major drug binding sites of HSA et al., We employed EPR spectroscopy to probe binding and protein in all groups as detailed in the Materials and methods of the 5-DSA and 16-DSA EPR spectra revealed changes in between control and COVID-19 groups (Figure and Figure Figure 4, 16-DSA in whole the in Figure and used to the protein packing order is to effects as interactions, on the spin probe that is in one of the HSA fatty acids binding the S/W ratio Figure to strongly bound populations of 16-DSA spin probe may reflect changes in protein that can Furthermore, the rotational correlation which is a measure of the spin probe rotational mobility is also Figure 5 Download asset Open asset of HSA transport function of HSA is assessed through the of fatty by following the rise in both strongly (A) and (B) bound SLFA with blood from representative subjects for each These results the fatty by HSA of critically ill patients relative to To the function of HSA, increasing volumes of were added to identical of all groups and the EPR spectra were acquired (C) to follow (D) and strongly (E) bound populations of of the fatty populations are through and increased of free fatty This is remarkably in critically ill patients reflecting and of fatty acids from HSA in those patients. HSA, human serum spin-labeled fatty acid. EPR parameters for all groups are in 1 including ANOVA and Tukey test p values with the of subjects 5-DSA and 16-DSA were used to probe the of local in sites the SLFA is in the protein mobility, and closer to the mobility due to with and and Indeed, 5-DSA reflected significantly values relative to 16-DSA both in plasma and in whole blood (Figure However, of the spin probe and both in plasma and whole blood samples, the order has been lower in Sev-R which was in Sev-D patients relative to the control group (Figure In whole similar results that showed more statistically robust differences have been of as described in Materials and methods showed rotational mobility of 16-DSA is significantly faster when bound with HSA from COVID-19 patients relative to that from control subjects in plasma or whole blood. similar trends have been observed for the S/W which of the strongly and bound of 16-DSA in different fatty acids these results that COVID-19 pathology is with structural changes in the HSA protein that the of of this critical into in whole blood of COVID-19 patients We followed the spin labels through analysis of the EPR by the et al., Figure experimental 16-DSA and in whole blood of control subjects remarkably faster when compared with both Sev-R and Sev-D group, and of the EPR of COVID-19 patients by the of the spin within the It is clear from these data that the HSA of COVID-19 patients is relative to control However, the of the HSA of both COVID-19 groups was not significantly different in of transport function of HSA in critically ill COVID-19 patients in severe patients relative to (Figure with the observed significant changes in the are to functional damage and 1990; et al., et al., et al., et al., In this changes in the are to functional changes in the solution of albumin due to of paramagnetic in

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75)demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Mapping the Interactions between the Alzheimer’s Aβ-Peptide and Human Serum Albumin beyond Domain Resolution

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.

High-field electron paramagnetic resonance (EPR) experiments to monitor binding of lipophilic Gd(III) complexes to human serum albumin (HSA) are described. It was observed that magnetic interactions between the nitroxide moiety ofn-doxyl-stearic acids bound to HSA and Gd(III) complexes resulted in broadening of nitroxide continuous-wave EPR spectra. The broadening effect can be well described by a one-parameter model of additional Lorentzian broadening At 95 GHz, continuous-wave EPR spectra from Gd(III) complexes are fully resolved from the nitroxide signal allowing for simultaneous and independent line shape analysis. Analysis of the line width broadening effects for spectra from a series ofn-doxyl-stearic acids bound to HSA indicated a progressive decrease of spin label-Gd(III) magnetic interactions along the fatty acid (FA) binding channel, consistent with binding of Gd-DOTAP complex in the vicinity of the main FA binding site. The substantial difference in spin label-metal interactions along the FA binding channel for lipophilic Gd(III) complexes with different chelates is indicative of binding to different sites. We also report measurements of dissociation constant for noncovalent binding of Gd(III) complexes to HSA on the basis of analyses of 95 GHz Gd(III) EPR line shapes.