Formation of a bovine serum albumin diligand complex with rutin for the suppression of heme toxicity

Biophysical Chemistry 258 (2020) 106327

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Formation of a bovine serum albumin diligand complex with rutin for the suppression of heme toxicity

T

Mengjuan Luo, Yinhua Sui, Rong Tian, Naihao Lu

⁎

Key Laboratory of Functional Small Organic Molecule, Ministry of Education; Key Laboratory of Green Chemistry, Jiangxi Province and College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, China

HIGHLIGHTS

GRAPHICAL ABSTRACT

complex was formed • BSA-diligand after the simultaneous addition of heme and rutin.

rutin in the BSA-diligand • Bioactive complex still kept strong antioxidant activity.

cytotoxicity of heme was de• The creased in the BSA-diligand complex. BSA-based carriers of • Designing bioactive compounds for biomedical applications was potential.

ARTICLE INFO

ABSTRACT

Keywords: Bovine serum albumin Rutin Heme Cytotoxicity

Serum albumin binds avidly to heme to form heme–serum albumin complex and can protect against the potentially toxic effects of heme. Rutin is a glycoside of the bioflavonoid quercetin with various protective effects due to its antioxidant ability. Clarification of the interaction mechanisms between serum albumin and bioactive components (such as heme and flavonoid) is important to develop effective carriers for encapsulation of heme and suppression of its toxicity. In this study, bindings of bovine serum albumin (BSA) to heme and/or rutin were investigated by experimental and molecular docking techniques. The fluorescence of BSA was quenched by both heme and rutin in static mode (i.e. formation of BSA-monoligand complexes), which was confirmed by Stern-Volmer calculations. Although heme showed higher affinity to BSA than rutin, the interactions of both components with BSA did locate within subdomain IIA (site I). BSA-diligand complexes were successfully formed after the simultaneous addition of heme and rutin. Bioactive rutin in the BSA-diligand complex still kept strong free radical scavenging activity compared to free rutin or BSA-monoligand complex. Hydrogen peroxide (H2O2)-induced heme degradation and free iron release was inhibited upon BSA binding and further decreased in BSA-diligand complexes. Consistently, the cytotoxicity of heme and oxidative stress in endothelial cells was decreased in the BSA-diligand complexes relative to those of heme or BSAheme complex, where the co-presence of rutin played an important role. These results suggest the possibility and advantage of developing BSA-based carriers for the suppression of heme toxicity in their biomedical applications.

⁎

Corresponding author. E-mail address: [email protected] (N. Lu).

https://doi.org/10.1016/j.bpc.2020.106327 Received 10 November 2019; Received in revised form 2 January 2020; Accepted 5 January 2020 Available online 07 January 2020 0301-4622/ © 2020 Elsevier B.V. All rights reserved.

Biophysical Chemistry 258 (2020) 106327

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1. Introduction

played an important role in suppressing the cytotoxicity of heme if a rutin-protein-heme complex could be generated. In this study, the generation of BSA-diligand complexes with rutin and heme was investigated. The inhibitive effect of bioactive rutin in BSA-diligand complexes on the cytotoxicity of heme was also investigated.

Free heme is mainly released from hemoglobin in severe hemolytic diseases and causes toxic effects [1–3]. Heme has been shown to accelerate protein oxidation, DNA damage and cellular injury. The cytotoxic effects of heme are usually related to the release of free iron and massive formation of highly reactive species by the interaction between extracellular heme and endogenous hydrogen peroxide (H2O2) [1–5]. Heme detoxification serves as a protective mechanism for cell survival in heme-associated disorders and is partly mediated by serum albumin [5–9]. Serum albumin can act as the heme scavenger by forming hemeprotein complex, and this binding is thought to protect against the toxic effects of heme. Serum albumin administration is recommended for the treatment of severe hemolytic disorders [5]. Therefore, the development of novel therapeutic tools and strategies will facilitate the management of heme-related diseases. Serum albumin, the most abundant protein in the blood serum as well as a dietary protein in milk, is a large globular protein having a molecular weight of 66 kDa. The protein functions primarily as a carrier of endogenous and exogenous compounds in the circulatory system [10–12]. Bovine serum albumin (BSA) containing 583 amino acid residues is composed of three homologous domains (I, II and III), each of which contains two subdomains (A and B). The two main binding sites are respectively located in the hydrophobic cavities of the subdomains IIA and IIIA, namely, site I and site II. BSA is able to bind a wide range of biologically active compounds including drugs, natural polyphenols and free heme [7–13]. This makes BSA a protein of great interest as a natural carrier for potential applications in the encapsulation and delivery of bioactive components. Clarification of the interaction mechanisms between serum albumin and bioactive heme is important to develop effective carriers for encapsulation of heme and suppression of its toxicity. Ferric heme binds strongly to human serum albumin (HSA) at a specific binding site and forms a non-toxic heme–HSA complex [5–9]. Serum albumin protects against the toxicity of heme by transferring the free radical to tyrosine residues in albumin, therefore protecting surrounding proteins from irreversible oxidation [5,9]. Besides, heme-serum albumin complex also serves as reactive species scavenger, which can facilitate the isomerization of peroxynitrite to nitrate and thereby protect more important targets [6,8]. As natural antioxidants, plant polyphenols have been widely used to attenuate oxidative damage in vitro and in vivo due to their strong activities in scavenging free radicals and chelating metal ions [14,15]. Quercetin has been reported as the most predominant flavonol and rutin is one of the most common flavonol glycosides in the human diet. The glycoside of quercetin is more abundant and stable than quercetin in medicinal herbs and plant foods, and has been reported to exert numerous biological, medicinal, and pharmacological activities [15–19]. Rutin can also bind to serum albumin with a relative high affinity and form serum albumin-rutin complex [20–22]. Moreover, some flavonoids (baicalin, baicalein) are also found to inhibit hemeinduced cytotoxicity [4]. However, the potential efficiency of flavonoids in the formation of heme-serum albumin complex and detoxification of heme in these complexes were scarcely reported. It is of great interest to develop the carriers that simultaneously contain several bioactive components, thus providing multiple health benefits [11,12]. It is thus necessary to design the effective carriers that can simultaneously encapsulate and protect different bioactive compounds. A multiligand complex of BSA with resveratrol, retinol and (−)-epigallocatechin-3-gallate is successfully generated, and thus the stability of these bioactive molecules is promoted in the complexes relative to free ones [11]. Although the generations of BSA-monoligand complexes are widely investigated [7,8,20–22], the formation of a multiligand complex of BSA with bioactive flavonoid and heme is still limited. Based on the antioxidant property of rutin and potential toxicity of heme, it was proposed that the co-presence of bioactive rutin

2. Materials and methods 2.1. Materials Ferriprotoporphyrin IX (hemin, which is referred to as heme), BSA, rutin (purity ≥95%), glucose oxidase (GO) and 2, 2′-Azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich. Warfarin (purity ≥98%) and ibuprofen (purity ≥98%) were purchased from J&K Scientific. 2.2. Fluorescence measurements Fluorescence intensity was measured by a fluorescence spectrophotometer. The intrinsic fluorescence emission spectra of BSA were recorded from 300 to 500 nm at an excitation wavelength of 280 nm [11,12,22]. Backgrounds of bioactive compounds were subtracted from the raw spectra. Fluorescence intensity was normalized relative to that of BSA at the emission maximum (λmax) in the absence of the second ligand unless indicated otherwise. Quenching of protein fluorescence in the presence of a bioactive component was expressed using the expression [(F0 − FL)/F0 × 100%], FL and F0 were the protein fluorescence measured at λmax in the presence and absence of the component, respectively. A high percentage indicated strong binding of the ligand [11,12]. In the site marker competitive experiments, backgrounds of warfarin and ibuprofen were subtracted from the raw spectra. 2.3. Molecular modeling and docking The 3D structures of rutin and heme were generated using Chemoffice software. Water molecules and ions were removed from the X-ray structure of BSA (PDB ID: 3V03). Rutin and/or heme were docked to the crystal structures of BSA by AutoDock software, as described previously [11,12,23,24]. All other parameters were set as default as defined by AutoDock software. A cubic box was created around protein with 88 × 60 × 74 points, and BSA was placed in the center. From the docking results, the best scoring (i.e., with the lowest docking energy) docked model was chosen to represent the most favorable binding mode. 2.4. Antioxidant activity of rutin The ABTS radical cation decolorization test was used to assess the antioxidant activity of bioactive rutin [25]. Free rutin, rutin with BSA and rutin with BSA-heme complex were incubated in PBS for 6 h. Then, stock solution of ABTS.+ was diluted to an absorbance of 0.7 ± 0.02 at 734 nm, and mixed with the obtained samples for 2 min. The decreased absorbance at 734 nm represented the anti-oxidant activity of tested component. 2.5. Heme depletion and free iron analysis Samples of heme (20 μM) were treated with H2O2 in the presence of rutin in PBS at room temperature (about 25 °C). After 10 min incubation, catalase was added to eliminate excess H2O2. The obtained reaction mixtures were used in latter assays. The relative heme content was determined by its typical Soret peak at 410 nm [26]. Free iron release from heme was measured using ferrozine [27]. 2

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2.6. Cytotoxicity of heme

between a quencher and a fluorophore (static quenching). Fluorescence quenching mechanism is studied by the well-known Stern–Volmer equation [11,12,22,30–33].

In the absence or presence of rutin, human umbilical vein endothelial cells (HUVEC) were cultured with heme, with or without BSAbinding in serum-free medium (containing glucose and/or glucose oxidase, glucose/glucose oxidase (GO) was used for H2O2 generation and similar to physiological circumstance) for 9 h. Then, cell viability was determined by the CCK-8 assay kits (Beyotime Company, China) [28]. As a widely used biomarker of oxidative stress, protein carbonyls were measured by the spectrophotometric method [29].

F0/F = 1 + KSV [Q] where F0 and F are the fluorescence intensities in the absence and presence of the quencher (heme or rutin), [Q] is the concentration of the quencher, and KSV is the Stern–Volmer fluorescence quenching constant. Consistent with previous studies [11,12,20–22], the SternVolmer plots appeared linear for both BSA-heme and BSA-rutin complex at low concentrations (Fig. 1C and D), and the linear Stern-Volmer plots were attributed to static quenching (i.e. compound formation). As shown in Table S1, Fig. 1C and D, the values of the fluorescence quenching constant (KSV) were 0.183 × 106 L mol−1 and 0.033 × 106 L mol−1 for BSA-heme and BSA-rutin complex, respectively. It indicated that KSV value, representing the binding strength of protein on quencher, was in the order BSA-heme > BSA-rutin. However, the Stern-Volmer plots at higher heme concentrations deviated from linearity to denote occurrence of additional quenching mechanisms, which was in accordance with the fact that dynamic collision between components and proteins should occur more frequently at high concentrations [12]. Therefore, the decrease of BSA fluorescence intensity caused by heme or rutin at low concentrations was attributed to static quenching and the formation of protein−ligand complexes, and heme showed higher affinity to BSA than rutin. Thus, the low concentrations of heme or rutin were selected to form the protein−ligand complexes in the following experiments. Warfarin and ibuprofen specifically bind to the subdomain IIA (site I) and the subdomain IIIA (site II) of BSA, respectively, and have been used as site markers to identify the binding of other ligands on serum albumins [11,12,30]. Fig. S1 showed the influence of the two site

3. Results and discussion 3.1. Interaction of BSA with a single bioactive component The bindings of bioactive components to protein were evaluated using the fluorescence quenching method. As shown in Fig. 1 A and B, BSA had an emission maximum (λmax) of 348 nm upon excitation at 280 nm, and the λmax in the case of heme or rutin was not significantly changed. The fluorescence intensity of BSA at λmax was gradually decreased when the concentrations of these components increased, demonstrating the conformational changes of the protein. This was also in accordance with previous studies that the decrease in fluorescence intensity was attributed to the formation of protein−ligand complexes [11,12,20–22]. In addition, fluorescence quenching caused by heme was more significant than that induced by rutin, suggesting that the affinity of bioactive components to BSA was in the order heme > rutin at these concentrations. Furthermore, the mechanisms for BSA fluorescence quenching were investigated. The fluorescence quenching is known to occur mainly by a collisional process (dynamic quenching) and/or formation of a complex

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markers on the fluorescence of BSA bound by heme or rutin. The change in the fluorescence quenching induced by these bioactive compounds was different in the absence and presence of warfarin (Fig. S1A and B), indicating that their binding to BSA did locate within subdomain IIA (site I). As the concentrations of heme or rutin in the warfarin–BSA system gradually increased, the fluorescence intensity decreased and tended to approach the fluorescence intensity of heme or rutin alone (Inset in Fig. S1A − B). This indicated that heme or rutin could replace warfarin binding to BSA at site I and lead to the dissociation of BSA–warfarin. In contrast, the BSA fluorescence quenching induced by heme or rutin was almost similar in the absence and presence of ibuprofen (Fig. S1C and D), suggesting that site II marker ibuprofen did not prevent the binding of ligand to its usual location and subdomain IIIA (site II) was not a main site for the binding of these components. Therefore, site marker competitive experiments illustrated that the binding of heme or rutin to BSA primarily took place in subdomain IIA (site I).

fluorescence by rutin was different in the absence and presence of heme (inset in Fig. S2A), suggesting that addition of rutin had impact on the affinity of heme to BSA. Similarly, rutin-induced protein fluorescence quenching was more significant in the presence of low concentrations of heme (inset in Fig. S2B), suggesting that heme improved the affinity of rutin to BSA or the synergistic effect was present in the combination of different components. These results indicated that the addition of rutin could influence the binding of heme to BSA, and addition of heme also affected the binding of rutin to BSA. 3.3. Formation of BSA-diligand complex The results above demonstrated that the addition of bioactive compounds was important for their binding to BSA. As shown in Fig. 2A, rutin decreased protein fluorescence intensity, and adding heme further decreased fluorescence intensity compared to rutin alone. Then, one question was proposed: Was the higher effect of diligands (heme + rutin) on fluorescence quenching than that of rutin attributed to the synergistic effects of both heme and rutin, or the competitive replacement of rutin by heme? Therefore, the content of BSA-unbound rutin was determined in the absence and presence of heme. After the centrifugation at 4000g for 5 min through Centricon filters (30,000 MW

3.2. Influence of rutin on the binding of heme to BSA Fig. S2A showed fluorescence spectra of BSA as the concentrations of rutin increased in the presence of heme. The quenching of protein

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Fig. 2. (A) Fluorescence spectra of BSA alone and in the presence of rutin and heme. (B) The content of BSA-unbound rutin in filtrate after its binding to BSA in the presence or absence of heme. Rutin alone without BSA binding was taken as control. (Values were means ± S.D. (n = 4), One-way ANOVA was used for statistical analysis, and P < .05 was considered as the statistical significant. *P < .05, groups versus Control group). Molecular modeling demonstrating possible interaction sites on BSA-heme (C), BSA-rutin (D) or BSA-heme-rutin (E) complexes. 4

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cut off, Millipore), the BSA-unbound rutin and heme was removed. The concentration of BSA-unbound rutin in filtrate was not significantly different from that after the addition of heme (Fig. 2B), and therefore rutin was not dissociated (or released) from BSA-rutin complex in the presence of heme. In other words, the addition of heme to BSA-rutin complex did form new complex rather than competitively replace the binding of rutin to BSA. BSA could possibly bind two bioactive components simultaneously to form protein-diligand complexes.

vicinity of subdomain IIA (site I) (Fig. 2C and D), and therefore, the binding sites investigated by molecular docking were in accordance with above site marker competitive experiments (Fig. S1). Second, the corresponding binding free energies were − 11.1 and − 7.9 kcal/mol for BSA-heme and BSA-rutin complex, respectively, and the theoretical result was also consistent with the experiments that heme showed higher affinity to BSA than rutin (Fig. 1). The binding energy for protein-diligand (BSA-heme-rutin) complexes was −11.2 kcal/mol and higher than those for the BSA-monoligand complexes. Thus, the two components could simultaneously bind to BSA with enough affinities. Therefore, the results from molecular docking were consistent with spectroscopic results (Fig. 1, S2, 2A and B), supporting the fact that BSA could bind the two bioactive components to form a stable protein-diligand complex.

3.4. Molecular docking Molecular docking studies were performed using AutoDock software to structurally investigate possible interaction sites between bioactive components and BSA. The predicted best docked conformations with lowest binding energy revealed strong binding of bioactive components to BSA (Fig. 2C-E). BSA contains some hydrophobic cavities as the potential sites for ligand binding [11,12]. The strongest binding site on BSA for heme was surrounded by ASP108, PRO110, ARG144, HIS145, LEU189, SER192, ALA193, ARG196, SER428, LEU454, ILE455 and ARG458; the binding site on BSA for rutin was surrounded by ARG194, ARG217, LYS221, GLU291, VAL292, GLU293, LYS294, PRO338, GLU339, LYS439, PRO446, CYS447, ASP450 and TYR451. All of these residues in the 4 Å binding pocket stabilized the binding site. In accordance with previous studies [11,12,20], electrostatic interaction and hydrogen bond played important roles in the formation of BSA-ligand complex. The binding of heme or rutin to BSA was mainly in the

3.5. Antioxidant activity of rutin Natural flavonoids exhibited strong free radical scavenging activity, which was attributed to its phenolic hydroxyl groups [14,15]. Different from the significant instability of quercetin [12], rutin was stable during storage and/or incubation [30]. The presence of BSA or heme alone did not significantly influence the free radical scavenging ability of bioactive rutin. However, the free radical scavenging ability of BSAdiligand complexes was almost same to that in free rutin and BSA-rutin complex (Fig. 3A). Moreover, the heme-BSA-rutin complex was separated after the centrifugation at 4000 g for 5 min through Centricon

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Fig. 3. (A) ABTS scavenging activities of rutin and rutin bound to BSA in the absence and presence of heme. (BeC) Rutin prevented H2O2-mediated heme depletion and free iron release. Heme (20 μM) was treated with H2O2 in the presence of rutin for 10 min in PBS. (Values were means ± S.D. (n = 4), **P < .01, *P < .05, groups versus H2O2-treated group, P < .01, P < .05, groups versus indicated group). 5

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filters (30,000 MW cut off). The heme-BSA-rutin complex in the concentrated protein solution had more significant antioxidant property than heme-BSA complex (Fig. 3A), further demonstrating the generation of BSA-diligand complex and the high antioxidant ability of this complex. Thus, BSA-diligand complex also kept the antioxidant activity of rutin compared to free rutin or BSA-monoligand complex. Excessive reactive oxygen species such as H2O2 are elevated in many inflammatory diseases and cause the accumulation of free iron [2,4,5]. Through the Fenton reaction, free iron has the ability to generate massive free radicals and subsequently leads to cellular oxidative stress. Fig. 3C and D showed the heme content and free iron release after H2O2 treatment in the presence and absence of BSA or rutin. Similar to previous studies [26], treatment of heme with H2O2 led to a noticeable decrease in the heme content and increase in free iron release. However, both BSA and rutin significantly prevented H2O2mediated heme destruction and free iron release, and the protective effects were more significant in the BSA-diligand complexes (Fig. 3C and D). The protective effects of BSA on H2O2-induced heme destruction were attributed to its encapsulation of heme and reduction of exposed heme. The inhibitory ability of rutin was related to its direct free radical scavenging and iron chelating. Therefore, H2O2-induced heme degradation and free iron release was inhibited upon BSA binding and further decreased in BSA-diligand complexes, demonstrating the antioxidant property of BSA-diligand complexes.

cells through the peroxidase activity and oxidative stress, the binding of BSA to heme would reduce the accessibility of heme to media and cells. Consistent with previous studies [5], heme with protein binding had less chance exposed to media, therefore, oxidative stress (reflected by the contents of protein carbonyls) and cytotoxicity were reduced (Fig. 4A and B). The simultaneous binding of rutin on the cytotoxicity of heme or heme-BSA complex was then investigated. The presence of rutin alone had insignificant influence on cell viability [30], and this compound did suppress cytotoxicity of heme/H2O2 system and oxidative stress (Fig. 4A and B), which was attributed to the antioxidant ability of bioactive rutin such as free radical scavenging and iron chelating. Moreover, the simultaneous presence of this compound further decreased cytotoxicity of BSA-heme complex (Fig. 4A). In other words, the binding of rutin further suppressed the cytotoxicity of heme when the carrier protein (BSA) was coexisting, which was related to that bioactive rutin in the BSA-diligand complexes still kept strong free radical scavenging activity compared to free flavonoid. Consistent with the high antioxidant activity of bioactive rutin in BSA-diligand complexes (Fig. 3A) and lower cytotoxicity of these complexes (Fig. 4A), oxidative stress in BSA-heme-treated cells was further reduced during the simultaneous presence of rutin (Fig. 4B). These results demonstrated that BSA-diligand complexes provided a better inhibition on the cytotoxicity of heme than did BSA-monoligand complexes, in which the simultaneous presence of rutin in the complexes played an important role.

3.6. Cytotoxicity of heme 4. Conclusion

Heme is high toxic to different cells and leads to serious pathological consequences. It has been reported that serum albumin can act as the heme scavenger by forming heme-serum albumin complex and reduces its toxic effects [5–9]. To mimic H2O2 formation in vivo, we used glucose oxidase (GO)/glucose to generate H2O2. The coexistence of heme and H2O2 significantly resulted in the loss of cell viability to ≈45% (Fig. 4A), demonstrating the cytotoxicity of heme peroxidase activity. As shown in Fig. 4A, the binding of BSA could reduce the cytotoxicity of heme/H2O2 system. Control experiments demonstrated that BSA at this concentration did not induce cytotoxicity. These results were also consistent with recent studies that the binding of serum albumin to heme reduced the biological toxicity of heme whereas albumin was oxidized and albumin-bound heme was less toxic [5,9]. Since heme caused toxicity effects in the immediate proximity to the

A

In conclusion, when heme and rutin was added into BSA solutions, BSA could simultaneously bind these two bioactive components to form protein-diligand complexes. Site marker competitive experiments demonstrated that both heme and rutin bound to the subdomain IIA (site I) of BSA. BSA-diligand complexes provided a better inhibition on the cytotoxicity of heme than did BSA-monoligand complexes, where the co-presence of bioactive rutin in the complexes played a crucial role in this protection. Therefore, these results herein suggest the possibility and advantage of developing BSA-based carriers for the delivery of bioactive components and suppression of heme toxicity in their biomedical applications.

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Fig. 4. Cytotoxicity of heme/H2O2 system (A) and oxidative stress (B) in cells with/without BSA binding in the presence or absence of rutin. HUVEC cells were treated with the indicated combination of heme (2 μM), H2O2 (1.5 mU/mL GO plus glucose), BSA and rutin (20 μM). After treatment for 9 h, the cell viability (A) and oxidative stress (B, reflected by the contents of protein carbonyls) were determined. Untreated cells were taken as control. (Values were means ± S.D. (n = 4), **P < .01, *P < .05, groups versus heme/H2O2-treated group, P < .05, groups versus indicated group). 6

Biophysical Chemistry 258 (2020) 106327

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Declaration of Competing Interest

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