Adsorption Induced Changes of Human Hemoglobin on Ferric

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Adsorption induced changes of human hemoglobin on ferric pyrophosphate nanoparticle surface probed by isotope exchange mass spectrometry: An implication on structure-function correlation Bindu Y Srinivasu, Beena Bose, Gopa Mitra, Anura V Kurpad, and Amit Kumar Mandal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01905 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 24, 2017

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Adsorption induced changes of human hemoglobin on ferric pyrophosphate nanoparticle surface probed by isotope exchange mass spectrometry: An implication on structure-function correlation

Bindu Y Srinivasu1, Beena Bose2, Gopa Mitra1, Anura V Kurpad2, Amit K Mandal1*

1

Clinical Proteomics Unit, Division of Molecular Medicine,

St. John’s Research Institute, St. John’s National Academy of Health Sciences, 100 ft road, Koramangala, Bangalore, India 2

Department of Physiology, St John's Medical College, and Division of Nutrition,

St. John’s Research Institute, St. John’s National Academy of Health Sciences, 100 ft road, Koramangala, Bangalore, India

* Corresponding author Amit Kumar Mandal Professor Clinical Proteomics Unit, Division of Molecular Medicine, St. John’s Research Institute, St. John’s National Academy of Health Sciences 100 Feet Road, Koramangala Bangalore - 560034, India Email: [email protected] Phone: +91-80-49467243 Fax: +91-80-25501088

ACS Paragon Plus Environment

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Abstract: In general, proteins in the biological system interact with nanoparticles (NPs) via adsorption on the particle surface. Understanding the adsorption at molecular level is crucial to explore NP-protein interactions. The increasing concerns about the risk to human health on NP exposure have been explored through the discovery of a handful protein biomarkers and biochemical analysis. However, detailed information on structural perturbation and associated functional changes of proteins on interaction with NPs is limited. Erythrocytes (red blood cells) are devoid of defense mechanism of protecting NP penetration through endocytosis. Therefore it is important to investigate the interaction of erythrocyte proteins with NPs. Hemoglobin, the most abundant protein of human erythrocyte, is a tetrameric molecule consisting of α- and βglobin chains in duplicate. In the present study, we have used hemoglobin as a model system to investigate NP-protein interaction with ferric pyrophosphate NPs [NP-Fe4(P2O7)3]. We report the formation of a bioconjugate of hemoglobin upon adsorption to NP-Fe4(P2O7)3 surface. Analysis of the bioconjugate indicated that Fe3+ ion of NP-Fe4(P2O7)3 contributed in the bioconjugate formation. Using hydrogen/deuterium exchange based mass spectrometry, it was observed that the amino termini of α- and β-globin chains of hemoglobin were involved in the adsorption on NP surface whereas the carboxy termini of both chains became more flexible in its conformation compared to the respective regions of the normal hemoglobin. Circular dichroism spectra of desorbed hemoglobin indicated an adsorption induced localized structural change in the protein molecule. The formation of bioconjugate led to functional alteration of hemoglobin, as probed by oxygen binding assay. Thus, we hypothesize that the large amount of energy released upon adsorption of hemoglobin to NP surface might be the fundamental cause of structural perturbation of human hemoglobin and subsequent formation of the bioconjugate with an altered function.


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Introduction: NPs, due to their unique physiochemical properties attract considerable attention in the domain of science and technology. Despite the rapid increase in the use of NPs in nanomedicine and in the environment, where living systems are exposed to NPs, it is alarming that very little is known about the effect of NPs on biomolecules and subsequent biological responses in the living system. Being small in size, NPs penetrate almost all parts of the body including cells and tissues via different pathways.1 The large surface to volume ratio of NPs offers an increased surface area for biomolecular adsorption.2 Upon entering into a biological system, the surface of the NPs gets adsorbed with different biomolecules, e.g. proteins and lipids, leading to the formation of two types of biomolecular interfaces viz hard and soft corona.3 The composition of protein corona is highly dynamic in nature that changes from high abundant proteins on its immediate formation to high affinity proteins at equilibrium.4 Recent studies on NP-protein interaction have shown that proteins on adsorption to the surface of NPs may accompany various events such as conformational changes, formation of molten-globule like intermediate and changes in the stability.5-8 The secondary structure of bovine serum albumin was reported to be perturbed upon adsorption to the surface of gold NPs.7 Billsten et al. reported that following adsorption on silica NP surface, genetically engineered form of human carbonic anhydrase II were shown to attain a molten globule like structure.6 In an another study it was shown that adsorption of myoglobin on silica NP surface lead to destabilization of C-terminal portion of the protein molecule.9 Indeed, protein function is defined by its three dimensionally active conformation. Studies have reported that on adsorption to NPs, proteins like lysozyme, catalase, trypsin and horseradish peroxidase showed a significant loss in their functions.10-13 In contrast, a study by Pandey et al. reported an increase in functional activity of glucose oxidase on its adsorption to the surface of


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gold NPs.14 Nevertheless, adsorption induced conformational changes of protein molecules and its effect on function may have profound consequences on cellular responses.15,16 Various kinds of NPs have been developed for the application of nanomedicine, especially in bioimaging, bioanalysis, drug delivery, therapy, etc.17-20 However there is a possibility that upon adsorption of proteins on the surface of NPs, biophysical and biochemical properties of proteins might change. Thus the modified proteins desorbed from the particle surface might cause an adverse effect to the biological system. Therefore, a detailed investigation on NP-protein interaction might facilitate the research on biosafety issues in the use of nanomaterials. The blood circulatory system is most likely the first one to interact with NPs on its exposure to a living system. In general, NPs enters the cell either through an active transport, endocytosis or by a passive transport, penetration. In fact, erythrocytes are devoid of endocytic machinery and therefore the entry of NPs across its cellular membrane would be possible through passive transport.21 This facilitates erythrocyte proteins to encounter the NPs that enters the blood stream.22,23 Hence it is important to investigate the NP-human hemoglobin interaction at the molecular level. In the present study we have explored the interaction of NP-Fe4(P2O7)3 with hemoglobin, a major abundant protein of erythrocytes, in vitro. Hemoglobin is a tetrameric protein molecule with a molecular mass of 64453 Da. It is composed of two types of globin chains α (141 amino acid residues) and β (146 amino acid residues) in duplicate along with four heme units.24 Following adsorption of hemoglobin on NP surface, the structure and composition of the desorbed hemoglobin were analyzed using electrospray ionization mass spectrometry (ESI-MS) and thermal ionization mass spectrometry (TIMS), respectively. The adsorption induced conformational changes of human hemoglobin was investigated using hydrogen/deuterium


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exchange based mass spectrometry (H/DX-MS) and circular dichroism spectroscopy (CD). In addition, oxygen binding equilibrium was monitored to assess any functional changes associated with the adsorption of human hemoglobin on NP surface.

Experimental: The study was approved by Institutional Ethical Committee (IEC Study Ref No. 194/2016) and written consent was obtained from volunteers.

Materials All the chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Stable isotope of 57Fe was purchased from Chemgas (Boulogne-Billancourt, France).

Preparation of hemolysate Venous blood was collected from healthy volunteers in an ethylenediaminetetraacetic acid (EDTA) coated vacutainers. The sample was centrifuged at 3000 rpm for 10 minutes at 25 °C, plasma was separated and the red blood cells (RBCs) were washed using 0.9% aqueous NaCl for 3 times. The obtained RBCs were then lysed with 3 volumes of ice cold distilled water. The hemolysate was centrifuged at 12000 rpm for 10 minutes, the clear supernatant was collected and the pellet containing the RBC membrane was discarded. Hemoglobin concentration in the supernatant was measured on a Shimadzu UV-1800 spectrophotometer at 548 nm with ε = 12.51 M-1 cm-1/ heme.


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Synthesis of labeled NP-57Fe(P2O7)3 57

Fe with 97.83% purity was dissolved in 6 N HCl to form isotopically labeled

57

FeCl3

(aq). The pH of the solution was adjusted within the range of 3-4 using ammonium hydroxide. The obtained 57FeCl3 (aq) was used for the synthesis of NPs as described in our previous study.25 In brief, 110 ml of 0.17 M Na4P2O7 (aq) was titrated against 100 ml of 0.23 M FeCl3 (aq) in presence of 15% ethylene glycol at 65 ̊C. The titration was carried out for six days with continuous stirring to avoid particle agglomeration. After the titration the reaction mixture was kept under stirring condition for 3 hrs. The obtained precipitate was washed four times with water and centrifuged at 3500 rpm for 30 minutes. The synthesized particle was filtered and vacuum dried.

Adsorption and desorption of hemoglobin on the surface of NP Hemoglobin obtained from RBC lysate was subjected to adsorption onto the surface of NP-Fe4(P2O7)3 at 4 °C for 60 minutes using molar stoichiometries of Hemoglobin: NPs = 1: 25. After incubation the sample was centrifuged at 12000 rpm for 15 minutes at 4 °C. The supernatant was discarded and the pellet was washed 3 times with water until a clear supernatant was obtained. The resulting red colored pellet was suspended in 10 mM ammonium acetate buffer pH 7.4 and kept at 37 °C for 45 minutes with intermittent gentle vortexing. The supernatant containing the desorbed hemoglobin molecules was collected by centrifugation at 3500 rpm for 5 minutes. The absorption spectra for both normal and desorbed hemoglobin molecule from the NP-Fe4(P2O7)3 surface have been provided as supplementary information (“see Figure S1”). The steps involved in preparation of the sample for different analysis is provided as a schematic representation in Fig. 1.


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Mass analysis of tetrameric hemoglobin using nano-ESI MS Hemoglobin obtained from normal hemolysate and the desorbed molecular pool were analysed using electrospray ionization (ESI) mass spectrometer with Q-TOF mass analyser (Synapt HDMS, Waters, USA), and a nano source (Z-spray) using gold-coated borosilicate glass needles. Cesium Iodide (2mg/ml) was used as a calibrant. The experimental condition was maintained such that the protein was in its tetrameric form throughout its acquisition. The typical operating parameters for the acquisition of the spectra was as follows: capillary voltage, 1.5 – 2.1 kV; sample cone voltage, 60 V; extraction cone voltage, 2 V; source temperature, 37 °C; deslovation temperature, 150 °C; backing pressure, 5.44 mbar; source pressure, 4.47e1 mbar; TOF analyzer, 8.63e-7mbar. Spectra were acquired in the range of 900–6000 m/z in positive-ion V mode. The recorded spectra were base line corrected and the molecular masses of the normal hemoglobin and the bioconjugate were calculated using MassLynx software (MassLynx version 4.1). To record tandem mass spectra, the parent ion was selected in the mass-resolving quadrupole and the collision energy of the trap cell was increased to 37 kV. Argon was used as a collision gas. In order to quantify the abundance of the bioconjugate, summation of peak intensities of multiply charged ions of the bioconjugate was expressed as a fraction of total intensities of the normal hemoglobin and the bioconjugate. It was hypothesized that the ionization probability of the bioconjugate was identical to that of normal hemoglobin. +16

+17

+18

(Iconj + Iconj + Iconj )

% Relative abundance of bioconjugate =

× 100 +16

+17

+18

[(Iconj + Iconj + Iconj )

+16 +18 +17 + (IHb + IHb + IHb )]


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Solubility measurement of NP-Fe4(P2O7)3 using AAS Fe3+ concentration in the supernatant solution of NP-Fe4(P2O7)3 was determined using iCETM 3000 series Atomic Absorption Spectrophotometer (AAS), Thermo Scientific, equipped with 100 mm air-acetylene burner, a single element hollow cathode lamp and a deuterium lamp for background correction. The wavelength was set to 248.3 nm. The iron standards ranging from 0.05 to 0.2 ppm (Titrisol AAS-standard, Merck Millipore) were used to draw the standard curve. 5.08 µM desorbed hemoglobin isolated from the surface of NPs containing NP56

Fe4(P2O7)3:NP-57Fe4(P2O7)3 = 9:1, 5.08 µM normal hemoglobin and NP-56Fe4(P2O7)3 in 10

mM ammonium acetate, pH 7.4 were diluted 20 times using 1 N HNO3 in two separate sets and subsequently acid digested at 140 °C. The Fe3+ concentrations in all the samples were analyzed in triplicates.

Analysis of (57Fe/56Fe) in the bioconjugate using TIMS The isotopic ratio measurement of desorbed hemoglobin molecules from the surface of labeled and un-labeled NP-Fe4(P2O7)3 was measured using negative ion mode thermal ionization mass spectrometry (Triton, Thermo Scientific, Bremen, Germany) following methods described previously.26 In brief, to isolate iron, the desorbed hemoglobin was subjected to acid digestion using 15 N HNO3 and 30% H2O2 at 150 °C. The dried sample was dissolved with 6 N HCl and heated to dryness at 150 °C. To purify Fe3+, the obtained digest was resuspended in 6 N HCl and loaded onto an anion exchange column using resin (200–400 mesh Ag 1-X8, Bio-Rad, California, USA). Fe3+ was eluted with 1 N HNO3 and concentrated by heating at 140 °C for 2 hours. The dried sample was reconstituted with 500 µl of 6 N HCl and subsequently mixed vigorously with 1ml of diethyl ether. The obtained supernatant was mixed with 10 µl of mili-Q


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water. This solvent extraction procedure was repeated thrice. The obtained sample was evaporated to dryness and mixed with 10 µl of concentrated hydrofluoric acid and used for mass spectrometric analysis. Isotope ratio measurement of iron was carried out using double filament arrangement (ionization and evaporation filaments) equipped with a dynode secondary electron multiplier and a multiple collector system of faraday cups for ion beam measurement simultaneously. 99.95% pure Rhenium was used as a filament. Barium fluoride, used as an emitter, was coated onto the etched surface of both the ionization and evaporation filaments. 20-30 µg of iron was then loaded on the BaF2 coated evaporation filament and dried at a current of 0.8 A. After drying, both the filaments were coated with a fluorinating agent, silver fluoride and evaporated at a filament current of 1.2 A. The filaments were then loaded onto the sample carrousel and introduced to the ion source. The optimum temperature used for ionization and evaporation filament was 1440 mA and 1200 mA respectively. For the isotopic measurements, the center -

fixed Faraday cup was used to collect 56FeF4 ions with mass number 132. Other three movable faraday cups were positioned to collect 54FeF4-,57FeF4- and 58FeF4- ions with mass numbers 130, 133 and 134 respectively.

H/DX analysis of adsorbed hemoglobin on NP-Fe4(P2O7)3 surface using MALDI mass spectrometry The hydrogen/deuterium exchange (H/DX) kinetics of peptide backbone amide hydrogens of adsorbed hemoglobin was used to investigate the conformation of adsorbed hemoglobin on the surface of NP-Fe4(P2O7)3. The isotope exchange was initiated by incubating hemoglobin-NP-Fe4(P2O7)3 pellet in 10 mM ammonium bicarbonate pD 7.8 (pD = pH + 0.4)


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buffered D2O. At different exchange time points, a part of the pellet was taken out and any trace amount of D2O was removed by centrifugation. This exchange kinetics was quenched by adding ice chilled 0.1% trifluoroacetic acid (TFA) solution and proteolytic digestion was performed in situ using pepsin:substrate = 1:10 (mol/mol). The digestion was carried out for 5 minutes at 4 °C. The obtained reaction mixture was centrifuged at 12000 rpm at 4 °C for 1.5 minutes. The supernatant containing proteolytic peptides were isolated and subsequently mixed with matrix, αCyano-4-hydroxy cinnamic acid (5 mg/ml) in 2:8:1 acetonitrile/ethanol/0.1% aqueous TFA. 1 µl of the mixture was spotted on a MALDI plate and dried rapidly in a vacuum desiccator. Samples were analyzed using MALDI mass spectrometer (Synapt HDMS, Waters, USA) in the positive ion- reflectron V mode, using 200 Hz solid-state laser λ= 355 nm. Calibration of the mass spectrometer was performed using PEG mix. The H/DX kinetics data of nine peptides obtained from α- and β-globin chains of hemoglobin were analyzed following procedure described previously.27 In brief, the kinetic parameters of isotope exchange reaction of backbone amide hydrogens were obtained from the best fit curve of the number of deuterium atoms incorporated at time t, [D(t)] vs. t plot for both the control and the experimental sets. The algebraic summation of the H/DX rates of normal and adsorbed hemoglobin pool were compared. The magnitude and the sign of the difference in exchange rates of a group of amide hydrogens in the adsorbed hemoglobin on NP surface and normal hemoglobin in solution phase indicated the degree and the direction of conformational change for the respective group of amide hydrogen associated with adsorption. The positive sign indicates increase in conformational flexibility of the respective region of hemoglobin molecule associated with adsorption. Similarly the negative sign indicates the increase in the conformational rigidity upon adsorption.


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Structural analysis of desorbed hemoglobin using CD The structure of the desorbed hemoglobin was investigated using JASCO, J-815 CD spectrometer. The spectra were recorded using 10 mm path length at 25 °C, with a scan speed of 50 nm/ minute in both the near UV (300 nm - 250 nm) and the far UV (250 nm -200 nm) region of the wavelength. The protein concentration of 12 µM and 1.2 µM in 10 mM phosphate buffer, pH 7.4 were maintained to record spectra at the near UV and the far UV regions of wavelength respectively. Each spectrum was an average of 5 scans.

Oxygen binding assay of desorbed hemoglobin Oxygen

binding

affinity

of

desorbed

hemoglobin

was

measured

by

the

spectrophotometric quantification of hemoglobin saturation at varying partial pressure of dissolved oxygen (pO2) using Hemox analyser (ABL 800 Basic, Radiometer). The hemolysate was prepared in the presence of 1 mM sodium thiosulphate, as a reducing agent to avoid oxidation of Fe2+ during adsorption followed by desorption of hemoglobin from the surface of NP-Fe4(P2O7)3. Following adsorption, desorption of hemoglobin was carried out in 100 mM phosphate buffer pH 7.6 at 37 °C for 45 minutes. Oxygen equilibration assay of 25 µM desorbed hemoglobin was carried out in the presence of 93% of hemoglobin concentration of 2,3-DPG, 50 mM sodium chloride, 1 mM EDTA, 2 mM sodium dithionite. Deoxygenation of hemoglobin was monitored spectrophotometrically using the ratio of absorbance at 577 nm and 548 nm. A known mixture of gases N2 (94.4% Nitrogen and 5.6% CO2) and O2 (94.4% Oxygen and 5.6% CO2) was allowed to bubble through the solution for 15 minutes and the saturation of hemoglobin with oxygen was measured. The percentage saturation of hemoglobin was fitted to sigmoidal curve by plotting against partial pressure of dissolved oxygen (pO2) using OriginLab 8.1 software. The


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partial pressure of oxygen at half saturation of hemoglobin (P50) and the Hill coefficient (n) were calculated from the oxygen equilibrium curve.


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Results and Discussion: The adsorption of hemoglobin on the surface of NP-Fe4(P2O7)3 was carried out by incubating the protein with the NPs at 4 °C for 1 hour and desorption in 10 mM ammonium acetate, pH 7.4 at 37 °C for 45 minutes. It was observed that 60.75% of the adsorbed hemoglobin on the surface of NPs was desorbed back in the solution phase. Subsequent structural analysis of hemoglobin was performed using this desorbed molecular pool. Analysis of hemoglobin structure on adsorption to NP-Fe4(P2O7)3 surface Intact mass analysis of tetrameric human hemoglobin was performed using nano-ESI source in presence of 10 mM ammonium acetate, pH 7.4. Under soft ionization condition, noncovalently bound four globin chains (2α, 2β) along with their heme units appeared with three predominant charge states, +16, +17 and +18. The charge state distribution as observed in the mass spectra indicates existence of the compact structure of tetrameric hemoglobin molecule.28 The observed molecular mass of normal hemoglobin was 64454 Da (Mtheo=64453) (Fig. 2A). Similarly, the mass spectra of intact tetramer, obtained by desorption of hemoglobin from NP surface was recorded and analyzed. In the desorbed molecular pool, besides normal hemoglobin with molecular mass of 64455 Da, an additional species at 65413 Da was observed with a mass difference of ∆M = +958 Da. Interestingly, the charge state distribution of this new molecular species was found to be identical compared to normal hemoglobin (Fig. 2B). Tandem mass spectra of the most abundant charge state, +17 of normal hemoglobin was recorded and the constituent globin chains, α and β without heme (apo α, apo β) and with heme units (holo α, holo β) appeared with masses 15126 Da (Mtheo= 15126), 15867 Da (Mtheo= 15867) and 15742 Da (Mtheo= 15742), 16481 Da (Mtheo= 16483) respectively (Fig. 3A). The tandem


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mass spectra of +17 charge state of additional molecular ion showed a series of product ions with masses 15126 Da (apo α), 15742 Da (holo α), 15868 Da (apo β) and 16483 Da (holo β) (Fig. 3B). This indicated that the additional molecular entity is a bioconjugate of normal hemoglobin formed during adsorption followed by desorption of hemoglobin from NP-Fe4(P2O7)3 surface. The relative abundance of the observed bioconjugate was quantified and expressed as a fraction of total hemoglobin. A consistent presence of 43% (approximately) of the bioconjugate in the desorbed molecular pool was observed.

Characterization of the bioconjugate The composition of the bioconjugate in the desorbed molecular pool with respect to iron content was determined using atomic absorption spectroscopy. Solubility measurement of NPFe4(P2O7)3 in 10 mM ammonium acetate, pH 7.4, showed that the concentration of Fe3+ to be 1.18 µM, indicating that the salt is sparingly soluble. Solutions containing 5.08 µM normal hemoglobin and 5.08 µM desorbed hemoglobin showed that the concentration of Fe3+ was 18.14 µM and 21.34 µM respectively. Theoretically, Fe2+ concentration in 5.08 µM hemoglobin is 20.32 µM. Thus, the observed decrease in Fe3+ concentration of normal hemoglobin solution from 20.32 µM to 18.14 µM might be due to loss of iron in the pre-analytical steps of AAS measurement. We assumed a similar loss of Fe3+ in the desorbed hemoglobin pool, however the observed Fe3+ concentration was more than the theoretically calculated value, 20.32 µM. Thus, the additional amount of iron [2.02 µM = {(21.34-18.14) - 1.18}] in the desorbed molecular pool might be due to incorporation of Fe3+, a conjugate ion of Fe4(P2O7)3, into the desorbed hemoglobin molecule. Excluding Fe2+ contribution of normal hemoglobin and considering 43% abundance of the bioconjugate in the desorbed molecular pool, the composition of the


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bioconjugate in terms of Fe3+/hemoglobin was 0.926 [2.02/(5.08×0.43)]. This suggested that the bioconjugate consisted of one Fe3+ ion per hemoglobin tetramer. Thus, sparingly soluble salt Fe4(P2O7)3 in its nano form probably contributed Fe3+ ion to the bioconjugate. To reconfirm the contribution of Fe3+ by the NPs in the bioconjugate, NP-Fe4(P2O7)3 was synthesized using 57Fe, an isotope of 56Fe. TIMS was used to measure the isotopic abundance of iron in the desorbed hemoglobin obtained from the surface of labeled NP-Fe4(P2O7)3. A mixture of NP-57Fe4(P2O7)3 and NP-56Fe4(P2O7)3 in a proportion of 1:9 was used during adsorption of hemoglobin molecule on NP surface. The desorbed molecular pool was subjected to iron extraction as described in the method section. The isotope abundance ratio of

57

Fe/56Fe was

analyzed in the negative ion mode of TIMS. 57Fe/56Fe ratio in the experiment was observed to be 0.039885. According to the natural abundance

57

Fe/56Fe is 0.023094. The observed

57

Fe/56Fe

ratio in normal hemoglobin and desorbed hemoglobin obtained from unlabeled NP-Fe4(P2O7)3 surface were 0.022849 and 0.025014 respectively (Table 1).Therefore, the isotopic composition of the desorbed molecular pool showed a definite shift of 57Fe/56Fe ratio from its natural isotopic abundance to 0.039885. This confirmed that Fe3+ in the bioconjugate was contributed by the NPFe4(P2O7)3.


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Table 1: Iron isotope ratio measurement by thermal ionization mass spectrometry. Data are represented as mean with respective standard deviation

Parameters Isotope ratio Molecules 56

Fe/ 54 Fe

SD

57

Fe/ 56 Fe

9.6 × Normal Hb

15.91

SD

58

Fe/ 56 Fe

3.1× 0.02284

10-3 Desorbed Hb from

SD 6.8×

0.002856 10-5

10-6

1.2×

1.4×

2.4 × unlabeled NP-

15.84

0.02501 10

10-5

0.003078

10-6

-3

Fe4(P2O7)3 Desorbed Hb from labeled NP-Fe4(P2O7)3

8.1 × 16.21

(unlabeled : labeled =

1.2× 0.03988

10-2

1.0× 0.003359

10-5

10-5

9:1)

Conformational analysis of hemoglobin adsorbed on NP-Fe4(P2O7)3 surface The conformation of human hemoglobin molecule adsorbed on NP-Fe4(P2O7)3 surface was analyzed using H/DX-MS by monitoring the exchange kinetics of backbone amide hydrogens of globin chains as described in methods. The obtained sequence coverage from pepsin digestion of hemoglobin measured through MALDI-MS was found to be 49.82%. The


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exchange kinetics parameters consisting of differential populations of exchangeable amide hydrogens of a peptide fragment and respective rate constants are given in the Table 2. Fig. 4 shows the shift in the envelope consisting of isotopic distribution upon deuterium incorporation of a representative peptide with m/z 1494.9 in both the sample and the control across varying time points and its best fit curve of D(t) vs. t plot of exchange kinetics. Compared to normal human hemoglobin, three peptides from β-globin chain with m/z 1494.9 (β 1-14), 1799.1 (β 1531), 1308.9 (β 32-41) showed decreased rate of deuterium incorporation whereas two peptides with m/z 1869.1 (β 130-146) and 931.6 (β 103-110) showed increased rate of deuterium incorporation. Three peptides of α-globin chain with m/z 2910.9 (α 1-29), 3327.1 (α 1-32) and 1585.9 (α 34-46) showed decreased rate of deuterium incorporation, whereas one peptide with m/z 3428.3 (α 110-141) showed increased rate of deuterium incorporation with respect to normal hemoglobin molecule. Fig. 5 shows the best-fit curves of D(t) vs. t plot of exchange kinetics for eight peptides of the normal and the adsorbed hemoglobin molecule. Isotope exchange envelopes obtained across different time points for all the peptides of adsorbed hemoglobin as well as normal hemoglobin have been provided (“see Figure S2-S9”). The spatial orientation of nine peptides in hemoglobin quaternary structure is shown in Fig. 6. H/DX based mass spectrometry is a powerful tool, in studying the conformational change of protein molecules associated with a particular event.29 Monitoring the H/DX kinetics of backbone amide hydrogens on exposure to solvent containing heavy water (D2O) provides the information on structural changes of the protein molecule on its interaction with other proteins and ligands.30,31 The rate of exchange depends on the differential solvent accessibility which is representative of conformational flexibility and/or rigidity of a protein structure. Thus, the dynamics of the protein conformation can be assessed through the flexibility and/or rigidity


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measured through the isotope exchange kinetics of backbone amide hydrogens of protein molecules.30,31 Upon adsorption on solid surface the binding interface of protein molecules gets buried at the interface leading to limited solvent accessibility and subsequent reduced isotope exchange with the solvent.32 Previously, a study has shown that the structural perturbation of protein molecule on adsorption to the surface of NPs might get reflected in the H/DX rate of respective peptide fragment compared to its native analogue.33 The reduced solvent accessibility as observed in the present study for the peptides in the amino termini of both α- and β-globin chains indicated that these peptides of hemoglobin are involved at the binding interface with NPFe4(P2O7)3. Previously, a study reported that the binding interface of cytochrome c on adsorption to gold NPs resembled the functional binding sites of its redox partners.34 However, an increased solvent accessibility at the regions of carboxy termini of both α- and β-globin chains in the present study indicated that these regions of protein experienced an increase in conformational freedom upon adsorption to the surface of NPs. A similar observation was reported by Buijs et al. where myoglobin on adsorption to silica NPs showed a structural heterogeneity and an altered stability.13 Adsorption is a spontaneous and an exothermic process. Thus in the present study, the energy released upon adsorption of hemoglobin on the surface of NP-Fe4(P2O7)3, might have contributed in the conformational perturbation of carboxy termini of both α- and β-globin chains of the hemoglobin molecule. A very small change in the rate of H/DX of the adsorbed hemoglobin for the peptide with m/z 1585.9 of α-globin chain indicated that there was no significant change in the conformation with respect to the normal hemoglobin.


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1 2 3 4 5 6 7 8 9 10 11 12Peptide, 13 14 m/z 15 16 1494.9 17 18 19 20 1799.1 21 22 23 24 1308.9 25 26 27 931.6 28 29 30 31 1869.1 32 33 34 35 2910.9 36 37 38 39 40 3327.1 41 42 43 44 1585.9 45 46 47 48 3428.3 49 50 51 52 53 54 55 56 57 58 59 60

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Table 2: H/D exchange kinetic parameters of 9 peptic peptides of human hemoglobin and from adsorbed hemoglobin on the NP-Fe4(P2O7)3 surface

Adsorbed hemoglobin Residue

β 1-14

Normal hemoglobin

Sequence

VHLTPEEKSAVTAL

Inference

0.01

- 61.77

Rigid

0.009

- 32.46

Rigid

Rigid

k2PB

k3PC

SSR

k1PA

k2PB

k3PC

SSR

45.79

0.59

0.67

0.02

108.14

0.67

0.02

56.32

1.28

0.02

89.40

0.67

WGKVNVDEVGGEALG β 15-31

Sum k1PA

1.2 ×

7.8 ×

10-3

RL

10-3

1.5 × β 32-41

LVVYPWTQRF

31.01

1.34

0.05

55.39

1.24

0.42

0.02

- 24.69

1.48

0.030

0.09

45.19

1.14

0.02

57.23

Flexible

10-2 β 103-110

FRLLGNVL

46.61

2.56

2.7 × 10-2

0.009

2.46

87.56

0.21

1.72

0.01

31.11

YQKVVAGVANALAHK β 130-146

Flexible

9.2 × 10-3

YH VLSPADKTNVKAAWG α 1-29

128.77

0.05

0.97

0.004

158.18

0.26

0.66

0.004

- 29.32

Rigid

KVGAHAGEYGAEALE 16.97

2.00

0.87

0.01

47.84

0.05

0.71

0.05

- 28.77

Rigid

39.45

2.44

0.12

0.002

42.39

0.42

0.25

0.02

- 1.06

Rigid

87.15

1.23

6.6 × 10-5

0.031

38.81

1.34

6.7 × 10-4

0.028

48.22

KVGAHAGEYGAEAL VLSPADKTNVKAAWG α 1-32

RM α 34-46

LSFPTTKTYFPHF AAHLPAEFTPAVHASL

α 110-141 DKFLASVSTVLTSKYR


Flexible

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The structure of the desorbed hemoglobin was analyzed using circular dichroism (CD) spectroscopy and compared with that of the normal hemoglobin. CD spectra of desorbed hemoglobin molecule recorded in the near UV and far UV region are shown in Fig. 7 and 8 respectively. It is evident from Fig. 7 that there was no noticeable difference in the ellipticity of desorbed and normal hemoglobin in the near UV range of spectrum, indicating the absence of any significant global change in the quaternary structure of the molecule, associated with adsorption.35 However, compared to normal hemoglobin, an increase in ellipticity was observed for the desorbed hemoglobin in the far UV region of the spectrum (Fig. 8, panel A). Percent changes in ellipticity at some representative wavelengths are shown in Fig. 8, panel B. This indicated that there might be a localized secondary structural change of the desorbed hemoglobin molecule upon its interaction with NP-Fe4(P2O7)3. Previously, Martin et al. reported an increase in CD for synthetic peptides upon its adsorption on silica NPs indicating an increased helical structure. The possibility that the positively charged side chains of the peptides gets closer on its interaction with negatively charged NP surface led to an increased helical structure of the peptides.36 It was also observed by Zoungrana et. al that α-chymotrypsin showed an increased heilicity upon adsorption to the Teflon surface.37 Another study by Alexey et al. reported that lysozyme on adsorption to silica NP surface resulted in a decreased α-helical content with a significant loss in its enzymatic activity.38 In the present study, the observed increase in the ellipticity of hemoglobin indicated that adsorption induced structural change might have led to an increase in the helical structure of desorbed hemoglobin molecule.


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Functional analysis of the desorbed hemoglobin To investigate whether the observed conformational change of hemoglobin upon adsorption to NP surface results in functional alteration of the molecule, oxygen binding assay of the desorbed hemoglobin was performed. The partial pressure of oxygen at half saturation of desorbed hemoglobin, P50, was found to be 32.2 mm Hg, with a Hill coefficient n=3.3 (Fig. 9). P50 of normal adult human hemoglobin was observed to be 26.7 mm Hg, with Hill coefficient n= 2.9 which were corroborated well with the respective literature values, P50 = 26.88 mm Hg with Hill coefficient n= 2.9.39,40 Thus, the increase in P50 and increase in hill coefficient of desorbed hemoglobin from NP surface indicated decreased oxygen affinity with increased cooperativity. A recent study by Devineau et al. reported a partial loss in secondary structure with a concomitant increase in oxygen affinity of porcine hemoglobin molecule on its adsorption to silica NPs.41 Thus, in our study the observed functional alteration might be due to adsorption induced structural change of hemoglobin molecule. In case of NPs, reduced particle size provides large surface area thereby indicating large amount of energy release during adsorption of protein on its surface. In the present study, we hypothesized that a part of the energy released during adsorption might be consumed by the adsorbent, hemoglobin molecules resulting in an increase in its rotational and vibrational degrees of freedom. Subsequently, the increase in the degrees of freedom might lead to a change in conformation of hemoglobin molecules adsorbed on NP-Fe4(P2O7)3 surface. Morteza et al. reported that adsorption of transferrin, an iron transporter, on superparamagnetic iron oxide NPs surface resulted in a conformational change leading to release of iron from the core of protein molecule.42 In the present study, the formation of the bioconjugate upon adsorption of hemoglobin on NP-Fe4(P2O7)3 surface might be attributed to the conformational change of


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hemoglobin molecule. This conformational change might have led to formation of the channel for the incorporation of conjugate ions, Fe3+and P2O74- of Fe4(P2O7)3. Therefore, the observed bioconjugate with mass addition of 958 Da might have formed due to the incorporation of conjugate ions of Fe4(P2O7)3 along with their water of solvation within the tetrameric hemoglobin molecule. Although nanotechnology finds extensive applications in several fields such as consumer products, medicine, nano imaging etc., it also accompanies the challenges regarding biocompatibility of NPs and its subsequent clearance from the living system. On exposure to NPs, it can enter the living system by various pathways such as inhalation, oral delivery or even by skin penetration, and can potentially interact with the biomolecules in vivo.43 Depending on the particle dimension and surface charge, they can be excreted from the system either through renal or hepatic clearances.44 However, NPs that escapes from the natural mechanism of excretion, gets retained in cells and tissues resulting in NP-induced toxicity.45 Furthermore, NPs that are administered through intravenous delivery enter the vascular system, thereby interacting with the blood proteins and blood cells.46 Proteins in the blood adsorb on the surface of NPs (opsonization) facilitating the process of phagocytosis through monocytes and macrophages resulting in its elimination from the circulatory system.25,47 However, erythrocytes, which make up 96% of the blood cells are devoid of phagocytic machinery. Therefore NPs follows a passive transport to penetrate into the RBCs.48 A study by Wang and co-workers showed the membrane penetration and intracellular internalization of D-penicillamine Quantum dots of 8 nm within RBCs occurs via a passive transport mechanism.21 Another study by Barbara et al. showed that titanium dioxide NPs of 0.2 µm dimension can penetrate RBC cell membrane.23 Therefore, it is crucial to understand the effect of NP on the erythrocyte proteins at the molecular level. In the


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present study, the conformational changes of the desorbed hemoglobin associated with its adsorption on the surface of NP-Fe4(P2O7)3 might have led to the functional abnormality.

Conclusion: Adsorption induced conformational change of human hemoglobin on its interaction with negatively charged NP-Fe4(P2O7)3 resulted in the formation of a bioconjugate. Compositional analysis of the conjugate showed the presence of Fe3+ contributed by Fe4(P2O7)3 salt in its nano form. The oxygen binding affinity of the bioconjugate was found to be significantly different from the normal hemoglobin. The obtained results in the present study might be useful in understanding the protein-NP interactions at the molecular level.


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Acknowledgement: We acknowledge Nano Mission, Department of science and technology, Govt of India for funding the project (SR/NM/NS-1068/2015) and Department of Biotechnology, Govt of India for Thermal ionization mass spectrometry facility. We also acknowledge Indian Council of Medical Research, Govt of India for providing senior research fellowship to Bindu Y Srinivasu. We would also like to acknowledge Mr. Charles Milton for his technical assistance.


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Figures and captions:

Figure 1:

Figure 1: Schematic representation of the method used to analyze the interaction of human hemoglobin with NP-Fe4(P2O7)3. (1) Adsorption of hemoglobin on NP-Fe4(P2O7)3 surface for 1 hour at 4°C (2) Isolation of the pellet of NPs with adsorbed proteins (3) Desorption of the hemoglobin from NP-Fe4(P2O7)3 surface at 37 °C for 45 minutes and its structure and function analysis (4) H/D exchange of hemoglobin in the adsorbed state followed by proteolysis and mass-spectrometric analysis of isotope exchanged peptides


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Figure 2:

Figure 2: A. ESI-mass spectrum of native hemoglobin with non-covalently bound four globin units with their respective heme units (holo-α2β2). Different charge states are labeled. Inset shows the deconvoluted mass spectrum of holo-α2β2 with mass of 64454 Da. B. ESI-mass spectrum of desorbed hemoglobin with non-covalently bound four globin units with their respective heme units (holo-α2β2). Different charge states are labeled. The additional peaks are labeled


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as ‘holo-α2β2-bioconjugate’ with its respective charge states. Inset shows the deconvoluted mass spectrum of holo-α2β2 and holo-α2β2-bioconjugate with mass of 64455 Da and 65413 Da respectively.


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Figure 3:

Figure 3: A. Tandem mass spectrum of holo-α2β2 (+17) m/z 3791.9 of intact hemoglobin. Different charge states of individual globin chain with and without heme units are labeled. Inset shows the deconvoluted peaks of apo-α, holo-α and apo-β, holo-β, globin chains with mass 15126 Da, 15742 Da and 15867 Da, 16481 Da respectively. B. Tandem mass spectrum of holo-α2β2-bioconjugate (+17) m/z 3846.2 of the desorbed hemoglobin. Different charge states of individual globin chain with and without heme units are labeled. Inset shows


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the deconvoluted peaks of apo-α, holo-α, and apo- β, holo-β globin chains with mass 15126 Da, 15743 Da and 15868 Da, 16481 Da respectively.


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Figure 4:

Figure 4: MALDI mass spectra for the peptide 1494.9 m/z obtained on H/D exchange kinetics of adsorbed hemoglobin molecule on the NP-Fe4(P2O7)3 surface (Panel A) and normal hemoglobin (Panel B) across varying time points. Panel C represents the best fit curves of D(t) versus t plot of adsorbed hemoglobin (red color, ) and normal hemoglobin (blue color,

) where D(t) represents

number of deuterium atoms incorporated with exchange time in minutes plotted along Y and X axis respectively. m/z, subunit of the globin chain, β, and the stretch of amino acid residues of the peptic


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peptide is indicated. Error bars represent the standard deviation of three replicates of the H/DX experiment


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Figure 5:

Figure 5: Panel A to H represents the best fit curve of H/D exchange kinetics for 8 peptic peptides of adsorbed molecule on the NP-Fe4(P2O7)3 surface (red color, ) and normal hemoglobin (blue color,

). D(t), along Y-axis represents the number of deuterium atoms incorporated and t along

X-axis represents the exchange time in mins. m/z, globin chain subunits, α and β and the stretch of


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Langmuir

amino acid residues of the respective peptic peptide is indicated in each of the panel. Error bars represent the standard deviation of three replicates of the H/DX experiment


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Figure 6:

Figure 6: Quaternary structure of oxyhemoglobin (PDB ID: 1gzx). Heme units are shown in red and Fe2+ ions are shown in black. Trace 1, 2, 3 and 6, 7, 8 shows the peptides that are involved in the adsorption on NP surface. Trace 4, 5 and 9 shows the peptides with increased conformational flexibility on its adsorption on NP surface. 1-Blue-m/z 1494.9 (β 1-14); 2-Cyan-m/z 1799.1 (β 15-31); 3-Purple-m/z 1308.9 (β 32-41); 4-Dark green-m/z 931.6 (103-110); 5-Light green-m/z 1869.1 (β 130146); 6-Violet m/z 2910.9 (α 1-29); 7-Violet & Yellow-m/z 3327.1 (α 1-32); 8-Orange- m/z 1585.9 (α 34-46); 9-Red- m/z 3428.3 (α 110-141)


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Figure 7:

Figure 7: Near UV circular dichroism spectra of the normal hemoglobin (blue color,

) and the

desorbed hemoglobin (red color, -----). The concentration of hemoglobin in both sets were 12 µM in 10 mM phosphate buffer, pH 7.4


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Figure 8:

Figure 8: Panel A represents far UV circular dichroism spectra of the normal hemoglobin (blue color,

) and the desorbed hemoglobin (red color,-----). The concentrations of hemoglobin in

both sets were 1.2 µM in 10 mM phosphate buffer, pH 7.4. Panel B represents the average percentage difference in CD of the desorbed hemoglobin and normal hemoglobin at some representative wave lengths


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Figure 9:

Figure 9: Oxygen equilibrium curves of normal adult hemoglobin (blue color, hemoglobin (red color,

) and desorbed

). 25 µM of hemoglobin in 50 mM phosphate buffer, pH7.6 was used in the

experiment. Y-axis represents the percentage of oxygen saturation of hemoglobin and X-axis represents the respective partial pressure of dissolved oxygen (pO2)


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For table of content only:


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Adsorption Induced Changes of Human Hemoglobin on Ferric

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