Journal of Photochemistry & Photobiology, B: Biology 169 (2017) 7–12

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Journal of Photochemistry & Photobiology, B: Biology

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Aggregation of trypsin and trypsin inhibitor by Al cation

P. Chanphai a, L. Kreplak b, H.A. Tajmir-Riahi a,⁎a Department of Chemistry-Biochemistry, Physics University of Québec, Trois-Rivières, C. P. 500, TR, Quebec G9A 5H7, Canadab Department of Physics and Atmospheric Science, Sir James Dunn Building Dalhousie University, Lord Dalhousie Drive, Halifax NS B3H 4R2, Canada

Abbreviations: Try trypsin, Tryi, trypsin inhibitor; FTAFM, atomic force microscopy.⁎ Corresponding author.

E-mail address: tajmirri@uqtr.ca (H.A. Tajmir-Riahi).

http://dx.doi.org/10.1016/j.jphotobiol.2017.02.0181011-1344/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 December 2016Received in revised form 16 February 2017Accepted 21 February 2017Available online 24 February 2017

Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerativediseases. We report the effect of Al cation on the solution structures of trypsin (try) and trypsin inhibitor (tryi),using thermodynamic analysis, UV–Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomicforce microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and vander Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin intolarger or more robust aggregates than trypsin inhibitor, with trypsin 5 ± 1 SE (n = 52) proteins per aggregateand for trypsin inhibitor 8.3±0.7 SE (n=118). Thioflavin T test showed nomajor protein fibrillation in the pres-ence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Al cationTrypsinTrypsin inhibitorAggregationAFMThermodynamicSpectroscopy

1. Introduction

Extensive studies have been reported on the effect of aluminum onhuman health and disease [1]. Al accumulation in tissues and organsresults in their dysfunction and toxicity, which can be correlated withthe local concentration of the Al cation [2–4]. Al is known to induceformation of beta-sheet-rich fibrils that disrupt tissue structure andcause disease. Although not fully proven, Al accumulation in the brainis proposed to be associatedwith neurodegenerative diseases, includingAlzheimer's dementia, Parkinson's disease [3]. It has been shown thatmetal ions including Al cation may trigger protein structural changessuch as aggregation and fibrillation [5,6]. This studywas designed to de-termine the effect of Al cations on the structural transformations oftrypsin and trypsin inhibitor.

Trypsin awater soluble globular protein is a proteolytic enzyme thatcleaves peptide bonds at the carboxylic groups of arginine and lysine[7]. Trypsin inhibitors are classified as small proteins or polypeptidesthat exhibit inhibitory activity against trypsin and can lead to certaindiseases in animals and humans [8]. The inhibitory role of trypsininhibitors comes from their bindings to trypsin and other proteins,causing major protein structural changes [9,10]. The hydrophobic andhydrophilic characteristics of trypsin and trypsin inhibitor are wellknown and their effects on enzyme-substrate interactions have been

IR, Fourier transform Infrared;

investigated [11–16]. Trypsin inhibitor with a large hydrophobic regionshowed different affinity than trypsin towards ligand interactions. Re-ports show hydrophobicity plays a major role in protein-protein andprotein-polymer interactions [10–17].

We report the results of thermodynamic analysis, spectroscopicstudies and AFM imaging for Al cation complexation with trypsin andtrypsin inhibitor in aqueous solution at physiological conditions. Struc-tural information regarding Al-protein binding and the effect of cationinteraction on protein aggregation and fibrillation are presented here.

2. Experimental Section

2.1. Materials

Trypsin from bovine pancreas (MW = 23.8 kDa) and trypsin inhib-itor type-1S (MW = 24 kDa) from glycine, max soyabean were pur-chased from Sigma Chemical Company (St-Louis, MO) and used assupplied.Hydrated AlCl3was fromAldrichChemical Co andused as sup-plied. Other chemicals were of reagent grades.

2.2. Preparation of Stock Solutions

Solutions of trypsin (in H2O) and trypsin inhibitor (in ethanol/H2O25/75%) 120 μM were prepared and diluted to various concentrationsin 10 mM Tris-HCl (pH 7.4). Hydrated AlCl3 was dissolved in 10 mMof Tris-HCl solution (pH 7.4) and diluted to various concentrations.

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2.3. AFM Imaging

1:1mixtures of aluminum chloride and trypsin and trypsin inhibitorat a concentration of 120 μM in 10mMTris-HCl, pH 7.4 were diluted 10times in nanopure water. Freshly cleaved mica disks around 1 cm in di-ameter were incubated with 30 μl of sample solution for 1 min andwashed thoroughly with several milliliters of nanopure water. Themica disks were then dried with compressed nitrogen and imaged inAC mode with an Agilent 5500 atomic force microscope (Keysight,USA). We used NTESP silicon cantilevers (Bruker, USA) with a typicalresonant frequency around 300 kHz and a nominal tip radius of10 nm. Images were acquired at a scan rate of 1 Hz and analyzedusing the free software Gwyddion. For the measurement of particlesizes, the particles were marked using a height threshold of 0.5 nmand the pixel size was 1 nm for all the images used. In order to removefalse-positive particles we only present results for particles with anequivalent radius above 3 nm.

To estimate the number of proteins per aggregate based on their dryvolume, we used the molecular weight of trypsin and trypsin inhibitorto estimate their hydrated volume assuming a protein density of1.37 g/cm3. Assuming all the water leaves the protein upon dryingwhich is an over simplification, the dry volume should be 37% of thewet volume, which gives us 10.7 nm3 for the trypsins.

2.4. UV–Visible Spectroscopy

The UV–Vis spectra were recorded on a Perkin-Elmer Lambda spec-trophotometer with a slit of 2 nm and scan speed of 400 nm min−1.Quartz cuvettes of 1 cm were used. The absorbance measurementswere performed at pH 7.4 by keeping the concentration of proteinconstant (60 μM), while increasing Al cation concentrations (1 μM to60 μM). The binding constants of Al-protein adducts were obtainedaccording to the published method [18,19].

2.5. FTIR Spectroscopic Measurements

Infrared spectra were recorded on a FTIR spectrometer (Impact 420model), equipped with deuterated triglycine sulphate (DTGS) detectorand KBr beam splitter, using AgBr windows. Solution of Al cation wasadded dropwise to the protein solution with constant stirring to ensurethe formation of homogeneous solution and to reach the target Al con-centrations of 15, 30 and 60 μM with a final protein concentration of60 μM. Spectrawere collected after 2 h incubation of trypsin and trypsininhibitor with Al solution at room temperature, using hydrated films.Interferograms were accumulated over the spectral range 4000–600 cm−1 with a nominal resolution of 2 cm−1 and 100 scans. Thedifference spectra [(protein solution + Al solution) − (protein solu-tion)] were generated using water combination mode around2300 cm−1, as standard [20]. When producing difference spectra, thisbandwas adjusted to the baseline level, in order to normalize differencespectra.

Fig. 1. AFM height images of the trypsin and trypsin inhibitor aggregates

Analysis of the secondary structures of trypsin and trypsin inhibitorand their Al complexes were carried out on the basis of the procedurepreviously reported [21,22]. The curve-fitting analysis was performedusing the GRAMS/AI Version 7.01 software of the Galactic IndustriesCorporation.

2.6. Fluorescence Spectroscopy

Fluorimetric experiments were carried out on a Varian Cary Eclipse.Solution containing thioflavin T 25 μM in Tris-HCl (pH = 7.4) was pre-pared at room temperature andmaintained at 24 °C. Solutions of trypsinand trypsin inhibitor containing 50 μM in 10 mM Tris-HCl (pH = 7.4)were also prepared at 24 °C. The fluorescence spectra were recordedat excitation 450 nm and emission from 480 to 520 nm.

3. Results and Discussion

3.1. AFM Analysis and Protein Aggregation by Al Cation

We observed the presence of aggregates on the mica surface for Al-protein complexes (Fig. 1). A protein concentration of 1.2 μMwas usedfor incubation to limit unwanted crowding of the aggregates on the sur-face. As a resultwewere able tomeasure the average volume of the pro-tein aggregates and estimate the number of protein per aggregatesassuming that all the water is removed upon drying. For trypsin we ob-tained 5±1SE (n=52) proteins per aggregate and for trypsin inhibitor8.3 ± 0.7 SE (n=118) (Fig. 1A–C). These numbers clearly indicate thataluminum ions are able to force trypsin and trypsin inhibitor into largeaggregates. The difference between trypsin and trypsin inhibitor is thepresence of elongated aggregates in the former but not in the latter(Fig. 1A–C).

3.2. Stability of Al-Protein Complexes by UV–Visible Spectroscopy

Al-protein binding results in changes in the absorption spectra of theprotein, and the observed changes can be used to calculate the Al-protein binding constants [18,19]. The UV spectra of Al-proteincomplexes are presented in Fig. 2. Al-protein complexation occurredwith a decrease in the intensity of trypsin and trypsin inhibitor absorp-tion band at 280 nm (Fig. 2).

The Al-protein binding constants were calculated as described earli-er inmaterials andmethods [18], usingplots of 1 / (A0−A) vs (1/Al con-centrations) (Fig. 2). The double reciprocal plot is linear and gives theoverall binding constant for each complex with KAl-trypsin = 5.24(±0.5) × 103 M−1 and KAl-trypsin inhibitor = 7.48 (±0.9) × 104 M−1

(Fig. 2 and Table 1). The calculated binding constants show stronger af-finity for Al trypsin inhibitor than trypsin and this correlate well withprotein hydrophobicity. The increase of protein surface hydrophobicityin trypsin inhibitor, results in an increase of the binding affinity for Al-protein complexes (Fig. 2 and Table 1). However, evidence regardinghydrophobic, hydrophilic or H-bonding contacts come from the ther-modynamic analysis of Al-protein complexes discussed below.

induced by aluminum cations, A and B) trypsin, C) trypsin inhibitor.

Table 1Variations of the binding constants for trypsin and trypsin inhibitor with Al cation at dif-ferent temperatures.

Complexes Temperatures (K) Binding constant Ka

Al - Trypsin 298.15308.15318.15

5.24 × 103

3.17 × 103

2.91 × 102

Al-Trypsin inhibitor 298.15308.15318.15

7.48 × 104

5.16 × 104

3.01 × 103

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3.3. Thermodynamic Analysis of Al-Protein Interactions

The interactions between protein and Al cation in aqueous solutioncan be driven by hydrogen bonding, hydrophobic and electrostatic in-teractions [23,24]. In order to characterize the nature of the drivingforces between Al and trypsin and trypsin inhibitor the thermodynamicparameters of the complexes are determined. The thermodynamic pa-rameters (standard enthalpy changes, ΔH; standard entropy changes,ΔS and standardGibbs free energy changes,ΔG) for the Al-protein inter-actions were calculated. According to the data of ΔH and ΔS, the natureof the Al-protein interaction can be characterized [24]. The thermody-namic parameters for the interaction of Al cation with trypsin and tryp-sin inhibitor at 298.15, 308.15 and 318.15 K are presented in Fig. 3 andTable 2. The negative sign of ΔG means that the binding process be-tween Al and proteins is spontaneous. Furthermore, the cation proteinadducts studied here have negative ΔH, which means the complexformation between enzymes and Al is an exothermic reaction. Thenegative ΔH and negative ΔS for protein-polymer complexes indicatethat H-bonding and van der Waals contacts are predominant in theseAl-protein adducts.

A detailed thermodynamic analysis of Al-protein interactions showsthe importance of the binding constant (K), ΔH, ΔS ΔG in determiningwhat type of interaction is predominant in Al-protein complexation[20,21]. In our study, the enthalpy provides more contribution to ΔGthan entropy, which indicates that the binding process is enthalpy driv-en (Table 2). It should be noted that the enthalpy contribution to thefree energy results from the formation of H-bonding and van derWaals interactions. Therefore, negative value indicates that enthalpy

Fig. 2. UV–Visible spectra of trypsin and trypsin inhibitor and their Al complexes fortrypsin (A) and trypsin inhibitor (B) with free protein 30 μM (a) and their complexeswith Al cation at 1, 5, 10, 20, 30, 40, 50 and 60 μM (b–i) and Al cation at 1, 5, 10, 20, 30,40, 50 and 60 μM (b–i). Inset: plot of 1 / (A − A0) vs (1/Al concentration) and bindingconstant (K) for Al-protein complexes.

changes favoring the Al-protein interactions. The order of negative en-tropy changes is trypsin-inhibitor N trypsin (Table 2), which is consis-tent with the stability of the Al-protein complexes (Table 1). However,at pH 7.4 the overall charge of the proteins is negative and is very likelythat the Al cations bind through ion pairing to negative sites of the pro-tein molecule. This is expected to contribute much stronger than Vander Waals interaction (since Al cation represents a very small mass). Itshould be realized that the changes in the thermodynamic quantities,ΔG, ΔH and ΔS, reflect the whole process, including Al-protein bindingand the accompanying conformational changes in the protein. Due tothe Al-binding the degree of random structure increases for these pro-teins and this is discussed in the IR discussion below.

3.4. FTIR Spectra of Al-Protein Adducts

Al-trypsin and trypsin inhibitor interactions were characterized byinfrared spectroscopy and its derivative methods. The spectral shiftingand intensity variations of protein amide I band at 1657–1656 cm−1

(mainly C_O stretch) and amide II band at 1555–1554 cm−1 (C\\Nstretching coupled with N\\H bending modes) [19,21,25] were moni-tored upon Al cation interactions. The difference spectra [(protein

Fig. 3. lnK vs. 1/T for Al-trypsin and Al-trypsin inhibitor systems.

Table 2Thermodynamic parameters for trypsin and trypsin inhibitor with Al cation.

Complexes Thermodynamic parameters

ΔH(KJ·Mol−1 ± 2)

ΔS(J·Mol−1·K−1 ± 3)

TΔS(KJ·Mol−1 ± 2)

ΔG(KJ·Mol−1 ± 1)

Al-trypsin −49.15 −132.79 −39.59−40.92−42.25

−9.56 (298.15)−8.23 (308.15)−6.90 (318.15)

Al-trypsin inhibitor −54.57 −141.01 −42.04−43.45−44.86

−12.53 (298.15)−11.12 (308.15)−9.71 (318.15)

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solution+Al solution)− (protein solution)] were obtained, in order tomonitor the intensity variations of these vibrations and the results areshown in Fig. 4. Similarly, the infrared self-deconvolution with secondderivative resolution enhancement and curve-fitting procedures [21]were used to determine the protein secondary structures in the pres-ence of Al cation (Fig. 5).

At low Al cation concentration (15 μM), decrease of intensity wasobserved for the protein amide I at 1657–1656 and amide II at 1555–1554 cm−1, in the difference spectra of the Al-trypsin and Al-trypsin in-hibitor complexes (Fig. 4A–B, diffs, 15 μM). The negative features locat-ed in the difference spectra for amide I and II bands at 1652 and1551 cm−1 (Al-trypsin) and at 1655 and 1554 cm−1 (Al-trypsin inhib-itor) are due to the intensity decrease of the amide I and amide II bandsupon Al interaction (Fig. 4A–B, diffs, 15 μM). This decrease of the inten-sity for the amide I and amide II bands is due to Al cation interactions

Fig. 4. FTIR spectra in the region of 1800–600 cm−1 of hydrated films (pH 7.4) for (A) freetrypsin (60 μM) and (B) free trypsin inhibitor (60 μM) and their Al cation complexes withdifference spectra (diff.) (bottom two curves) obtained at different trypsin concentrations(indicated on the figure).

with protein C_O, C\\N and N\\H groups (hydrophilic contacts). Aspolymer concentration increased to 60 μM, strong negative featureswere observed for protein amide I band at 1651 and 1554 cm−1 (Al-trypsin-) and at 1654 and 1554 cm−1 (Al-trypsin inhibitor), upon Alcomplexation (Fig. 4A–B, diffs, 60 μM). The observed spectra changesfor the amide I and amide II bands are due to Al binding to proteinC_O and C\\N groups and alterations of protein secondary structure,which is discussed below.

A quantitative analysis of the protein secondary structure for the freetrypsin and trypsin inhibitor and Al cation adducts in hydrated films hasbeen carried out and the results are shown in Fig. 5. The free trypsin con-tains 25% α-helix (1647 cm−1), β-sheet 15% (1632 and 1611 cm−1),turn structure 43% (1672 cm−1) and random coil 17% (1635 cm−1)(Fig. 5A) consistent with the spectroscopic studies of trypsin [26–28].The free trypsin inhibitor contains α-helix 27% (1659 cm−1), β-sheet45% (1631 and 1617 cm−1), turn 16% (1675 cm−1) and random coil

Fig. 5. Second derivative resolution enhancement and curve-fitted amide I region (1700–1600 cm−1) for free trypsin (A) and free trypsin inhibitor (C) at 60 μM and their Alcomplexes (B and D) with 60 μM Al cation concentration.

Fig. 6. Thioflavin T assay to study the fibrillation of trypsin and trypsin inhibitor in thepresence of Al cations. Fluorescence emissions were monitored in the wavelength range460–600 nm and excitation at 450 nm for A) Al-trypsin B) Al-trypsin inhibitor.

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12% (1645 cm−1) (Fig. 5C) consistent with the conformation of trypsininhibitor reported [29–30]. Upon Al interaction, a decrease of α-helixfrom 25% (free trypsin) to 22% (Al-trypsin) with a major decrease inβ-sheet from 15% (free trypsin) to 24% (Al-trypsin) and an increase ofrandom structure from 17% (free trypsin) to 47% (Al-trypsin) were ob-served (Fig. 5B). Similarly, a decrease of α-helix from 27% (free trypsininhibitor) to 19% (Al-trypsin inhibitor) with a major decrease of β-sheet from 45% (free trypsin inhibitor) to 12% (Al-trypsin inhibitor)and increase of random coil from 12% (free trypsin inhibitor) to 39%(Al-trypsin inhibitor) were observed (Fig. 5D). It should be noted thatthe spectral changes were more pronounced for Al-trypsin inhibitor-than Al-trypsin adducts. The major conformational changes are due todestabilization of protein structure by Al cation as protein aggregationoccurred.

3.5. Thioflavin T Binding Study

The fluorescence of thioflavin T is a marker signature of amyloid fi-bril formation as it shows a significant increase in fluorescence intensityaround 485 nmupon binding to theβ-sheet structure of fibrilswhen ex-cited at 450 nm [31]. Our results showed nomajor enhancement of fluo-rescence intensity for thioflavin T band around 480 nm, indicating noprotein fibrillation occurred in the presence of Al-protein complexes(Fig. 6). This is consistentwith our AFM study that showedmajor aggre-gation of trypsin and trypsin inhibitor uponAl interaction (Fig. 1). Fibril-lation induces major spectroscopic and morphological changes toprotein structure [32–39].

4. Conclusions

Al-protein bindings are mainly via H-bonding and van der Waalscontacts for trypsin and trypsin inhibitor. More stable complexesformed with trypsin inhibitor than trypsin. Major protein aggrega-tion was observed in the presence of Al cations. Al-protein interac-tions induced more alterations of trypsin inhibitor conformationthan trypsin, leading to protein aggregation and a partial proteinunfolding.

Acknowledgments

Thiswork is supported by grant fromNatural Sciences and Engineer-ing Research Council of Canada (NSERC).

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