Accepted Manuscript

Self-association of hypericin analyzed by light absorption and fluorescencespectroscopy and molecular dynamics simulations

Monika Pietrzak, Maciej Maciejczyk, Mariusz Szabelski, Adam Kasparek,Zbigniew Wieczorek

PII: S0009-2614(14)00236-XDOI: http://dx.doi.org/10.1016/j.cplett.2014.03.076Reference: CPLETT 32067

To appear in: Chemical Physics Letters

Received Date: 29 January 2014Accepted Date: 26 March 2014

Please cite this article as: M. Pietrzak, M. Maciejczyk, M. Szabelski, A. Kasparek, Z. Wieczorek, Self-associationof hypericin analyzed by light absorption and fluorescence spectroscopy and molecular dynamics simulations,Chemical Physics Letters (2014), doi: http://dx.doi.org/10.1016/j.cplett.2014.03.076

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Title

Self-association of hypericin analyzed by light absorption and fluorescence spectroscopy and

molecular dynamics simulations

Authors:

Monika Pietrzak, Maciej Maciejczyk, Mariusz Szabelski, Adam Kasparek and Zbigniew

Wieczorek

1Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn,

Oczapowskiego 4, 10-719 Olsztyn, Poland;

Corresponding author:

Monika Pietrzak

telephone/fax number: +48 89 523 34 32/+48 089 523 38 61

e-mail: pietrzak@uwm.edu.pl

Abstract:

Self-association of hypericin was investigated with absorption and fluorescence spectroscopy

methods. Constants of hypericin association in a 50% aqueous solution of DMSO were

determined in the function of temperature, and absorption spectra of the monomer and

molecules participating in the complex were examined. Changes in enthalpy ∆HӨ and entropy

∆SӨ in the process of hypericin association accounted for -46 kJ⋅mol

-1 and -55 J⋅mol

-1⋅K-1,

respectively. Molecular dynamics simulations methods were applied to determine

conformations of the hypericin complex, and “head-to-head” and “head-to-tail” models of

interaction were proposed. The “head-to-head” conformation turned out to be the most stable

one.

Key words: hypericin, self-association, absorption, fluorescence, molecular dynamics

simulations

Introduction

Hypericin is a natural substance synthesized by plants of the Hypericaceae family. It is

derived in the highest quantities from common St. John’s wort (Hypericum perforatum L.).

Different extracts from St. John’s wort have for years been applied in herbal therapy as

cholagogic and digestion-enhancing agents. Hypericin has also other medical applications. It

is widely used as a natural, herbal anti-depressant which is often offered as an alternative to

synthetic agents (Mennini and Gobbi 2004, Kubin et al. 2005). Hypericin is a photo-

sensitizing substance, susceptible to the action of sunrays, hence avoidance of sharp sun is

recommended while taking hypericin-containing medicines. Hypericin is a potential drug in

photodynamic therapy, however its properties, poor solubility in water in particular, curb its

medical applicability (Skalkos et al. 2006).

Hypericin may affect metabolism of other orally-administered drugs, e.g. cyclosporin, HIV

protease inhibitors, or oral contraceptives, by reducing their therapeutic concentration in

plasma and thus debilitating their efficacy (Henderson et al. 2002). Mechanisms of hypericin

interaction with other molecules have not been entirely explored so far. The characteristic

structure of this compound, i.e. high contribution of condensed rings, predisposes hypericin to

act as an interceptor molecule (Hartman and Shankel 1992). Hypericin molecules have been

shown capable of forming stacking complexes with other flat, aromatic molecules, thereby

decreasing their bioavailability likewise chlorophyllin or resveratrol (Pietrzak et al. 2003,

Pietrzak et al. 2006, Osowski et al. 2010). Studies on the intercepting role of hypericin are,

however, impaired by its poor solubility in water. The presence of water in a solution

facilitates the formation of dimers and, probably, higher aggregates. The higher the water

content in the solution, the greater the self-association of hypericin is (Falk and Meyer 1994).

Many authors (Beshnova and Evstigneev 2007, Evstigneev et al. 2008, Buchelnikov et al.

2012) have postulated that during determining a correlation between the interceptor molecule

and the intercepted molecules consideration should be given to any possible objects formed in

the analyzed system, including dimers and higher order aggregates of both the trapping and

the trapped compound. When discussing heteroassociation of hypericin and other molecules it

is also necessary to take account of the self-association mechanism and to determine

association constants at specified experimental conditions, considering the specific character

of the measuring methods applied.

The methods used in our previous investigations addressing heteroassociation of

chlorophyllin and xanthines with DNA intercalators allowed neglecting the effect of self-

association of these compounds. In the case of hypericin, however, the use of even relatively

low concentrations as enabled by spectroscopic methods, does not justify this negligence.

This manuscript describes a tentative study introducing investigations on the potential

classification of hypericin as an interceptor molecule. For the first time we present the self-

association process of hypericin in water solution containing 50% DMSO. We used

absorbance and emission spectra to calculate the self-association constant. Additional we

perform theoretical simulation to visualize hypericin-hypericin dimer in aqueous solutions of

DMSO by using molecular dynamics methods. Our results indicate that interpretation of

previously published data was not correct e.g. Falk and Meyer (1994) observed the same

changes in absorbance spectra in case of concentration changing of hypericin or DMSO. In

this paper we proved that increasing concentration of hypericin generate hypochromic and

batochromic effects.

Experimental

2.1. Reagents

Hypericin (1,3,4,6,8,13-hexahydroxy-10,11-dimethylphenanthro[1,10,9,8-opqra]perylene-

7,14-dione) (HWI Analytic GmbH, Ruelzheim, Germany), DMSO (dimethyl sulfoxide pure

p.a.) (PPH Polskie Odczynniki Chemiczne, Gliwice, Poland), Tris (Tris-(hydroxymethyl)-

aminomethane) (Fluka Chemie AG, Buchs, Switzerland).

2.2. Solutions

Hypericin was dissolved in pure DMSO. The resultant stock solution was then mixed with 30

mM Tris–HCl buffer (pH 7.4) until reaching solutions with desired concentrations of

hypericin and appropriate percentage content of DMSO.

2.3. Absorption measurements

Measurements of absorption spectra were carried out using a Cary 5000 spectrophotometer

(Varian, Australia). Hypericin absorption spectra were measured in the function of

concentration (in a concentration range of: 1.3×10-6M – 2.7×10-5M) and temperature (283 K,

293 K, 303 K, 313 K, 323 K, and 333 K) in solutions with various percentage contents of

DMSO. The measurements were carried out in a 1-cm cuvette, in a wavelength range of 300 –

790 nm.

The self-association constants Ka and the absorption spectra of hypericin in the monomeric

form and in complexes were estimated numerically. Analyses were conducted using a

fragment of spectrum in a wavelength range of 400 – 700 nm. The isodesmic model was

applied for non-linear regression analysis according to Eq. 1.

�(λ, �) = ��() − ��() ��������������

�����

+ ��()� (1)

where A(λ,C) – absorbance at wavelength λ of the measured sample at total concentration of

hypericin C in solution; �() and ��() – the extinction coefficients for the monomeric and

the aggregated hypericin, respectively; and Ka – self-association constants.

2.4. Fluorescence measurement

Fluorescence measurements were carried out using a Cary Eclipse spectrophotometer (Varian

Australia), in 0.4 × 1 cm cuvettes, using excitation wavelength of 570 nm with a 2.5 and 5 nm

excitation and emission spectral width. Concentration and temperature ranges were as in the

absorption measurements.

Fluorescence measurements enabled determining association constants Ka. Fluorescence

intensities at various temperatures were corrected considering the relative quantum yield

achieved based on fluorescence lifetimes. Analyses were carried out using a fragment of

spectrum in a wavelength range of 650 – 660 nm.

2.5. Time-resolved fluorescence

Measurements were performed using Fluo Time 200 (PicoQuanta, Germany). Hypericin

fluorescence lifetimes were measured in solutions with different concentrations of hypericin

and DMSO and at various temperatures. Fluorescence was excited with a 574 nm diode and

observed at 600 nm with a 2 nm excitation and emission spectral width.

2.6. Molecular dynamics simulations

All molecular dynamics simulations were performed with Desmond package as implemented

in the Maestro Suite from Schrödinger. All parameters, including partial charges, of hypericin

were assigned from OPLS2005 force-field (Banks et al. 2005).

Vacuum simulation was performed in the NVT ensemble with 2fs time-step and no cutoffs

applied to electrostatic interactions. The geometry of molecule was optimized by energy

minimization and initial velocities were drawn from Maxwell distribution and the system was

coupled to 300 K thermal bath with Nose-Hoover thermostat (Nose 1984, Hoover 1985).

The following protocol was used for simulations of hypericin dimerization process in a water-

DMSO mixture. Two hypericin molecules were immersed in 37 A cubic box filled with

DMSO molecules. The overlapping DMSO molecules were removed. Then the system was

immersed in 46.6 A cubic box filled with TIP3P water molecules and overlapping water

molecules were removed. The system was heated up and equilibrated with the standard

Desmond Maestro protocol. All simulations were run in NPT ensemble with Nose-Hoover

thermal bath (coupling time 1.0 ps, T=300 K) and Martyna-Tobias-Klein pressure coupling

(coupling time 2.0 ps, p=1 atm). Equations of motion were integrated numerically with

multiple time-step RESPA algorithm (Tuckerman et al. 1992). Bonded and near non-bonded

interactions were computed every 2.0 fs and far non-bonded interactions were integrated

every 6.0 fs. The 9.0 A cutoff was applied to electrostatic interactions and particle-mesh

Ewald summation (Ewald 1921, Darden et al. 1999) was applied for particles placed beyond

this distance. The snapshots were recorded every 4.8 ps. Total length of gas-phase simulation

was 1µs. A single hypericin molecule in a water-DMSO mixture was simulated for 200 ns.

The simulation of hypericin dimerization in a water-+DMSO mixture was 55 ns-long.

Metadynamics method (Laio and Parinello 2002) was applied to two hypericins placed in the

small water+DMSO box in the “head-to-head” conformation. Two collective variables (CV)

were defined as follows: dihedral angle responsible for the relative rotation of molecules

around the axis perpendicular to their contact surface and the distance between two central

carbon atoms of hypericins. The later CV was restricted by the wall-potential placed at 6.5 A,

preventing dissociation of molecules. Gauss kernels of height of 0.03 kcal/mole were dropped

every 0.09 ps. Widths of gauss kernels were 50 for dihedral angle CV and 0.05 A for distance

CV. The simulation was 70ns-long.

Results and discussion

Many works have shown interactions of hypericin with different pharmaceuticals and

therefore the need of their cautious application (Henderson et al. 2002). The analysis of

interactions with biologically-active compounds usually requires water presence in the

solution. Hypericin is insoluble in water, but DMSO appears a good solvent to introduce it

into the water medium. Falk and Mayer (1994) demonstrated that the higher the water content

in the solution, the more aggregated hypericin is, however they did no determine values of

association constants.

Spectroscopic properties of hypericin

At constant temperature, fluorescence extinction was mono-exponential and did not depend

on hypericin concentration (data not shown). Both these facts confirm explicitly that only one

type of object was fluorescing, namely hypericin monomers. Fluorescence intensities are not

proportional to hypericin concentrations but to the concentration of the monomeric form. As

the fluorescence lifetime is proportional to the fluorescence quantum yield (Lakowicz 2006),

it was feasible to determine the relative quantum yield of hypericin (Tab. 1). The measured

fluorescence spectra were corrected considering the determined relative quantum yield and in

this form served to determine association constants Ka, for different temperatures (Tab. 2).

Simultaneously, the fluorescence lifetime was observed to shorten along with measurement

temperature increase, i.e. from 5.84 ns (��� 0.962) at 288 K to 5.18 ns (��

� 0.975) at 333 K,

which is a typical phenomenon for most chromophores. Hypericin emission spectra are two-

banded and constitute a mirror image of monomer absorption spectra. The fluorescence band

at λmax 598 nm strongly overlaps the absorption band at λmax 594 nm, which is the reason

behind reabsorption and internal filter phenomena (Wieczorek et al. 1993). In order to neglect

these effects in association constants determination (Tab. 2), used was made of a short

spectrum fragment at a spectrum edge.

In absorption spectra measured in the function of both DMSO concentration (data not shown)

and hypericin concentration, changes were observed in the intensity of absorption bands, and

especially noticeable isosbestic points were noticed at ca. 554 nm, 569 nm and 602 nm. The

determined molar coefficients of absorption are consistent with literature data (Falk and

Mayer 1994). In absorption spectra we also observed clear batochromic effects (Fig. 1a). This

results indicate that interpretation of previously published data was not correct e.g. Xia et al.

(1998) published that the absorption spectrum of the aggregate was the same as the monomer

except the lower intensity of absorption. Our measurements indicated that the spectrum of

aggregated molecules was shifted to longer wavelength compared to the absorption spectrum

of monomer.

Association constants of hypericin complexes (Tab. 2) were determined with the use of the

Equal K (EK) model which assumes equal association constants in every consecutive step

(Martin 1996). The author recommends this model as the simplest and the most useful one.

Sizes of absorption spectra of the monomer molecules, obtained from measuring series run at

various temperatures, were very similar and could be averaged, whereas spectra of the

molecules of complexes differed in band intensity depending on temperature and, therefore,

could not be averaged (Fig. 1b). From the view point of the analysis of results achieved with

spectroscopic methods, it is significant to determine spectra of a pure monomer and of

aggregated hypericin molecules. Numerical methods enabled explicit determination of the

spectrum of a pure hypericin monomer, however the spectrum of molecules from the complex

was observed to change in the function of temperature. Probably, the spectrum of aggregated

molecules differs depending on molecule location in the complex (Martin 1996). The plotted

spectrum seems to be a sum of “external” and “internal” molecules, where dimers and

“external” molecules tend to predominate at higher temperatures, whereas aggregates and

“internal” molecules – at lower temperatures.

Effect of pH has been tested on the range 5.5 - 8.9 and was observed any influence on

interaction constant. Additionally it was checked the effect of ionic strength by addition a

proper amount of sodium chloride. The results confirm a linear dependence of Ka on ionic

strength in the range 0 – 0.3 M of NaCl concentration. It can be shown that: Ka = (1.4 + 2.9

[Na+]) × 105 M

-1. The accuracy of Ka determination was ± 2×10

4 M

-1.

Critical aggregation concentration (CAC) defined as the minimum concentration of molecules

at which the intermolecular hydrogen bonding, micelles, or other aggregates start forming was

determined according to the method published by Yu at al. (2012). Estimated value of CAC in

50% DMSO solution at temperature 297 K is 5×10-8

M.

The calculated thermodynamic changes (Fig. 2) are typical of the formation process of

stacking complexes. Changes of enthalpy ∆HӨ and entropy ∆S

Ө in the process of hypericin

association reached ∆HӨ = -46 kJ/mol and ∆SӨ = -55 J/mol×K. Other authors (Lonnberg at al.

1984, Martin 1996, Wieczorek et al. 1997) investigated the thermodynamic quantities of the

self-associaton process for other compounds. Negative enthalpy and entropy changes are

typical of those found for stacking interactions. Our results are consistent with stacking being

attributed solely to hydrophobic interactions. The relatively great change in ∆SӨ is indicative

of the high strength of the stacking between individual molecules of hypericin.

Conformational equilibrium of hypericin molecule based on molecular dynamics

simulations.

A hypericin molecule is not flat and can exist in four degenerated macrostates as shown in

Fig. 3. Four different macrostates of the molecule are defined by values of two dihedral

angles. χ� defines the geometry of methyl-bay region and is marked blue in Fig. 3a. χ� defines

the geometry of the hydrogen-bond-bay region and is marked red in Fig. 3a. The “Methyl UP”

and “Methyl DOWN” macrostates are defined by χ� ≈ −40! and χ� ≈ 40! , respectively.

The geometries of cis and trans conformations are defined by the positions of hydroxyl

groups with respect to methyl groups. For cis conformations χ� ≈ χ� and for trans

conformation χ� ≈ −χ�. The degeneracy of cis and trans macrostates is a direct consequence

of symmetry of hypericin molecule. Both gas phase and water-DMSO solution molecular

dynamics simulations of single hypericin shows preference of trans over cis macrostates. This

effect, which seems to be a result of mechanical stress inside the molecule, is slightly reduced

by interactions of hypericin with a water-DMSO mixture. Trans macrostate was also localized

as a ground state by Guedes and Eriksson (2005) using density functional theory-based

optimization method. These authors analyzed three conformations of hypericin with different

arrangement of protons. All conformations represented trans macrostate. The Hyp I

conformation (see Fig. 2 in Guedes and Eriksson (2005)) forms a hydrogen bond in the bay

region. In the Hyp II conformation the hydrogen bond is broken and protons are exposed to

solvent. It was found out that energy of Hyp II conformer is ~3.7 kcal/mol higher than energy

of hydrogen-bonded Hyp I conformer (Guedes and Eriksson 2005), which means that a

population of h-bonded conformer should be around 3 orders of magnitude larger than the

population of Hyp II conformers at the temperature of 300 K. Both vacuum and solvent

simulations confirm this result as the populations of Hyp II conformers were negligible and

hydrogen-bonded conformations were clearly dominant. Deprotonated Hyp III conformation

analyzed by Guedes and Eriksson (2005) is beyond the scope of this paper.

During molecular dynamics simulations switching between cis and trans conformations was

observed. Fig. 4 shows free energy profiles for χ� dihedral angle obtained from distributions

determined from free dynamics simulations in vacuum and water+DMSO mixture. The free

energy difference between cis and trans conformation for gas phase simulation equals

≈ 2.4 kcal/mol, which is around 1 kcal/mol larger than the same difference for simulation of

hypericin in the water-DMSO solution. As the χ� dihedral angle remained in the vicinity of its

starting value during both simulations, the free energy barrier between “Methyl UP” and

“Methyl DOWN” macrostates seems to be very high, which is a consequence of the steric

clash between methyl groups.

Spatial arrangement of hypericin molecules in a dimmer based on molecular dynamics

simulations.

The simulation of dimerization process started from two hypericins separated by 18 A

immersed in the cubic box with a 1:1 mixture of water and DMSO. Two molecules came into

contact after 20 ns and assumed stacking conformation, which remained stable during the rest

of the simulation (35 ns). Molecules assumed “head-to-tail” conformation in which methyl

groups of one molecule overlapped hydroxyl groups of the other one (Fig. 5a). Stacked

molecules oscillated around “head-to-tail” conformation and never reached another possible

conformation - “head-to-head”, which maximizes contact surface area between two molecules

(Fig. 5b). Its stability was confirmed by another 50 ns-long simulation, which was started

from stacked “head-to-head” conformation and remained stable during the whole simulation.

The relative stability of “head-to-head” and “head-to-tail” conformations was estimated by

70 ns long metadynamics simulation. The free energy surface as a function of two collective

variables (CV’s) was generated. First CV describes relative rotation of two hypericins around

axis perpendicular to their surfaces. Second CV describes separation between hypericins. The

wall potential, which was applied when the distance between two hypericins is greater than

6.5 A, prevented their dissociation. The free energy surface, shown in Fig. 6, has five minima.

Two of them correspond to “head-to-head” conformation (+ ≈ 0,) and the remaining three

lay in the vicinity of “head-to-tail” conformation (+ ≈ 180,). The “head-to-head”

conformation seems to be the most stable one.

Conclusions

Summarizing, in 50% water-DMSO mixture (v/v) hypericin forms stable dimers and higher

aggregates. Spectroscopic data indicate that absorption spectra of aggregated molecules have

distinct batochromic effects compared to the absorption spectrum of monomers. Based on

calculated absorption spectra of the monomeric and complexed hypericin at different

temperature we can conclude that absorption spectra of “internal” and the “external”

molecules in the aggregates are different. Moreover, self-association constant of hypericin is

linearly dependent on the ionic strength but there is no influence of solution pH on Ka in the

studied range. Our experimental results are supported by molecular dynamics simulations

revealed conformational equilibrium within single monomer and the most stable conformation

of hypericin dimer. These MD results can not be directly (quantitatively) compared with our

experimental data and therefore should be treated as supplement, which gives more insight

into the phenomena. Theoretical simulations indicated that the most stable conformation of a

hypericin dimer in water-DMSO mixture is the “head-to-head” conformation.

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Figure captions

Figure 1. a) Apparent molar extinction coefficients of hypericin with a concentration range of

1.3 µM – 27 µM (solid lines) and determined molar extinction coefficients of hypericin

monomer (dashed line) and molecule in a complex (dotted line) at 293 K in 50% DMSO

solution. b) Molar extinction coefficients of hypericin monomer (dashed line) and molecule in

a complex (solid lines) at temperatures of 283 K, 293 K, 303 K, 313 K, 323 K and 333 K in

50% DMSO solution.

Figure 2. Van’t Hoff plot achieved from the analysis of absorption spectra (circles) and

fluorescence (triangles).

Figure 3. Hypericin molecule (a). Dihedral angles related to internal conformational changes

are marked red (hydrogen-bond bay region) and blue (methyl bay region). Four distinct

macrostates of hypericin molecule are: Methyl-UP trans (b), Methyl-DOWN trans (c),

Methyl-DOWN cis (d), and Methyl-UP cis (e).

Figure 4. Two binding modes of hypericin dimer. a) “head-to-head” conformation, b) “head-

to-tail” conformation. Methyl groups are marked blue.

Figure 5. Free energy profiles of �� dihedral angle for gas-phase hypericin (black line) and

hypericin in 50% water – DMSO solution (red line).

Figure 6. Free energy profile of “Methyl UP”-“Methyl UP” hypericin dimer. The deepest

minimum corresponds to “head-to-head” conformation.

Tables captions

Tab.1. Fluorescence lifetimes and relative quantum yields of hypericin determined in 50%

aqueous solution of DMSO.

Tab.2. The self-association constants Ka of the hypericin complexes estimated respectively by

spectrophotometry and spectrofluorometry in 50% DMSO solution.

Figure

Figure

Figure

Figure

Figure

Figure

Tab.1. Fluorescence lifetimes and relative quantum yields of hypericin determined in 50%

aqueous solution of DMSO.

T [K] τ [ns] �/2

Relative quantum

yield of fluorescence

283 4.672 1.001 1

293 4.527 1.003 0.968964

303 4.382 1.014 0.937928

313 4.254 1.002 0.910531

323 4.12 0.983 0.881849

333 3.992 0.975 0.854452

Tab.2. The self-association constants Ka of the hypericin complexes estimated respectively by

spectrophotometry and spectrofluorometry in 50% DMSO solution.

Absorption Fluorescence

T [K] Ka (M-1) SD (M-1) Ka

(M-1) SD (M-1)

283 3.2×105 5×10

4 3.5×10

5 3×10

4

293 1.6×105 4×104 2.2×105 5×104

303 8.4×104 3×10

4 1.3×10

5 3×10

4

313 4.5×104 2×104 6.8×104 1×104

323 2.4×104 7×10

3 3.6×10

4 6×10

3

333 1.4×104 6×103 1.7×104 3×103

Graphical abstract

Highlights

• Self-association constants of hypericin was determined.

• Changes in enthalpy ∆H and entropy ∆S in the process of hypericin association was

accounted.

• Two models of conformations of the hypericin dimer was proposed.