An Anionic Porphyrin Binds b-Lactoglobulin A at a SuperficialSite Rich in Lysine Residues

Ivan Silva Æ Samuel Sansone Æ Lorenzo Brancaleon

Published online: 6 January 2009

� Springer Science+Business Media, LLC 2008

Abstract Binding of small ligands to globular proteins

remains a major research topic in biophysics. We have

studied the binding of several photoactive dyes to b-lacto-

globulin (BLG), as a model to investigate the photoinduced

effects of porphyrins on proteins. A combination of optical

spectroscopies (fluorescence, circular dichroism) and

molecular docking simulations were used to estimate the

pH-dependence of the binding parameters and the docking

location for meso-tetrakis(sulfonatophenyl)-porphyrin

(TPPS). We have observed that the binding of TPPS is not

modulated by the pH-mediated conformational transition of

the protein (i.e., Tanford transition). Binding of TPPS

appears to occur with some degree of negative cooperativ-

ity. Moreover, TPPS remains bound even upon partial

denaturation of the protein. These results are consistent with

a superficial binding site at a location removed from the

aperture of the interior b-barrel. Binding occurs through

electrostatic interactions between the negative SO3- groups

of TPPS and positively charged Lys and Arg residues. This

is the first study that explores the interaction of an anionic

porphyrin with BLGA in a pH range that spans across the

Tanford transition. Establishing the location of the binding

site will enable us to explain the photoinduced conforma-

tional effects mediated by TPPS on BLG.

Keywords Lactoglobulin � Porphyrin �Fluorescence spectroscopy � Binding �Photodynamic therapy (PDT)

Abbreviations

PPIX Protoporphyrin IX

b-lg b-Lactoglobulin

GI Gastrointestinal tract

DMSO Dimethylsulfoxide

KI Potassium iodide

S–V Stern–Volmer

Trp Tryptophan

TPPS4 Meso-tetrakis(sulfonatophenyl)-porphine

ANS 1-Anilinonaphthalene-8-sulfonate

FRET Fluorescence resonance energy transfer

CD Circular dichroism

1 Introduction

Because of its small size (162 amino acids) and its well

characterized structure [1, 2], b-lactoglobulin (BLG) rep-

resents a viable protein model for the investigation of the

effects of photoactive ligands (such as porphyrins) on

globular proteins. Following our study on the binding of

protoporphyrin IX (PPIX) to BLG [3], we have recently

shown that irradiation of the porphyrin in the PPIX/BLG

complex induces partial unfolding of the protein [4].

Light-induced processes have been extensively applied

to study the dynamic structure of proteins [5, 6]. The direct

photoinduced, porphyrin-mediated conformational effects

[4] could also have consequences for biomedical applica-

tions since porphyrins have been used as experimental and/

or clinically useful [7, 8] photosensitizing drugs in photo-

dynamic therapy (PDT). The direct targeting of proteins in

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10930-008-9158-8) contains supplementarymaterial, which is available to authorized users.

I. Silva � S. Sansone � L. Brancaleon (&)

Department of Physics and Astronomy, University of Texas at

San Antonio, One UTSA Circle, San Antonio, TX 78249, USA

e-mail: lorenzo.brancaleon@utsa.edu

123

Protein J (2009) 28:1–13

DOI 10.1007/s10930-008-9158-8

PDT has been historically overlooked despite the mounting

evidence that proteins are indeed directly targeted during

cell irradiation in the presence of various photosensitizers

[9–11] and that such damage is not confined to membrane

proteins [10, 12]. Understanding light-induced, porphyrin-

mediated conformational changes of model globular pro-

teins will therefore be useful to understand some of the

mechanisms that produce protein damage during PDT, but

also provide a new approach that could intentionally target

specific proteins, thus providing a potential paradigm shift

in the PDT protocol.

A fundamental element in the study of photoinduced,

porphyrin-mediated conformational changes of proteins is

establishing the location of the binding site for the porphyrin.

Binding of meso-tetrakis(sulfonatophenyl)-porphine

(TPPS) to BLG has been investigated near neutral and

acidic pH values [13] where the porphyrin is a tetra-anion

(TPPS4-) and a di-anion (TPPS2-), respectively. In the

acidic regime where BLG is a monomer and TPPS2- tends

to form J-dimers, it was found that the protein itself can

induce the formation of J-aggregates [13]. Near neutral pH

where BLG is dimerized and TPPS4- is monodispersed the

signature band of the bound J-aggregate disappeared [13].

The study, however, did not include a wider pH range that

could probe the effects of the conformational changes of

BLG (the so-called Tanford transition) [14].

We analyzed the binding of TPPS4- to b-lactoglobulin

variant A (BLGA) at 6 B pH B 9. In such pH range BLGA

is a dimer and each monomer undergoes a conformational

transition near pH 7.5 [14, 15]. In this range the presence of

TPPS2- or its J-aggregates is negligible [16]. The com-

bined used of emission spectroscopy, fluorescence lifetime

measurements, circular dichroism spectroscopy (CD) and

computational docking simulations, provided a likely sce-

nario for the binding site of TPPS4- to BLGA and revealed

the docking to a superficial protein location stabilized by

electrostatic interactions between the negatively charged

SO3- groups of TPPS4- and positively charged amino acid

residues. This superficial binding site is in agreement with

what had been suggested previously [13].

2 Materials and Methods

2.1 Chemicals

TPPS and bovine lactoglobulin (genetic variant A, BLGA)

were purchased from Sigma–Aldrich (St. Louis, MO) and

used without further purification. Electrophoretic analysis

of BLGA under denaturing conditions revealed [99%

purity. In addition we analyzed the BLG sequence using

capillary liquid chromatography/mass spectrometry (LC/

MS) which confirmed the purity of the protein and revealed

a mass of 18,278 Da for BLG which is within 1 Da of

the NCBI sequence gi|6729725. This sequence differs from

the expected BLGA sequence for eight amino acids in the

C-terminal region of the protein.

2.1.1 Buffers

Aqueous phosphate buffers were prepared using deionized

water. One tablet of phosphate buffered saline tablets

(Sigma–Aldrich) was dissolved in 200 mL of water to pro-

duce a 10 mM buffer at pH 7.4. Buffers were subsequently

adjusted to the correct pH by addition of small aliquots of a

0.5 M solution of HCl or a 0.1 M solution of NaOH. In order

to investigate the effects of the BLGA conformational

transition [14] on the porphyrin binding, the pH of the buffer

was adjusted from 6 to 9 at increments of one pH unit.

2.2 Sample Preparation

2.2.1 TPPS UV–Vis Absorption and Fluorescence

The porphyrin was dissolved directly in buffer and the

concentration adjusted to 0.5 lM using absorption

spectroscopy, assuming e413 = 5.10 9 105 M-1 cm-1 in

aqueous solution [17]. The fluorescence of TPPS was

recorded with excitation at 413 nm and emission in the

580–750 nm range. A stock solution of BLGA at the same

pH as the porphyrin solution was prepared at a concentration

of 30 lM by dissolving it directly in buffer. The concen-

tration of the protein in solution was always determined by

spectrophotometric analysis assuming e280 = 1.76 9 104

M-1 cm-1. The LC/MS data did not detect any aromatic

impurity in our sample, therefore we assumed that the

content in aromatic amino acids (Tyr ? Trp) was identical

to the one expected for other BLG molecules whose molar

extinction coefficient, e280, is the one shown above [13, 18].

Increasing aliquots (from 10 to 100 lL) of the protein

solution were added directly into 1.5 mL of the aqueous

solution of TPPS. The fluorescence spectrum of TPPS was

recorded after each addition of the protein. The absorption

spectra of the same solutions were also recorded from 250 to

500 nm and used to correct for sample dilution and to nor-

malize the emission spectra [3] but also to survey the

possible formation of J- or other aggregates.

2.2.2 BLGA Fluorescence Quenching

The solutions for the TPPS-induced quenching of BLGA

contained 7 lM of the protein (OD295 & 0.1) [19]. Exci-

tation of protein fluorescence was carried out at 295 nm

which selectively excites the Trp residues of BLGA. A

20 lM stock solution of TPPS in buffer was prepared and

added at increasing aliquots to a 1.5 mL volume of the

2 I. Silva et al.

123

BLGA solution. The intrinsic fluorescence of the protein

was recorded between 300 and 450 nm after each por-

phyrin addition. Absorption values at the excitation

wavelength were recorded for all samples and used to

normalize the emission spectra of the protein and correct

for the dilution of the sample [3].

2.2.3 FRET, Fluorescence Anisotropy and Circular

Dichroism Spectroscopy

All these experiments were carried out at a single

BLGA:TPPS molar ratio where the fluorescence spectra

indicated the maximum shift of the emission (thus binding

saturation). FRET experiments were carried out using

steady state and time resolved fluorescence.

2.3 Instrumentation

Absorption spectra were recorded on a dual beam spectro-

photometer (Evolution 300, Thermo Scientific, Waltham,

MA). All spectra were recorded at a rate of 240 nm/min and

a spectral resolution of 2 nm. Appropriate baseline and

reference cells were used for each scan. Circular Dichroism

(CD) experiments were carried out using a J-810 spectro-

polarimeter (Jasco Inc., Easton, MD).

Steady state fluorescence and fluorescence anisotropy

were recorded using a double-monochromator fluorimeter

(AB2, Thermo Scientific, Waltham, MA). Spectra were

recorded at a speed of 1 nm/s and spectral resolution of

4 nm in both emission and excitation.

Fluorescence lifetime was carried out using an IBH

5000U instrument (Horiba JobinYvon, Edison, NJ). The

fluorescence decay of TPPS was recorded upon excitation

with a pulsed diode laser at 405 nm (NanoLED-405L,

pulsewidth * 150 ps); the fluorescence decay of BLGA

was recorded upon excitation with an LED source at 295 nm

(NanoLED-295, pulsewidth & 750 ps). Both sources are

manufactured by IBH Ltd. (Glasgow, UK) and are operated

at a repetition rate of 1 MHz.

2.4 Methods

2.4.1 Fluorescence of TPPS

The fluorescence spectra of the porphyrin as a function of

the addition of the protein were analyzed using Gaussian

fitting of the spectra as reported in detail in a previous

manuscript [17]. In summary, each spectrum of the tetra-

anion TPPS4- was analyzed according to.

IðkÞ ¼ Afe�ðk�Kf Þ2

Df þ Abe�ðk�KbÞ2

Db þX

j

Aje�ðk�KjÞ2

Dj ð1Þ

where I(k) is the overall fluorescence spectrum, Af and Ab are

proportional to the concentration and the emission quantum

yield of the free and bound porphyrin, respectively, and Kf

and Kb are the peak position of the free and bound Gaussian,

respectively [17], calculated for the Q0(0,0) peak [20, 21]

(Fig. 1). The last summation of Eq. 1 fits the region of the

Q0(0,1) peak [20, 21] (Fig. 1), which is less sensitive to the

binding of porphyrins to proteins and is produced by an

unknown number of bands. This assumption enabled us to fit

the emission spectra with a minimum number of Gaussian

components [17]. The quality of the fitting was determined

by visual inspection and by using the value of the reduced v2.

The amplitude of each Gaussian yields the products

Uf[porphyrin]f and Ub[porphyrin]b [17].

In order to determine the binding parameters we

assumed the modified Hill model [13] which after the

introduction of the Gaussian fitting becomes [17]

½TSPP�b½TSPP�Tot

¼ cðK½BLGA�Þn

1þ ðK½BLGA�Þn ð2Þ

where the parameter n represents the Hill coefficient [24,

25], [TPPS]b and [TPPS]Tot are the concentrations of bound

and the maximum concentration of bound TPPS4-,

respectively, under the conditions of the experiments, K is

the equilibrium binding constant and c is a function of the

emission quantum yield for free and bound ligands. The

non-linear fitting of Eq. 2 yields the values of K and n, c.

2.4.2 Stern–Volmer (S–V) analysis

We assumed that quenching of BLGA fluorescence by

TPPS4- is due to the binding between the two molecules;

thus static quenching is responsible for the decrease of

protein fluorescence upon addition of TPPS4-. Stern–

Volmer analysis for static quenching predicts that the ratio

F0/F between the fluorescence of BLGA in the absence of

TPPS (F0) and the fluorescence of BLGA in the presence of

TPPS (F), is, for small amounts of quencher, a linear

function of the concentration of the ligand [26]

F0

F¼ 1þ K½TSPP� ð3Þ

where both F0 and F are corrected for the absorption at the

excitation wavelength as well as the potential filter effects

with the equation [26]

F ¼ 10AexþAem

2 Fraw ð4Þ

In this case Fraw is the uncorrected emission intensity and

Aex and Aem are the optical densities of the sample at the

excitation and the maximum of the emission wavelength,

respectively.

The slope obtained from the linear regression of F0/F vs.

[TPPS] provides the static quenching constant, K, which in

An Anionic Porphyrin Binds b-Lactoglobulin A 3

123

the case of ground state quenching reduces to the equilib-

rium binding constant. However, since the BLGA dimer is

a multi-Trp complex (two residues per BLGA monomer), it

is necessary to establish the fraction of fluorescence

quenching induced by binding of TPPS4- in order to cor-

rect the values of F0 and F in Eq. 3. Static quenching

theory assumes that the formation of a chromophore-

quencher ground state complex eliminates the fluorescence

contribution of the complex from the fluorescence signal

[26]. If the fluorescent molecule is a multi-Trp protein,

binding of a ligand may quench some but not all of the Trp

residues in the protein. Thus a residual fluorescence is left

from the protein-quencher complex that, if unaccounted,

would lead to an underestimate of the binding constant

[27]. The fraction of BLGA fluorescence quenched by

binding of TPPS4- was retrieved by applying and

extending the classic method introduced by [28] since,

formally, the linearity of Eq. 3 is true for both static and

dynamic quenching. In this case we have considered m

independent Trp residues each contributing F0i, to the

overall emission intensity, proportional to the quantum

yield U0i. Therefore, the fluorescence quantum yield in the

absence of quencher (TPPS4-) can be written as

U0 ¼1

m

Xm

i¼1

U0i ð5Þ

Upon addition of the quencher (Q), the fluorescence

detected is [28]

U ¼ 1

m

Xm

i¼1

U0i

1þ KiðQÞð6Þ

where Ki is the static quenching constant for each Trp

residue. From Eqs. 5 and 6

U0 � U ¼ DU ¼ 1

m

Xm

i¼1

U0iKiðQÞ1þ KiðQÞ

ð7Þ

and

U0

DU¼Xm

i¼1

1þ KiðQÞU0iKiðQÞ

ð8Þ

So if we assume that the BLGA–TPPS4- complex

quenches only a fraction of accessible Trp residues (i.e.,

Trp residues in close proximity to the porphyrin), then for

those Trp residues that are not quenched Ki = 0 and Eq. 8

can be simplified to [28]

0

0.2

0.4

0.6

0.8

1

600 650 700 750

Wavelength (nm)

Q'(0,0)Q'(0,0)

Q'(0,1)

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

Wavelength (nm)

600 650 700 750

600 650 700 750

No

rmal

ized

Flu

ore

scen

ce In

ten

sity

No

rmal

ized

Flu

ore

scen

ce In

ten

sity

No

rmal

ized

Flu

ore

scen

ce In

ten

sity

No

rmal

ized

Flu

ore

scen

ce In

ten

sity

600 650 700 750

(a) (b)

(d)(c)

Fig. 1 Fluorescence of TPPS4-

and Gaussian fitting of the

spectra upon addition of BLGA.

Solid black corrected emission

spectrum; dashed red fitting

from Eq. 1; dotted blackGaussian of free TPPS4-; solidred Gaussian of bound TPPS4-;

dotted green, dotted orange,

dotted blue Gaussians of the last

term of Eq. 1. a pH 6,

[BLGA] = 0.13 lM; b pH 6,

[BLGA] = 2.30 lM; c pH 9,

[BLGA] = 0.13 lM; a pH 9,

[BLGA] = 2.30 lM. The plot

shows that at increased

concentration of BLGA the

Gaussian associated with bound

TPPS4- increases relative to the

one associated with the free

porphyrin. The increase is

similar at pH 6 and 9 thus, it is

not affected by the

conformational transition of the

protein. In (a) the two emission

peaks are labeled according to

Refs. [22, 23]

4 I. Silva et al.

123

U0

DU¼ 1

aKQðQÞþ 1

að9Þ

where a is the fraction of Trp residues proximal (quenched)

to the bound TPPS4- and KQ is the quenching constant

assumed to be identical for all Trp residues. In multi-Trp

proteins this assumption may be often considered an

oversimplification; however, in BLG this condition may be

realistic. Of the two Trp residues in each monomer, it has

been shown that Trp61 does not contribute to the fluores-

cence of the protein, likely because of its proximity to the

Cys66–Cys160 disulfide bond [19, 29]. Therefore, in each

monomer only Trp19 contributes to the emission which

implies that only two Trp resides contribute to the fluo-

rescence in each dimer.

Since U and the corrected fluorescence of Eq. 4 are related

by an instrumental multiplication constant which cancels out

in the left-hand side ratio of Eq. 9, this equation can be

employed to obtain the corrected fluorescence of Eq. 3 as

F0

aF¼ 1þ K½TSPP� ð10Þ

where a operates as a correction factor due to the fact that

the BLGA/TPPS4- complex may retain residual fluores-

cence. As a result, without the correction by a the

fluorescence F would be overestimated and the value of K

would be underestimated.

2.4.3 Fluorescence Resonance Energy Transfer

The occurrence of fluorescence resonance energy transfer

between Trp and TPPS4- was probed qualitatively, using

fluorescence excitation and emission spectroscopy [3], as

well as quantitatively, using fluorescence lifetime to

establish FRET efficiency [30]. Because of the artifact

introduced by static quenching in the emission and exci-

tation spectra [3], fluorescence lifetime is more reliable in

establishing FRET efficiency.

2.4.4 Fluorescence Anisotropy

Steady-state fluorescence anisotropy was recorded by

selecting the excitation light and the emitted fluorescence

with manually rotating polarizers and recording the emis-

sion spectra of TPPS and the BLGA/TPPS complex at

various temperatures. The anisotropy was calculated

according to [31]

r ¼ IVV � GIVH

IVH � 2GIVH

ð11Þ

where I is the intensity of the emission (integral of the

spectrum) and the subscripts indicate, in spatial order, the

orientation of the excitation and emission polarizers

(V, vertical, H, horizontal). G is the factor that accounts

for the differential sensitivity of the instrument to light

polarized parallel and perpendicular, and is calculated as

G ¼ IHV

IHH

ð12Þ

Each value of intensity was calculated as the average of

three spectra. By varying the temperature of the sample

(using a circulating water bath connected to the water-

jacketed sample holder of the fluorimeter) we retrieved

data for the Perrin equation

1

r¼ 1

r0

þ sRT

gVr0

ð13Þ

which yields the value of the limiting anisotropy (r0) and

the hydrodynamic volume of the rotating molecule (V),

when plotting 1/r vs. T/g ratio between the temperature of

the solution and the viscosity of the medium [26]. R is the

gas constant. The Perrin equation was used to retrieve V

and establish, whether BLGA formed aggregates (larger

than the dimer) either alone or induced by the binding of

TPPS4-.

2.4.5 Effects of Urea

The effects of TPPS on the stability of BLGA was inves-

tigated using the dissociation coupled unfolding model

(DCU) proposed for BLG [32]. According to the model the

free energy of denaturation is calculated as DGDCU =

- RT ln KDCU, where KDCU is given by

KDCU ¼ 4½BLGA� F2D

1� FD

ð14Þ

and FD is the fraction of denaturated protein calculated as

FDEN ¼Y � Ymin

Ymax � Ymin

ð15Þ

where Y is an observed property of the protein [32] which

in this case is the position of the emission maximum, as a

function of urea. The standard free energy of denaturation

extrapolated at zero urea concentration is derived from the

equation

DGDCU ¼ DG0DCU � m½urea] ð16Þ

where m is the denaturant index [33].

2.4.6 Docking Simulations

Docking simulations were carried out using the software

package Arguslab 4.0 (Planaria Software Inc., Seattle,

WA). The TPPS4- structure, generated and optimized with

Chimera (University of California San Francisco) [34], was

docked to one of the available structures of BLG dimers

An Anionic Porphyrin Binds b-Lactoglobulin A 5

123

obtained from the Protein Data Bank (1BEB.pdb). The

protein was stripped of the water molecules encoded in the

structure. The porphyrin ligand was ‘‘manually’’ brought

into the vicinity of a possible binding site and the docking

computation was carried out using different initial orien-

tations of TPPS4-. Several possible binding sites on each

monomer as well as at the monomer/monomer interface

were probed.

3 Results

3.1 Absorption Spectroscopy

At all pH values the addition of BLGA to a stock solution

of TPPS4- produces a small bathochromic shift of the

Soret band (from 413 to 416 nm) and simultaneous hypo-

chromicity (Fig. 2). The same effect occurs at every pH

probed in this study. In agreement with what was suggested

by previous results [13] there is no evidence for the for-

mation of J-aggregates of TPPS4- (appearance of a band

near 490 nm) either before or after the addition of the

protein. Moreover, there is no clear shift of the Q-bands

induced by the presence of BLGA [16].

3.2 Fluorescence of TPPS4- bound to BLGA

At all pH values the addition of BLGA prompts a shift of

the emission maximum to longer wavelengths (Fig. 3a, b).

Such shift is larger ([4 nm) and occurs much earlier than

the one observed in absorption (Fig. 2). The red-shift is

more pronounced at higher pH values (Fig. 3c).

However, Fig. 3a, b also show that the normalized and

corrected intensity of the TPPS4- emission, changes only

slightly in the presence of BLGA. Such small change

would not allow the application of fluorescence intensity

methods to establish the amount of bound porphyrin [35,

36]. Therefore, the contribution of free and bound por-

phyrin to the emission spectrum must be retrieved via

Gaussian analysis of the emission spectra with Eq. 1

(Fig. 1). The plots of [TPPS4-]b/[TPPS4-]Tot vs. [BLGA]

obtained from the Gaussian fitting show the relative

increase of the fraction of bound porphyrin (Fig. 4). The

range of protein and TPPS concentration has to be kept

relatively low because of the restriction on the optical

density (O.D.) of the samples imposed by the fluorescence

method; in particular, signal linearity requirements impose

that the O.D. at the excitation wavelength be \0.2. Nev-

ertheless, fitting of the isotherms in Fig. 4 with Eq. 2,

yields an estimate of the binding constant, K, the coeffi-

cient n, and the factor c, which are summarized in Table 1.

The data show that the binding constant increases by

approximately 20% with increasing pH. The value of n

remains \1 at all pH values, indicating a negative coop-

erativity, and decreases by about 16% as the pH increases.

A student’s t-test analysis reveals that differences in the

value of K between pH are not statistically significant

(p-value C 0.2). Similarly, an analysis of the value of n

reveals that, although its decrease with increasing pH is

consistent with electrostatic surface binding of TPPS4 [37],

the differences in its value at various pH are not statisti-

cally significant (p-value C 0.1). Thus we are led to

conclude that that approximately one TPPS molecule binds

to each BLGA unit and that the binding constant is

approximately constant across the range of pH.

A large increase with the pH occurs in the value of c(Table 1). This factor depends on the emission quantum

yields for bound and free TPPS4-. Individual quantum

yields for free and bound porphyrins at various pH values

could not be experimentally calculated and are not avail-

able from the literature; therefore, our fitting only indicates

that the large change in the binding isotherm of Fig. 4 is

due to changes of the emission quantum yields of the free

and bound porphyrin at the different pH values.

3.3 Quenching of BLGA by TPPS

Each BLGA monomer has two Trp residues, thus each

BLGA dimer has potentially four Trp residues that can be

quenched. The contribution of each residue to BLGA

fluorescence is still under debate but some convincing

studies suggested that only Trp19 at the bottom of the

interior barrel contributes to the fluorescence of the protein

0

0.05

0.1

0.15

0.2

350 400 450 500

Wavelength (nm)

Op

tica

l Den

sity

Fig. 2 Absorption spectra of TPPS4- upon addition of BLGA. Black[BLGA] = 0 lM; Red [BLGA] = 0.73 lM; Blue [BLGA] =

2.48 lM; Green [BLGA] = 4.02 lM; Yellow [BLGA] = 5.5 lM

6 I. Silva et al.

123

[19, 29]. We, therefore, used Eq. 9 to analyze whether the

BLGA/TPPS4- complex retains some residual fluores-

cence. The results show (Fig. 5a) that the porphyrin

quenches 80–100% of the monomer fluorescence and that

this ratio is basically independent of the pH of the solution

Table 2. Fluorescence spectra of BLGA as a function of

added porphyrins (supplemental material) show that the

decrease of protein fluorescence is not accompanied by a

shift in its emission maximum. The values of the binding

constants retrieved from S–V plots (Fig. 5b; Table 2) are

numerically in good agreement with the ones retrieved

from TPPS4- fluorescence (Table 1) and they do not

appear to be pH-dependent.

3.4 Fluorescence Resonance Energy Transfer

Steady state and fluorescence lifetime experiments rule out

any significant FRET between the Trp residue and the bound

TPPS4-. Excitation of Trp residues does not produce

0

0.2

0.4

0.6

0.8

1

600 650 700 750

Wavelength (nm)

11 2

3

0

0.2

0.4

0.6

0.8

1

1.2

12 3

Wavelength (nm)

640

641

642

643

644

645

646

647

648

0 5 10-7

750

1 10-6

1.5 10-6

2 10-6

2.5 10-6

[BLGA] M

Flu

ore

scen

ce In

ten

sity

(a.

u.)

Flu

ore

scen

ce In

ten

sity

(a.

u.)

600 650 700 750

emm

axλ

(a) (b)

(c)

Fig. 3 Fluorescence spectra of

TPPS4- upon addition of

BLGA. 1 [BLGA] = 0 lM; 2[BLGA] = 1.33 lM; 3[BLGA] = 2.07 lM. a pH 6;

b pH 9. The spectra show the

red-shift of the emission upon

binding to the protein. The

spectra also show little change

of the emission intensity.

c Position of the emission

maximum of TPPS4- as a

function of the concentration of

BLGA. d pH 6, j pH 7, r pH

8, m pH 9. The red-shift occurs

nearly at the same rate at all pH

values

0

0.05

0.1

0.15

0.2

0 5 10 -7 1 10 -6 1.5 10 -6 2 10 -6

[BLGA] µM

[TP

PS

4-] b

/[T

PP

S4-] to

t

Fig. 4 Plot and fitting of bound/total TPPS4- according to Eq. 3.

d pH 6, j pH 7, r pH 8, m pH 9

Table 1 Binding parameters obtained from the fluorescence of

TPPS4- using Gaussian fitting and Eq. 2

c K 9 10-5 (M-1) n

pH 6 0.67 ± 0.12 0.63 ± 0.14 0.85 ± 0.04

pH 7 0.72 ± 0.14 0.75 ± 0.17 0.86 ± 0.06

pH 8 0.85 ± 0.13 0.76 ± 0.16 0.69 ± 0.10

pH 9 0.95 ± 0.17 0.78 ± 0.18 0.71 ± 0.08

An Anionic Porphyrin Binds b-Lactoglobulin A 7

123

emission by the ligands and, likewise, excitation spectra

recorded at the maximum of the emission wavelength of the

porphyrin do not show any contribution from Trp. Fluo-

rescence decay experiments also reveal that there is no

change in BLGA emission lifetime upon addition of

TPPS4-. Such result is also in strong support of the occur-

rence of static quenching due to binding of the porphyrin to

the protein. Lack of FRET is consistent with what had been

observed in other systems [3, 17] and is a direct consequence

of the very small overlap between the emission spectrum of

BLGA and the absorption spectrum of TPPS4-.

3.5 Circular Dichroism

At all pH values, CD experiments in the region of the Soret

band did not show any optical activity of TPPS4- which

indicates that binding to BLGA does not distort the struc-

ture of the porphyrin ring.

At the same time the binding of the porphyrin did not

produce any change in the dichroic signal of BLGA either

in the region of the aromatic amino acids (260–320 nm) or

in the region of the amide (180–250 nm).

3.6 Effects of Urea

In order to probe whether the porphyrin binds to sites at the

monomer/monomer interface we studied the effects of the

addition of urea to solutions containing the BLGA/TPPS4-

non-covalent complex. It is known that, within a certain

range of urea concentrations, which depends on the pH of

the solution [32], BLGA dimers dissociate without any

appreciable change in the tertiary structure of the individual

monomers [32]. Thus if TPPS4- binds to the dimer at the

interface between the two monomers, the addition of urea

may cause the porphyrin to return in solution upon disso-

ciation of the dimers. This in turn would cause the emission

maximum of the porphyrin to blue-shift towards the position

of free TPPS4-. Our results, however, show (Fig. 6) that

addition of urea up to 8 M (a concentration where urea is

known to partially unfold the monomeric form of BLGA

[32, 38]) does not cause any shift of fluorescence of BLGA-

bound TPPS4-. Addition of the same aliquots of urea to

solutions containing only TPPS produced only a slight

(2 nm) red-shift of the emission of the porphyrin, probably

due to the sequestration of water molecules by urea. Since

the red-shift resulting from binding to BLG is 7 nm, the lack

of blue-shift of BLG-bound TPPS upon addition of urea

indicates that TPPS4- remains attached to the protein even

when this is partially unfolded.

The dissociation and unfolding of BLGA at increasing

urea concentration was probed by the red-shift and the

increase in intensity of the intrinsic fluorescence of the

protein [19] (supplemental material).

The comparison of urea-induced denaturation of BLGA

alone and BLGA in the protein/porphyrin complex shows

that the presence of bound TPPS4- stabilizes BLGA, as the

sigmoidal transition to the unfolded state appears to be

delayed (Fig. 7a, b). Calculation of DG0DCU and m with

Eq. 16 produces the values shown in Table 3. By com-

parison with the data obtained in the absence of TPPS4- it

can be seen that while the denaturant index, m, remains

virtually unchanged, DG0DCU of the BLGA/porphyrin

complex increases, at all pH values. The increased stability

2

4

6

8

10

2 105

4 105

6 105

8 105

1 106

1/[TPPS] M-1

1

1.2

1.4

1.6

1.8

0 1 10-6 2 10-6 3 10-6 4 10-6 5 10-6 6 10-6 7 10-6

[TPPS] M

F0/∆

F

F0/F

(a) (b)Fig. 5 a Plot F0/DF according

to Eq. 10 (model of

fractional quenching of [32]).

b Stern–Volmer plot of BLGA

fluorescence corrected

according to Eq. 11. d pH 6,

j pH 7, r pH 8, m pH 9

Table 2 BLGA fluorescence quenching. Fraction of quenched fluo-

rescence and binding constant obtained from Eqs. 9 and 10

a K 9 10-5 (M-1)

pH 6 0.89 ± 0.09 1.56 ± 0.33

pH 7 1.10 ± 0.05 1.10 ± 0.25

pH 8 0.91 ± 0.03 1.37 ± 0.29

pH 9 0.96 ± 0.08 1.14 ± 0.18

8 I. Silva et al.

123

against denaturation induced by binding of TPPS4- is

larger at smaller pH (Fig. 7c).

3.7 Fluorescence Anisotropy

Perrin plots from Eq. 13 are linear and show, as expected, a

steeper slope for free TPPS than for the BLGA/TPPS

complex at all pH (Fig. 8a, b). From the slope of the plot

and upon measurements of free and bound TPPS fluores-

cence lifetime (9.8 and 11 ns, respectively) we estimated

the radius of the free ligand to be\1 nm and the radius of

the complex to be 2.5–3 nm. The radius of the BLGA/

TPPS complex is in excellent agreement with the size of

the BLGA dimer reported by others [39, 40]. This in turn

confirms that in the pH and concentration range investi-

gated, binding of TPPS4- does not induce aggregation of

BLGA.

3.8 Docking Simulations

A survey of the BLGA dimer structure (PDB file 1BEB.pdb)

provides several potential binding sites for TPPS4-. There

are several Lys and Arg residues on the surface as well as in

0

0.5

1

1.5

600 650 700 750

Wavelength (nm)

[urea] = 0 M

[urea] = 2 M

[urea] = 4 M

[urea] = 6 M

[urea] = 8 M

Flu

ore

scen

ce In

ten

sity

Fig. 6 Fluorescence of TPPS4- in the protein/porphyrin complex as

a function of urea concentration. The spectra are normalized and

offset with respect to each other to show that the emission maximum

of bound TPPS4- is not affected by the addition of up to 8 M urea

0

0.2

0.4

0.6

0.8

1

-2 0 2 4 6 8 10

[urea] M

1

1.1

1.2

1.3

1.4

1.5

5.5 6 6.5 7 7.5 8 8.5 9 9.5

pH

0

0.2

0.4

0.6

0.8

1

-2 0 2 4 6 8 10

[urea] M

FD

EN

FD

EN

∆G0 D

CU(B

LG

A/T

PP

S4-

)/∆G

0 DC

U(B

LG

A)

(a)

(c)

(b)Fig. 7 Denaturation curves.

FDEN calculated according to

Eq. 18 using the wavelength of

the emission maximum as the

parameter Y. a pH 7: d BLGA/

TPPS4- complex, s BLGA

alone; b pH 9: d BLGA/

TPPS4- complex, s BLGA

alone. The data are

representative of the trend at the

other pH values. c Relative

increase of DGDCU induced by

the presence of bound TPPS4-

as a function of pH

An Anionic Porphyrin Binds b-Lactoglobulin A 9

123

the grove formed between the 3-turn helix and the outside of

the b-barrel [14, 22, 41]. In addition we investigated the

docking at intradimeric sites on the side of the 3-turn helices

as well as on the side of the aperture of the barrel [42]. We

attempted the docking to all these locations by approaching

TPPS4- at different orientations to each site. Our results

show that there is no stable binding configuration for either

the interior of the b-barrel or the outside grove. In addition,

docking at the monomer-monomer interface did not yield

stable binding configurations.

Our docking computation revealed that the most likely

binding site for TPPS4- is on the surface of BLGA at a

location which involves a near interdigitation of three of

the four SO3- groups of TPPS4- and the positively charged

side chains of Lys14, Lys47, Lys100 and Arg124 (Fig. 9a).

This location also places the ligand in proximity (*7.2 A´

)

of the fluorescent Trp19 residue (Fig. 9b) which would

explain the static quenching produced by the binding of

TPPS4- to BLGA. Thus, the computed binding site

involves regions that include the N-terminus, the B-strand,

the FG-loop, and the loop between strand I and the 3-turn

a-helix [14, 41]. Because of the large number of surface

Arg and Lys it is likely that other surface regions may also

offer docking sites to the porphyrin.

Table 3 Thermodynamic

parameters for the urea-induced

unfolding of BLGA in the

complex with TPPS4- and alone

at different pH values

m BLGA/TPPS4-

complex

m BLGA alone DG0DCU (kJ/mol)

BLGA/TPPS4- complex

DG0DCU (kJ/mol)

BLGA alone

pH 6 6.38 ± 0.52 4.44 ± 0.91 74.4 ± 4.1 51.1 ± 5.1

pH 7 4.46 ± 0.78 2.86 ± 0.40 53.3 ± 4.5 38.0 ± 4.3

pH 8 3.38 ± 0.42 2.79 ± 0.27 45.3 ± 4.3 35.8 ± 4.5

pH 9 2.53 ± 0.61 2.53 ± 0.22 40.1 ± 4.0 34.8 ± 5.0

3.2

3.4

3.6

3.8

4

4.2

4.4

2.5 104

3 104

3.5 104

4 104

4.5 104

5 104

5.5 104

6 104

6.5 104

pH 9

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

2.5 104

3 104

3.5 104

4 104

4.5 104

5 104

5.5 104

6 104

6.5 104

T/η (K/Pa) T/η (K/Pa)

1/r

1/r

(a) (b)Fig. 8 Perrin plots of the steady

state fluorescence anisotropy

(Eq. 16). a pH 6: d BLGA/

TPPS4- complex, j TPPS4-

alone; b pH 9: d BLGA/

TPPS4- complex, j TPPS4-

alone. The data are

representative of the trend at the

other pH values and show a

larger slope for TPPS alone than

for the porphyrin bound to the

proteins

Fig. 9 Simulation of the

docking of TPPS4- to BLGA. aThe most stable docking

configuration produces TPPS4-

(red) laying flat on the surface

of BLGA with its four SO3-

groups interacting with Lys

(yellow) and Arg (blue)

positively charged residues. bDifferent view of the same

binding site which shows that

TPPS4- (red) is \8 A´

away

from the indole ring of Trp19

(blue)

10 I. Silva et al.

123

4 Discussion

Despite its solubility in aqueous solutions, it had been

previously established that TPPS is able to bind small

globular proteins such as BLG and tubulin [13, 17]. This

investigation introduces, however, the analysis of the

effects of the pH-dependent conformational changes of

BLG [14, 15] on the binding of TPPS4- and establishes a

possible location for the docking of the porphyrin.

Assuming that the contribution of TPPS2- is negligible in

the pH range investigated (the pKa for TPPS is \4.5 [16,

43]), we considered TPPS4- as the only significant ligand.

First of all our results show that for 6 B pH B 9 there is

no formation of the porphyrin J-aggregate either before

binding or induced by BLGA.

The binding parameters obtained from the fluorescence

of the porphyrin and the quenching of the protein

(Tables 1, 2) show that the numerical values of K

retrieved from S–V plots (Fig. 5b) is higher than the one

obtained from the curves of Fig. 4. If the data obtained

with the two methods are compared with a student’s

t-test, the analysis reveals that 0.05 B p B 0.09. Although

the difference can be considered somewhat statistically

significant, one has to consider the very small sampling

associated with our measurements which makes the dif-

ferences between the values of K retrieved with the two

methods, difficult to interpret. Therefore, we can assume

that the data obtained from TPPS fluorescence and BLGA

quenching (with Eqs. 2, 10, respectively) yield the same

qualitative results that the affinity of the porphyrin for

BLGA does not change with the pH of the solution. Since

across the pH range probed with this study, BLGA

undergoes a major conformational change that involves

the access to the interior protein pocket (b-barrel) [14],

our results rule out that the pocket, or its proximity,

represents a binding site for the porphyrin. This is

somewhat expected since the insertion of one or more

SO3- substituents into the hydrophobic pocket would

carry a very unfavorable entropic contribution [14, 44].

The unsuitability of this site as a binding location for

TPPS4- is also confirmed by the absence of stable

binding configurations in our docking simulations inside

or in proximity of the interior pocket of the protein.

Possible alternative binding sites could be present on

both sides of the ‘‘seam’’ at the monomer/monomer inter-

face of the BLGA dimer [42]. However, our experimental

and computational data rule this site out. Urea experiments

show (Fig. 6) that even upon substantial denaturation of the

protein, the porphyrin remains attached to BLGA, thus

ruling out that the separation of the two monomers deta-

ches the porphyrin from the protein. In agreement with this,

computational docking did not yield any stable binding

conformation at the monomer/monomer interface.

All our experimental results instead seem to point in the

direction of a superficial site. The fact that the pH does not

affect binding constant of TPPS4- is in agreement with

such model. Because of the small size of BLGA, if the

binding site is not inside or in proximity of the aperture of

the interior barrel, then the binding possibilities for a rel-

atively large ligand, such as TPPS4- (Fig. 9), is limited.

Since our computational docking simulations rule out

binding to the grove formed between the outside of the

barrel and the 3-turn a-helix, the remaining binding sites

are on the surface of the protein. A superficial binding site

is also consistent with the lack of dichroic signal from the

porphyrin. The lack of CD signal signifies that the ring of

the porphyrin remains substantially undistorted as it would

if the whole porphyrin laid on the surface of the protein.

The urea data (Fig. 6) also support a superficial binding site

since even extensive (albeit not total [32, 45]) unfolding of

BLGA does not prompt the release of TPPS4- in solution.

Such an effect would be unlikely if the porphyrin was

bound to a ‘‘fold’’ or a pocket of the protein through non-

Coulombic interactions.

How would TPPS4- bind to the surface of BLGA? In

fact, why should a water soluble porphyrin such as TPPS4-

bind to a protein in the first place? We have shown that the

same porphyrin binds another, larger, globular protein

(tubulin) also at the surface [17] and that such interaction is

stabilized by the Coulombic interaction between one of the

SO3- groups and positively charged amino acids (His and

Arg in that case [17]). Thus we suggest that the interaction

between TPPS4- and BLGA occurs through Coulombic

interactions between the positively charged amino acids on

the surface of the protein and the negatively charged

groups on the porphyrins. Such interaction would substitute

the weaker ion-dipole interaction between the SO3- groups

and water molecule (solvation), thus explaining why a

water-soluble ligand would bind a protein in aqueous

solution. Even a substantial denaturation of the protein

would still keep the porphyrin ‘‘sticking’’ to the surface of

the protein since urea does not affect the protonation of

amino acid residues.

This scenario would also explain why the fitting with

Eq. 2 yields values of n \ 1. BLGA is not a specific

‘‘receptor’’ for TPPS thus, one would expect a set of n-

independent, non-interacting binding sites for the porphyrin

(i.e., there is no reason, and no CD evidence that BLGA

changes its conformation upon binding of one TPPS mol-

ecule to accommodate or inhibit the binding of additional

porphyrin molecules). One would expect a value of n = 1

according to the Hill theory of cooperative binding [46]. A

value of n \ 1, however, indicates some degree of coop-

erativity but because there is no evidence to support the

presence of interacting sites for TPPS4-, a likely explana-

tion for the values of n is that the binding of one porphyrin

An Anionic Porphyrin Binds b-Lactoglobulin A 11

123

molecule to the surface of the protein leads to a decrease of

the available surface charges and effectively reduces the

probability of binding of additional TPPS4- molecules.

Finally, the superficial binding is confirmed by our

docking simulations (Fig. 9a) which place the negative

groups of the porphyrin in close proximity (in fact almost

perfectly interdigitated) with positively charged amino

acids such as Lys and Arg residues. Although, as explained

above at the pH values of our experiments both molecules

carry an excess of negative charge, it is very likely that

many of the basic amino acids, Lys and Arg in particular,

still carry a net positive charge [23] making the interaction

with TPPS4- possible.

According to our anisotropy data (Fig. 8) the electro-

static ‘‘shielding’’ provided by the binding of TPPS4- is

not sufficient to cause aggregation of BLGA as seen

instead for other proteins [47].

Finally a superficial binding site such as the one sug-

gested in this work would explain also the stabilization

effect that TPPS4- produces on BLGA. The delay in the

urea-dependent unfolding and the higher free energy of

unfolding (Fig. 7) can be explained by the fact that TPPS4-

could act like ‘‘glue’’, linking different domains of the

protein [e.g., the N-terminus, the B-strand, the FG-loop,

and the loop between strand I and the 3-turn a-helix

(Fig. 9)]. The Coulombic interactions between the negative

porphyrin and the positive amino acid residues would

stabilize this region and as a consequence the whole protein

against urea denaturation. At lower pH values the anionic

porphyrin would be more effective in stabilizing the pro-

tein as the surface of the polypeptide contains a larger

number of positively charged residues. As shown by the

data in Table 3 the increase in DG0 between the BLGA/

TPPS complex and BLGA alone is larger at smaller pH.

The difference in the values of DG0 between complex and

protein alone is statistically significant (p-value B 0.01) at

pH 6 and 7 while it becomes smaller and statistically

insignificant at pH 8 and 9 (p-value C 0.05). Thus is fur-

ther indication of the stabilization effect that tetra-anionic

TPPS produces on BLGA at low pH.

Acknowledgments The research was supported by the 2006 Faculty

Research Award of the University of Texas at San Antonio (to L.B.)

and by the AFRL/HE grant # FA8650-07-1-6850 (to L.B.). The

author would also like to thank Dr. Markandeswar Panda for the use

of the CD spectrometer.

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