Complex of nicosulfuron with human serum albumin: A biophysical study

Journal of Molecular Structure 975 (2010) 256–264

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Complex of nicosulfuron with human serum albumin: A biophysical study Fei Ding a, Wei Liu a,b, Nan Li a, Li Zhang c,*, Ying Sun a,** a

Department of Chemistry, China Agricultural University, Beijing 100193, China College of Economics & Management, China Agricultural University, Beijing 100083, China c Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193, China b

a r t i c l e

i n f o

Article history: Received 26 February 2010 Received in revised form 23 April 2010 Accepted 23 April 2010 Available online 29 April 2010 Keywords: Nicosulfuron Human serum albumin Fluorescence spectroscopy Circular dichroism Molecular modeling Protein denaturation

a b s t r a c t Nicosulfuron is a sulfonylurea herbicide developed by DuPont that has been used successfully for weed control in maize. The binding mechanism and binding site identified in human serum albumin (HSA) with the use of fluorescence, circular dichroism (CD) and molecular modeling is the subject of this paper. From the CD, synchronous and three-dimensional fluorescence results, it was apparent that the interaction of nicosulfuron with HSA caused secondary structure changes in the protein. Fluorescence data revealed that the nicosulfuron induced the fluorescence quenching of HSA through a static quenching procedure. Thermodynamic analysis results implied the role of hydrophobic and hydrogen bonds interactions in stabilizing the nicosulfuron–HSA complex. Site marker competitive experiments showed the binding of nicosulfuron to HSA primarily took place in subdomain IIA (Sudlow’s site I), this corroborates the guanidine hydrochloride (GuHCl) induced denaturation of HSA, hydrophobic probe ANS displacement and molecular modeling results. In this work, the presented binding research extends our knowledge of the binding properties of sulfonylurea herbicides to the important plasma protein HSA. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Sulfonylurea herbicides were first introduced in 1982 by DuPont Agricultural Products. Due to its high herbicidal activity and low toxicity for mammals, they have widespread control over broad-leaved weeds and some grasses in cereals and are widely used for a variety of crops [1]. Among sulfonylurea products, nicosulfuron (2-[[[[(4,6dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-N,N-di methyl-3-pyridinecarboxamide) is very effective even applied at low rates (<100 g ha1) and is widely used for selective post-emergence control of annual and perennial grasses in maize [2]. It inhibits the enzyme acetolactate synthase (ALS), which is a key enzyme in the biosynthesis of branched chain amino acids viz. valine, leucine and isoleucine, and hence inhibiting cell division [3]. The highest use of nicosulfuron is on maize and approximately 200,000 lb annually [4], after the foliar application, the herbicide permeates into soil and can subsequently run off from cropland into rivers, ponds and lakes, causing surface and groundwater contamination. Since European Union indicates 0.1 lg L1 as the maximum limit value for a single pesticide in drinking water destined to human consumption, several analytical methods have been proposed for the determina-

* Corresponding author. Address: Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193, China. Tel./fax: +86 10 62737071. ** Corresponding author. Tel./fax: +86 10 62737071. E-mail addresses: [email protected] (L. Zhang), [email protected] (Y. Sun). 0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2010.04.033

tion of nicosulfuron, such as chromatography, mass spectrometry, immunoassay, capillary electrophoresis [1,3]. Sulfonylurea (e.g. nicosulfuron) has been proved to induce hypoglycemia in humans [5,6]. Recently, Simpson and colleagues [7] have clearly demonstrated that sulfonylureas increase the risk of cardiovascular disease; moreover, their result adds endorsement to a casual connection by identifying a dose-related effect on the risk of death. It is worth noting that the free concentration available for the toxic action can be obviously reduced for pesticides with high affinity to proteins, for the reason that proteins can dominate the extent and duration of toxicological effect in the body, although bindings of pesticide to proteins are often lower than that to the target enzyme [8]. However, no reports in the literature have appeared regarding the binding mechanism and binding site of nicosulfuron on protein at molecular level. Owing to the widespread use of nicosulfuron in fields around the world, which implied the potential toxicological risk to humankind, the investigation on the interaction of nicosulfuron with protein is of great importance. Human serum albumin (HSA) is the most abundant protein in blood plasma, which constitutes about 60% of the total plasma protein, and providing about 80% of the blood osmotic pressure [9]. The exceptional feature of HSA is its ability to bind and transport of many endogenous and exogenous substances, for example, fatty acids, metabolites, drugs, dyes and pesticides [10,11]. Also, it is widely accepted that the degree of affinity between ligand and HSA can dominate its distribution into target tissue, affect its elimination from the body, and finally influence its therapeutic

F. Ding et al. / Journal of Molecular Structure 975 (2010) 256–264

or toxic effects, biotransformation and biodistribution etc. of ligands [12]. Besides, there is testimony of conformational changes of protein caused by its interaction of ligands, and these changes seem to affect the secondary and tertiary structure of protein [12]. Thus, it is significant to study the interaction of nicosulfuron with HSA, this sort of studies may supply pivotal information on the structural characteristics that determine the toxic effect of nicosulfuron, since it has been proved that the distribution and free concentration of various ligands can be drastically altered as a result of their binding to HSA, and therefore become a focal research area in chemistry, life science and clinical medicine. Numerous methods have been utilized to study the ligand–protein interactions, including ultrafiltration, ultracentrifugation, equilibrium dialysis, fluorescence, UV/vis, capillary electrophoresis, NMR, surface plasmon resonance, chromatography, etc. [10,11,13,14]. Among them, fluorescence spectroscopy has been widely used due to its inimitable sensitivity, rapidity, and simplicity [15]. At the same time, rigorous literature survey divulges that attempts have not yet been made to investigate the binding mechanism and binding site of interaction of nicosulfuron with HSA. The intent of the study was to determine the binding mechanism and binding site of nicosulfuron with HSA by fluorescence, circular dichroism (CD) and molecular modeling methods. Great essays were made to investigate the binding properties pertain to the secondary structure changes of HSA, quenching mechanism, thermodynamic functions, specific binding site and binding patches. This work should give more understanding on realizing the transport and metabolism process of nicosulfuron, the relationship of structure and function of HSA, and the chemical essence of the interaction between biomacromolecule and ligand. 2. Materials and methods 2.1. Materials HSA (fatty acid free <0.05%) and nicosulfuron were obtained from Sigma–Aldrich Chemical Company, and used without further purification. All other reagents were of analytical reagent. Milli-Q ultrapure water was applied throughout the experiments. NaCl (1.0 M) solution was employed to maintain the ionic strength at 0.1. Tris (0.2 M)-HCl (0.1 M) buffer solution containing NaCl (0.1 M) was used to keep the pH of the solution at 7.4. The pH was checked with a suitably standardized Orion-868 pH meter (Orion, USA). Dilutions of the HSA stock solution (1.0  105 M) in Tris–HCl buffer were prepared immediately before use, and the concentration of HSA was determined spectrophotometrically using E1 cm1% of 5.30 at 280 nm [16]. The stock solution of nicosulfuron was prepared in Tris–HCl buffer.

257

the MRE values at 208 nm using the following equation described by Greenfield and Fasman [17]:

%a-helix ¼

MRE208  4000 33; 000  4000

ð2Þ

2.2.2. Fluorescence spectra Steady state fluorescence spectra were performed on a F-4500 spectrofluorometer (Hitachi, Japan) equipped with 1.0 cm quartz cuvette and a thermostat bath. Fluorescence quenching spectra were recorded at 291, 297, 303 and 309 K in the range of 250– 450 nm. The width of the excitation and emission slits was set to 5.0 nm for all the measurements. An excitation wavelength of 295 nm was chosen and very dilute solution was applied in the experiment (HSA 1.0  106 M, nicosulfuron in the range of 0–9.0  106 M). Fluorescence spectra were the average of five scans with the baseline corrected by Tris–HCl buffer as the control. 2.2.3. Three-dimensional fluorescence spectra Three-dimensional fluorescence spectra were measured under the following conditions: the emission wavelength was recorded between 200 and 500 nm, and the initial excitation wavelength was set to 200 nm with increment of 10 nm, the number of scanning curves was 16, and other scanning parameters were identical to those of the steady state fluorescence spectra. 2.2.4. Site-specific probe Site marker competitive experiments: binding location studies between nicosulfuron and HSA in the presence of four site markers (phenylbutazone, ibuprofen, digitoxin and hemin) were measured using the fluorescence titration method. The concentrations of HSA and site markers were held in equimolar (1.0  106 M), then nicosulfuron was added to the HSA-site markers mixtures. An excitation wavelength of 295 nm was chosen and the fluorescence emission wavelength was registered from 250 to 450 nm. 2.2.5. Hydrophobic probe ANS Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement experiments: in the first series of experiments, HSA concentration was kept fixed at 1.0  106 M, and nicosulfuron/ ANS concentration was varied from 1.0 to 9.0  106 M, fluorescence emission spectra of HSA was recorded (kex = 295 nm, kem = 334 nm). In the second series of experiments, the nicosulfuron was added to solutions of HSA and ANS held in equimolar concentrations (1.0  106 M), the concentration of nicosulfuron was also varied from 1.0 to 9.0  106 M and ANS fluorescence was measured (kex = 370 nm, kem = 465 nm).

2.2. Apparatus and methods 2.2.1. CD spectra CD spectra were recorded with a Jasco-810 spectropolarimeter (Jasco, Japan) using a 0.2 cm path length quartz cell. Measurements were taken at wavelengths between 200 and 260 nm with 0.1 nm step resolution and averaged over five scans recorded as a speed of 20 nm min1. All observed CD spectra were baseline subtracted for buffer solution and the results were taken as Mean Residue Ellipticity (MRE) in deg cm2 dmol1 which is defined as

MRE ¼

hobs 10  n  l  C p

ð1Þ

where hobs is the CD in millidegree, n is the number of amino acid residues (585), l is the path length of the cuvette, and Cp is the HSA molar concentration. a-helical content was calculated from

2.2.6. Molecular modeling Molecular modeling of the HSA–nicosulfuron interaction was performed on SGI Fuel Workstation. The crystal structure of HSA was downloaded from Brookhaven Protein Data Bank (entry codes 1H4 K, resolution 2.4 Å, http://www.rcsb.org/pdb). The twodimensional structure of nicosulfuron was downloaded from PubChem (http://pubchem.ncbi.nlm.nih.gov). The potential of the three-dimensional structure of HSA was assigned according to the AMBER force field with Kollman all-atom charges. The initial structure of nicosulfuron was generated by molecular modeling software Sybyl 7.3. The geometry of the molecule was subsequently optimized to minimal energy using the Tripos force field with Gasteiger-Hückel charges, and the Surflex docking program was applied to calculate the possible conformation of the ligand that binds to the protein [18].

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2.2.7. Statistical analysis All experiments were performed in n = 3 repetitions, the mean values, standard deviations, and statistical differences were evaluated using analysis of variance (ANOVA). The mean values were compared using student’s t-test, and all statistic data were processed using the OriginPro Software (OriginLab Corporation, USA). 3. Results and discussion 3.1. HSA structural alterations 3.1.1. Synchronous fluorescence spectra Synchronous fluorescence spectroscopy was introduced by Lloyd in 1971 [19], which involves simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. It can provide the information about the molecular environment in a vicinity of the chromophore molecules. According to the theory of Miller [20], when the D-value (Dk) between excitation and emission wavelength was stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues (Tyr) or Trp residues. The effect of nicosulfuron on HSA synchronous fluorescence spectra were displayed in Fig. 1. As shown in Fig. 1, the maximum emission wavelength has a slight red shift at the investigated concentration range when Dk = 60 nm, but has no shift when Dk = 15 nm. The red shift effect explains that the conformation of HSA was altered, the polarity

Fluorescence Intensity

(A)

Δλ = 15nm 250

a 200 150

j

100 50

280

320

300

Wavelength (nm)

3.1.3. Three-dimensional fluorescence spectra Three-dimensional fluorescence spectroscopy is a vigorous method for providing conformational and structural information of proteins. The three-dimensional fluorescence spectra of HSA and HSA–nicosulfuron system are shown in Fig. 3, and the corresponding characteristic parameters are collected in Table 1. Peak a is the Rayleigh scattering peak (kex = kem), peak b is the secondorder scattering peak (kem = 2kex). Peak 1 (kex = 280.0 nm, kem = 329.0 nm) mainly reveals the spectral feature of Tyr and Trp residues. Because when HSA is excited at 280 nm, it primarily displays the intrinsic fluorescence of Trp and Tyr residues, and the phenylalanine residue fluorescence can be negligible [25]. Besides peak 1, peak 2 (kex = 230.0 nm, kem = 329.0 nm) chiefly exhibits the fluorescence spectral behavior of polypeptide chain backbone structures. The fluorescence intensity of peak 2 decreased a lot

700

MRE (deg cm2 dmol-1)

Δλ = 60nm

600

Fluorescence Intensity

3.1.2. CD spectra CD is an extremely convenient technique for detecting and monitoring the extent of conformational changes that may be associated with the activity or regulation of a protein. To further verify the possible influence of nicosulfuron binding on the secondary structure of HSA, we have performed far-UV CD studies in the absence and presence of 2.0  106 M and 4.0  106 M sulfonylurea [23]. The raw CD spectra of HSA in the absence and presence of nicosulfuron were displayed in Fig. 2. It was apparently observed that CD spectra of HSA exhibited two negative bands in the UV region at 208 and 222 nm, characteristic of a-helical structure of protein. A legitimate interpretation is that the negative peaks between 208 and 209 nm and 222 and 223 nm are both contributed by n ? p transition for the peptide bond of a-helix [17]. In addition, the band intensities of HSA at 208 and 222 nm decreased with the negative Cotton effect through the binding of nicosulfuron without causing conspicuous shift of the peaks, clearly indicating that nicosulfuron induced a slight decrease in the a-helical structure content of HSA. The a-helical structure content of HSA was calculated from Eq. (2), and the results displayed a reduction of a-helical content from 45.37% to 40.34% and 36.29% at a molar ratio of HSA to nicosulfuron of 1:2 and 1:4, respectively. From the above results, it was evident that the effect of nicosulfuron on HSA caused secondary structure changes of the protein and with the loss of helical stability [24].

300

0 260

(B)

around the Trp residue was increased and the hydrophobicity was decreased [21,22].

a

500 400 300

j 200

0

c -10000

100

a 0 240

260

280

300

320

Wavelength (nm) Fig. 1. Synchronous fluorescence spectra of HSA (1.0  106 M) in the absence and presence of nicosulfuron (pH = 7.4, T = 291 K), (a) ? (j): 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0  106 M. (A): Dk = 15 nm, (B): Dk = 60 nm.

-20000 200

220

240

260

Wavelength (nm) Fig. 2. CD spectra of the HSA–nicosulfuron system. (a) 1.0  106 M HSA; (b) 1.0  106 M HSA in the presence of 2.0  106 M nicosulfuron; (c) 1.0  106 M HSA in the presence of 4.0  106 M nicosulfuron pH = 7.4.

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(A)

3.2. Fluorescence quenching of HSA

peak 2 peak 1a b 400

200

Fluorescence Intensity

600

0350

500 450

300 400

250

350

300

EM (n m)

(B)

250

200

peak 1

) nm

( EX

200

a

3.2.1. Steady state fluorescence Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore, which is induced by a variety of molecular interactions, such as excited state reactions, energy transfer, ground state complex formation and collisional quenching [25]. From a quantitative perspective, fluorescence quenching of HSA could be utilized to derive protein–ligand binding information, such as quenching mechanism, association constant, the number of binding site, thermodynamic parameter etc. In order to explore quenching mechanism and various association constants, the fluorescence quenching spectra of HSA with increasing concentrations of nicosulfuron in the different molar ratios were registered with the excitation wavelength at 295 nm (Fig. 4). HSA shows a strong fluorescence emission peak at 334 nm due to its sole Trp residue (Trp-214), and the fluorescence intensity of HSA decreased regularly with the increase of nicosulfuron concentrations. Under the same conditions, there was no fluorescence emission for nicosulfuron at the investigated concentration range. These phenomena enunciated that the nicosulfuron could interact with HSA and thus quenching its intrinsic fluorescence [26].

b

400

200

Fluorescence Intensity

600

peak 2

0350

500 450

300 400

350

250 300

EM (n m)

250

200

200

EX

) (nm

Fig. 3. Three-dimensional fluorescence spectra of HSA (A) and the HSA–nicosulfuron system (B). (A): c(HSA) = 1.0  106 M, c(nicosulfuron) = 0; (B): c(HSA) = 1.0  106 M, c(nicosulfuron) = 1.0  106 M. pH = 7.4, T = 291 K.

after the addition of nicosulfuron, which implied that the peptide chain structure of HSA was changed and this result was consistent with CD spectra. Analyzing from the fluorescence intensity changes of the two peaks, which decreased evidently but to a different degree: the fluorescence intensity of peak 1 has been quenched of 4.70%, while peak 2 of 19.42%. The decrease of the intensity of peak 1 and 2 in combination with the synchronous fluorescence and CD results, demonstrated that the binding of nicosulfuron to HSA induced the slight unfolding of the polypeptide chain of HSA, which led to conformational change of HSA and increased the disclose of some hydrophobic regions that had been buried before. All the above phenomena and analyses confirmed that the interaction of nicosulfuron with HSA caused conformational and microenvironmental alterations in HSA molecule.

3.2.2. Binding property of nicosulfuron to HSA Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and the quencher, or static, resulting from the formation of a ground state complex between the fluorophore and the quencher. In both cases, molecular contact is required between the fluorophore and the quencher for fluorescence quenching to occur. Fluorescence quenching is described by the well-known Stern–Volmer equation [25]:

F0 ¼ 1 þ kq s0 ½Q  ¼ 1 þ K SV ½Q F

ð3Þ

where F0 and F are the fluorescence intensities before and after the addition of the quencher, respectively, kq is the bimolecular quenching constant, s0 is the lifetime of the fluorophore in the absence of the quencher (s0 = 108 s), [Q] is the concentration of the quencher, and KSV is the Stern–Volmer quenching constant. Hence, Eq. (3) was applied to determine KSV and kq by linear regression of F0/F versus [Q]. The fluorescence intensities were corrected for absorption of exciting light and reabsorption of the emitted light to decrease the inner filter effect using the relationship [25]: Aex þAem 2

F cor ¼ F obs  e

ð4Þ

where Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the absorption of the systems at the excitation and the emission wavelength, respectively. The fluorescence intensity used in this paper is the corrected intensity. Fig. 5 shows the Stern–Volmer plots for the HSA fluorescence quenching by the nicosulfuron and the calculated KSV and kq values are summarized in Table 2. The results display that the KSV values decreased with the increase of temperature and the kq values were much greater than 2.0  1010 M1 s1 [27], therefore, the probable quenching mechanism of the intrinsic fluorescence of HSA was not initiated by dynamic collision but by nicosulfuron– HSA complex formation.

Table 1 Three-dimensional fluorescence spectral characteristic parameters of HSA and HSA–nicosulfuron system. Peaks

Fluorescence peak 1 Fluorescence peak 2

HSA

HSA–nicosulfuron

Peak position kex/kem (nm/nm)

Stokes Dk (nm)

Intensity F

Peak position kex/kem (nm/nm)

Stokes Dk (nm)

Intensity F

280.0/329.0 230.0/329.0

49.0 99.0

635.9 606.5

280.0/330.0 230.0/331.0

50.0 101.0

606.0 488.7

260

F. Ding et al. / Journal of Molecular Structure 975 (2010) 256–264

3. The results show that Ka values were inversely correlated with temperatures, and this is in accordance with KSV’s dependence on temperature as mentioned above, which also coincides with static quenching mechanism. Dufour and Dangles [29] demonstrated that most ligands bound reversibly and display moderate affinities for serum albumin (association constants in the range 1–15  104 M1), thus, the Ka values in this work manifest the binding between nicosulfuron and HSA was moderate, so the nicosulfuron can be stored and carried by this protein in body.

800 OCH

a

N

O

Fluorescence Intensity

S

600

N NH

O

C

NH N

O

CON(CH )

400

OCH

j

3.3. Thermodynamic analysis of nicosulfuron binding

200

x 0 300

350

400

450

Wavelength (nm) Fig. 4. Fluorescence emission spectra of HSA–nicosulfuron system. c(HSA) = 1.0  106 M, c(nicosulfuron) = 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0  106 M (a) ? (j); (x) 9.0  106 M nicosulfuron. pH = 7.4, T = 291 K. Inset shows the molecular structure of nicosulfuron.

1.16

291 K

1.14

297 K 1.12

303 K

F0 / F

1.10

Considering the dependence of association constant on temperature, a thermodynamic process was considered to be responsible for this interaction. So, the thermodynamic parameters depended on temperatures were analyzed in order to depict the acting force between nicosulfuron and HSA. The noncovalent interactions between ligand and biomacromolecule primarily include hydrogen bonds, van der Waals forces, electrostatic and hydrophobic forces [30]. The thermodynamic parameters, enthalpy change (DH°), entropy change (DS°) and free energy change (DG°) are the main testimony for confirming the binding mode. The temperatures chosen were 291, 297, 303 and 309 K so that HSA did not undergo any structural degradation. If the DH° does not vary significantly over the temperature range studied, its value can be calculated from the van’t Hoff equation:

ln K ¼

309 K

 DH  DS þ RT R

ð6Þ

where K is analogous to the static quenching constant and R is gas constant. The values of DH° and DS° were obtained from linear van’t Hoff plot (Fig. 7), and DG° value was gained from the following relationship:

1.08 1.06 1.04

DG ¼ DH  T DS

1.02

Fig. 7 exhibits the linear relationship between ln K and 1/T, and the corresponding DH°, DS° and DG° values were presented in Table 3. The negative sign for DG° connotes that the binding process was spontaneous and the formation of the nicosulfuron–HSA complex was an exothermic reaction accompanied by a positive DS° value. Ross and Subramanian [30] have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction that may take place in protein association processes. From the point of view of water structure, a positive

1.00 0

2

4

6

8

10

[Q] (10-6 M) Fig. 5. Stern–Volmer plots for the quenching of HSA by nicosulfuron at different temperatures, pH = 7.4. Data are mean values ± standard deviations of three independent experiments, some error bars are within symbol.

In addition, the fluorescence quenching data obtained at different temperatures were further examined using Lineweaver–Burk equation [28]:

0.14

291 K

0.12

1 1 1 ¼ þ F 0  F F 0 K a F 0 ½Q

297 K

ð5Þ

(F0 - F )-1

0.10

where F0 and F are the fluorescence intensities in the absence and presence of quencher, Ka is the static quenching constant, and [Q] is the concentration of quencher. Fig. 6 displays the Lineweaver–Burk plots and the corresponding results of Ka values were listed in Table

ð7Þ

303 K 309 K

0.08 0.06 0.04

Table 2 Stern–Volmer quenching constants at four different temperatures, expressed as mean values (n = 3) ± standard deviations of three determinations.

a

T (K)

KSV (104 M1)

kq (1012 M1 s1)

Ra

291 297 303 309

1.420 ± 0.003 1.309 ± 0.003 1.141 ± 0.003 1.043 ± 0.002

1.420 ± 0.003 1.309 ± 0.003 1.141 ± 0.003 1.043 ± 0.002

0.9966 0.9974 0.9968 0.9989

R is the correlation coefficient.

0.02 0.00 0

200000

400000

600000

800000

1000000

-1

1/[Q] (M ) Fig. 6. Lineweaver–Burk plots for the HSA–nicosulfuron system at different temperatures, pH = 7.4. Data are mean values ± standard deviations of three independent experiments, some error bars are within symbol.

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F. Ding et al. / Journal of Molecular Structure 975 (2010) 256–264 Table 3 Lineweaver–Burk quenching constants Ka and relative thermodynamic parameters of the HSA–nicosulfuron system. T (K)

Ka (104 M1)

Ra

DH° (kJ mol1)

DG° (kJ mol1)

DS° (J mol1 K1)

291 297 303 309

3.167 2.693 2.191 1.929

0.9995 0.9997 0.9994 0.9991

21.10

25.07 25.19 25.18 25.35

13.66

a

R is the correlation coefficient for the Ka values.

10.5 -0.8 10.4

ln K = 1.643 + 2538 / K , R = 0.9971, S.D. = 0.021

-1.0

log[( F0 - F )/ F ]

lnK

10.3 10.2 10.1

291 K 297 K 303 K

-1.2

309 K

-1.4 -1.6

10.0 -1.8 9.9 -2.0 9.8 0.00320 0.00325 0.00330 0.00335 0.00340 0.00345 0.00350

-6.0

-5.8

Fig. 7. Van’t Hoff plot for the interaction of HSA and nicosulfuron in Tris–HCl buffer solution, pH = 7.4.

DS° value is regularly taken as a typical evidence for hydrophobic interactions. Moreover, the negative DH° value (21.10 kJ mol1) observed can not be mainly attributed to electrostatic forces since for electrostatic forces DH° is very small, almost zero [30]. A negative DH° value is observed whenever there are hydrogen bonds in the association reaction. The negative DH° and positive DS° values for the interactions between the nicosulfuron and HSA indicated that both hydrophobic and hydrogen bonds interactions played major roles in the binding reaction and made contributions to the stability of the complex. 3.4. Association constant and binding site of the nicosulfuron 3.4.1. Binding data analysis For ligand molecules that bind independently to a set of equivalent sites on a macromolecule, the equilibrium between the free and bound molecules is given by the equation [31]:

F0  F ¼ log K b þ n log½Q  F

-5.4

-5.2

-5.0

log[ Q]

1/T (K-1)

log

-5.6

ð8Þ

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, Kb and n are the association constant and the number of binding site, [Q] is the concentration of the quencher. Fig. 8 shows the plots of log(F0–F)/F versus log[Q], and the calculated Kb and n values were displayed in Table 4. The values of Kb suggested that the association constants decreased with the increase in temperature, resulting in the destabilization of the nicosulfuron–HSA complex. Additionally, the values of n were approximately 1, which attested that there is just a single binding site in HSA for nicosulfuron. The intrinsic fluorescence of HSA primarily originates from Trp-214 residue in subdomain IIA [32], therefore, it may be inferred from the data of n that the nicosulfuron is most likely to bind to the hydrophobic cavity located in subdomain IIA.

Fig. 8. The plots of log(F0–F)/F versus log[Q] at different temperatures for HSA pH = 7.4. Data are mean values ± standard deviations of three independent experiments, some error bars are within symbol.

Table 4 The association constants Kb and binding sites n at different temperatures, expressed as mean values (n = 3) ± standard deviations of three determinations.

a

T (K)

Kb (104 M1)

n

Ra

291 297 303 309

1.358 ± 0.013 1.026 ± 0.007 0.7482 ± 0.012 0.4457 ± 0.016

0.99 0.97 0.95 0.92

0.9992 0.9998 0.9993 0.9986

R is the correlation coefficient.

3.4.2. Effect of guanidine hydrochloride (GuHCl) To receive more clues on the binding region for nicosulfuron on HSA, the GuHCl initiated protein unfolding experiments were carried out. Ahmad et al. [33] reported that the unfolding of HSA by GuHCl occurred in multiple steps, at 1.4 M GuHCl, only domain III is entirely unfolded, the presence of a molten globule-like state of domain III around 1.8 M GuHCl and at 3.2 M GuHCl, domain I is detached from the domain II, domain I is totally unfolded while domain II is partly. In this experiment, samples of varied GuHCl concentration were prepared by blending different molar ratios of 6.0 M GuHCl stock solution and Tris–HCl buffer of pH 7.4. The final sample was incubated with various GuHCl concentrations for 12 h at room temperature before fluorescent measurements. By means of linear regression of a plot of log(F0–F)/F against log[Q], the association constants in the presence of different concentrations of GuHCl were calculated in Table 5. As evident from Table 5, when domain III is wholly unfolded, the association constant between HSA and nicosulfuron does not alter distinctly. While at 3.2 M GuHCl, the association constant was drastically reduced. This outcome signified that nicosulfuron binding site does not locate in domain III, but in domain I or II. The consequences of this study will be further discussed in the next section.

F. Ding et al. / Journal of Molecular Structure 975 (2010) 256–264

3.4.3. Competitive binding of site markers and nicosulfuron In order to determine the specificity of nicosulfuron binding sites on HSA molecule, competitive experiments were performed. Crystallographic analysis of HSA [32] reveals that the globular protein consists of a single polypeptide chain of 585 amino acid residues, and has many central physiological functions. It comprises three homologous domains (I, II and III): I (residues 1–195), II (196–383) and III (384–585), which are assembled to form a heart-shaped molecule. Each domain contains two subdomains called A and B to form a cylinder, respectively, and is stabilized by 17 disulfide bonds and 1 free thiol at Cys-34. The principal regions of ligand binding sites of HSA are located in hydrophobic cavities in subdomains IIA and IIIA, which are consistent with site I and site II according to the terminology proposed by Sudlow et al. [34]. Site I is known as the warfarin–azapropazone site formed as a pocket in subdomain IIA and involves the lone Trp of the protein (Trp-214), ligands binding in this site are bulky heterocyclic anions with a negative charge localized in the middle of the molecule, such as warfarin, phenylbutazone, and azapropazone [35]. While site II corresponds to the cavity of subdomain IIIA, which is known as the indole-benzodiazepine site, drugs binding to site II are aromatic carboxylic acids with negatively charged acidic group at the end of the molecule, e.g. ibuprofen, diazepam, and flufenamic acid [36]. Subsequently, Sjöholm et al. [37] pointed out that digitoxin binding in HSA is independent from Sudlow’s site I and site II, and perch on what was nominated as site III. In this work, the competitors used included phenylbutazone, a characteristic marker for site I, ibuprofen for site II, digitoxin for site III, and hemin for domain I. Salient information about the nicosulfuron binding site can be obtained by monitoring the changes in the fluorescence of HSA-bound nicosulfuron brought out by these site markers. According to Eq. (8), the association constants at the presence of site markers were gauged from the fluorescence data and were found to be K 0b : (0.5808  104 M1), (1.167  104 M1), (1.265  104 M1) and (1.306  104 M1) for phenylbutazone, ibuprofen, digitoxin and hemin, respectively. Compared with protein unfolding experiments, at 3.2 M GuHCl, the association constant was sharply reduced, an explanation to this phenomenon is the loss of compactness of domain structure [38]. While in the competitive experiments, site markers were employed so as to acquire insight into specific site or region on HSA. The association constant was remarkably decreased with the addition of phenylbutazone, but only changed a little after the addition of other site markers. The reason is that the HSA–nicosulfuron complex was mostly affected by adding phenylbutazone, namely, nicosulfuron shares the identical binding site with phenylbutazone in HSA molecule, whereupon the association constant between nicosulfuron and HSA was considerably decreased. From these analyses, it is distinct that nicosulfuron has one active site of HSA, that is, high-affinity site (Sudlow’s site I).

3.4.4. ANS binding studies Additional evidence regarding the nicosulfuron binding region on HSA comes from the hydrophobic probe ANS displacement

experiments. According to the protocol, the relative fluorescence intensity (F/F0) against ligand concentration ([Ligand]) plots is shown in Fig. 9. Both nicosulfuron and ANS quench the HSA fluorescence, but the degree of quenching by nicosulfuron was much less when compared with ANS. The binding of ANS to HSA occurs at one high-affinity site while at least three sites of lower-affinity have been reported by Sudlow et al. [34,39,40]. Thus, from the unequal level of HSA fluorescence quenching, it is proposed that the nicosulfuron probably shares the different site with ANS in HSA. When nicosulfuron is added to HSA–ANS system, it can compete with ANS for hydrophobic portions of HSA molecule, then it would substitute the binding site of ANS and the fluorescence intensity decreases. The reason is that ANS is essentially non-fluorescent in aqueous solution and becomes appreciably fluorescent in apolar environments after binding to the hydrophobic region of proteins [41]. As shown in Fig. 9, about 14.32% of ANS fluorescence is decreased, demonstrating that nicosulfuron can compete against ANS for its lower-affinity binding site in HSA. The above experimental results and analyses confirmed that hydrophobic patch (Sudlow’s site I) is the major site for nicosulfuron binding on HSA molecule. Similar research has also been reported by Klajnert et al. [42] for the binding of polyamidoamine dendrimers with bovine serum albumin. 3.5. Molecular modeling Molecular modeling simulations were also exploited to reconfirm the experimental data, and the best docking energy result is illustrated in Fig. 10A. As can be seen, nicosulfuron was situated within subdomain IIA in Sudlow’s site I formed by six-helices, pyrimidine ring and the peptide bond of nicosulfuron were deeply buried in the pocket, while sulfur atom and pyridine ring were located at the entrance to the cavity. Furthermore, the nicosulfuron molecule has two planes which are composed of pyridine ring, pyrimidine ring and peptide bond, link sulfur atom with single bond, and the angle of the two planes continuous variation via single bond rotation. The certification of the binding mode as per amino acid residue is forecasted to be the part of the binding site in Fig. 10B. Where the heterocyclic ring in His-242 and Trp-214 residue, and the hydrophobic pocket at the bottom of subdomain IIA have the tendency to make hydrophobic interactions with pyrimidine ring of the nicosulfuron molecule. The binding between HSA and nicosulfuron is not exclusively hydrophobic in nature, owing

1.2 1.0 0.8

F / F0

262

0.6 0.4 0.2

Table 5 The association constants K 00b between HSA and nicosulfuron in the presence of GuHCl, expressed as mean values (n = 3) ± standard deviations of three determinations.

a

c(GuHCl) (M)

K 00b (104 M1)

Ra

1.4 1.8 3.2

1.312 ± 0.028 1.476 ± 0.014 0.008629 ± 0.007

0.9965 0.9993 0.9993

R is the correlation coefficient.

0.0 0

2

4

6

8

10

-6

[Ligand] (10 M) Fig. 9. Fluorescence quenching profile of HSA and HSA–ANS system. Binding isotherm of nicosulfuron (j) and ANS (d) induced quenching of HSA fluorescence and quenching of HSA–ANS system fluorescence by nicosulfuron (N). pH = 7.4, T = 291 K.

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to the two polar residues (Lys-199 and Arg-257) in the propinquity of the bound ligand, which play vital function in stabilizing nicosulfuron through hydrogen bonds interaction (Fig. 10C). For example, Arg-257 is in apposite position involved in making hydrogen bonds with oxygen atom of the carboxy group in pyridine ring. The hydrogen bonds serve as an ‘‘anchor”, which extremely determines the three-dimensional space position of nicosulfuron in the subdomain IIA, and stimulates the hydrophobic interactions of the pyrimidine ring and peptide bond with the side chain of HSA [43]. This corroborates with the result of GuHCl induced denaturation of HSA and fluorescence probe studies, which lay the nicosulfuron on subdomain IIA, and verify conformational changes of HSA upon interaction with nicosulfuron. 4. Conclusions The present work reports the binding mechanism and binding site between a sulfonylurea herbicide, nicosulfuron, and the transport protein, HSA. Fluorescence measurement gives information about the molecular environment in the vicinity of the chromophore molecule. The experimental results demonstrate that the fluorescence quenching of HSA was primarily resulted from the static mechanism, fluorescence and CD study throws some light on the conformational alterations of HSA induced by the binding of nicosulfuron. Thermodynamic data exhibit the hydrophobic and hydrogen bonds interactions played major roles in stabilizing the HSA–nicosulfuron complex. Based on fluorescence probe, protein denaturation and molecular modeling results we can identify nicosulfuron was bound to subdomain IIA, which was the same as that of warfarin-azapropazone site (Sudlow’s site I) [35]. In brief, the combination of the different spectroscopic technique with computational modeling method utilized in this work were decisive in the depiction of HSA–nicosulfuron complex and can be interpreted the biochemical and biophysical property already accumulated on sulfonylurea herbicides binding to HSA and will also supply structural information for construction of a pesticide biosensors. Acknowledgements We thank Dr. Li Zhang of Department of Chemistry, China Agricultural University, for her constant support during the CD measurements. Thanks also go to Miss Xi Zhang for her fruitful discussions and assistance. We gratefully acknowledge funding from the Postgraduate Research and Innovation Project, China Agricultural University. References

Fig. 10. Molecular modeling of nicosulfuron bound HSA. Panel (A) shows docked nicosulfuron into HSA at subdomain IIA. Subdomain IIA of HSA, represented in surface colored in green, to nicosulfuron, represented in stick, colored as per the atoms. Panel (B) displays the hydrophobic cavity at the bottom of subdomain IIA. Subdomain IIA, represented in surface colored in green, to nicosulfuron, represented in ball-and-stick model. Panel (C) depicts the amino acid residues involved in binding of nicosulfuron. The ball-and-stick model represents nicosulfuron molecule, colored as per the atoms. The amino acid residues around nicosulfuron have been displayed in red color stick model, and the hydrogen bonds interactions between HSA and nicosulfuron represented using yellow dash line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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