A spectroscopic and thermal stability study on the interaction between putrescine and bovine trypsin

International Journal of Biological Macromolecules 94 (2017) 145–153

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A spectroscopic and thermal stability study on the interaction between putrescine and bovine trypsin Lida Momeni a , Behzad Shareghi b,∗ , Ali A. Saboury c,d , Sadegh Farhadian b , Fateme Reisi e a

Department of Biology, Faculty of Science, University of Payam Noor, Iran Department of Biology, Faculty of Science, University of Shahrekord, Shahrekord, P.O. Box 115, Iran Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran d Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran e Department of Biology, Faculty of Sciences, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran b c

a r t i c l e

i n f o

Article history: Received 25 July 2016 Received in revised form 2 October 2016 Accepted 5 October 2016 Available online 6 October 2016 Keywords: Trypsin Putrescine UV–vis spectroscopy Fluorescence spectroscopy Circular dichroism Molecular docking

a b s t r a c t The interaction of putrescine with bovine trypsin was investigated using steady state thermal stability, intrinsic fluorescence, UV–vis spectroscopy, far and near- UV circular dichroism and kinetic techniques, as well as molecular docking. The Stern-Volmer quenching constants for the trypsin- putrescine complex were calculated revealing that putrescine interacted with trypsin via the static fluorescence quenching. The enthalpy and entropy change values and the molecular docking technique revealed that hydrogen bonds and van der Waals forces play a major role in the binding process. Upon putrescine conjugation, the Vmax value and the kcat /Km values of the enzyme was increased. The results of UV absorbance, circular dichroism and fluorescence techniques demonstrated that the micro environmental changes in trypsin were induced by the binding of putrescine, leading to changes in its secondary structure. The thermal stability of trypsin- putrescine complex was enhanced more significantly, as compared to that of the native trypsin. The increased thermal stability of trypsin- putrescine complex might be due to the lower surface hydrophobicity and the higher hydrogen bond formation after putrescine modification, as reflected in the increase of UV absorbance and the quenching of fluorescence spectra. It was concluded that the binding of putrescine changed trypsin structure and function. © 2016 Published by Elsevier B.V.

1. Introduction A considerable interest has been focused on studying the interaction of stabilizers with organic and biological molecules, promising to be an exciting field of basic and applied research [1]. Putrescine (1,4 diaminobutane) is an aliphatic diamine belonging to the group of biogenic amines with a low-molecular-weight nitrogenous base. The basic amino groups carry a positive charge at the physiological pH of 7.4, making them appropriate for a wide range of functions in different cell types. Putrescine is found in decomposed animal materials, different kinds of foodstuffs, viruses, semen and all cell types [2]. This polyamine shows many physiological functions and is a precursor in the synthesis of other polyamines (spermine and spermidine) and the formation of carcinogenic Nnitrosamines. Putrescine, due to its polycationic nature, interacts

Abbreviations: CD, circular dichroism; BAEE, N␣ -benzoyl-l-arginine ethyl aster. ∗ Corresponding author. E-mail address: b [email protected] (B. Shareghi). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.009 0141-8130/© 2016 Published by Elsevier B.V.

with negatively charged molecules such as proteins, phospholipids, DNA and RNA [3]. The importance of negatively charged, aromatic and polar residues in relation to the interaction of polyamines with proteins has been confirmed [4]. The charge distribution is important for molecular recognition and the hydrophobic polymethylene backbone of them confers structural flexibility [5]. The effects of putrescine in decreasing membrane fluidity and increasing the resistance to fragmentation can be due to the stabilization of the membrane skeleton and deletion of the free radicals as luminal growth factors for intestinal maturation and growth [6,7]. It can play a significant role in the prevention of food allergies [8]. Putrescine also has direct effects on several ion channels and receptors, resulting in the regulation of Ca2+ , Na+ and K+ homeostasis [9]. Polyamines have the kosmotropic properties [10,11]. Kosmotropes cause water molecules to favorably interact, thereby stabilizing the intramolecular interactions in macromolecules such as proteins [12]. Bovine trypsin (T8003, E.C. 3.4.21.4), a serine protease, is a soluble globular protein that cleaves peptide bonds at the carboxylterminal end of lysine, arginine and ornithine residues, which are

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widely employed in biomedicine, biotechnology and food industry, as well as in protein analysis [13,14]. Trypsin consists of a single chain polypeptide of 223 amino acid residues. X-ray crystallography of trypsin indicates that it has two nearly equal size domains. It has six antiparallel strands making a ␤-sheet unit through a network of hydrogen bonds [15]. The amino acid sequence of trypsin is cross-linked by six disulfide bridges. The active site amino acid residues of trypsin include His 46 and Ser 183 [16]. It has an isoelectric point, pI, of around 10.5 [15]. To this date, there is not any report showing the effect of putrescine on the stability and conformation of trypsin. In addition, the explanation of enhanced stability, in terms of conformational change in trypsin, has not been provided. Thus, the aim of this study was to investigate the effect of putrescine on the stability and conformation of trypsin, and to discuss the stability improvement of the modified trypsin in terms of the conformational change. 2. Materials and methods 2.1. Materials Trypsin from bovine pancreas (catalogue no. T8003) was obtained freeze – dried from Sigma and used without further purification. Tris–HCl buffer, N␣ -benzoyl-l-arginine ethyl aster (a substrate used as a probe for the activity of trypsin, BAEE, catalogue no. B 4500) and putrescine were also bought from Sigma Aldrich Co. All trypsin solutions were made in the 50 mM Tris–HCl buffer at the pH 8. 2.2. Thermal stability assays The thermal stability of free enzyme and enzyme- putrescine complexes was investigated at different temperatures using a Pharmacia 4000 UV–vis spectrophotometer equipped with an external thermostat. According to the method developed by Zhang et al., thermal stability of trypsin (0.1 mg/mL) was estimated by incubating enzyme solutions in buffer for 10 min, at 280 nm and pH 8 [17]. The absorbance changes were recorded at 280 nm in the temperature range of 293 to 373 K, as well as the heating rate 1 K/min. The absorption data were plotted as a function of temperature. 2.3. Fluorescence spectroscopy measurements Fluorescence spectroscopy was used to monitor the binding of putrescine to the protein molecules and the tertiary structure changes in trypsin induced by putrescine. Steady-state fluorescence spectra of trypsin were measured by exciting 280 nm with scanning between 300 and 450 nm, at 298 and 308 K. The intrinsic fluorescence of enzyme was recorded using a Shimadzu RF-5301 fluorescence spectrophotometer with a temperature adjustable cell holder. A 1-cm quarts cuvette was used for these studies. Emission and excitation slits were set to 3 and 5 nm, respectively. The structural changes of trypsin (0.1 mg/mL) were monitored in the absence and presence of different concentrations of putrescine (0–7 mM). The mixture of trypsin with putrescine was incubated for 10 min to form the protein-putrescine complex before the measurement of the fluorescence intensity. To obtain the binding parameters, data were analyzed by the modified Stern-Volmer equation [18–20]. 2.4. Circular dichroism (CD) measurements The UV-CD studies were performed by an Aviv model 215 Spectropolarimeter (Lakewood, New Jersey, USA), using quartz cells of 1 mm and 10 mm path length for the Far-UV (200–260 nm) and Near-UV (260–320 nm) regions, respectively. Dry nitrogen gas was supplied to purify the equipment before and during the course of

Fig. 1. Structure of putrescine.

the measurements. The data was expressed as the mean residue ellipticity [] in degree cm2 d mol−1 , defined as [] = 100   MRW/cl, where   is the observed ellipticity in degrees, MRW is the mean molecular weight of amino acid residue, c is the trypsin concentration in mg/mL, and l is the length of the light path in cm [21]. CD samples were prepared with a concentration of trypsin (0.8 mg/mL for far UV-CD, and 1.6 mg/mL for near UV-CD) and different concentrations of putrescine (0, 2 and 4 mM). The suspension trypsin, Tris–HCl buffer and putrescine were allowed to equilibrate for 15 min at the room temperature. All spectra were also subtracted with putrescine solutions and smoothed. The different secondary structures were performed by the CDNN program, version 2.1.0.223, using a network trained with 33 complex spectra at the reference set. 2.5. UV absorption spectra analysis The Pharmacia 4000 UV–vis spectrophotometer (Japan) was applied to record the UV–vis absorbance spectra over the wavelength of 260–310 nm. The quarts cells used for these studies were 1 cm in length. The spectrophotometer was baseline with the Tris–HCl buffer as the relevant blank. The same volumes of putrescine (1–4 mM) and trypsin at 0.1 mg/mL were equilibrated for 15 min at 37 ◦ C and then the spectra of the enzyme were obtained at ␭280 . The resulting absorbance change in the absence and presence of putrescine was plotted versus putrescine concentration. 2.6. Enzymatic activity assay In order to investigate the effects of putrescine on the enzyme activity, the catalytic activity of trypsin was determined by UV–vis spectrophotometer at pH 8.0 and the temperature of 37 ◦ C. Various concentrations of putrescine were incubated with trypsin (25 ␮g/mL) in a 50 mM Tris–HCl buffer solution (pH 8) for 15 min and the reaction mixture was used to monitor the change in absorbance at 253 nm and 37 ◦ C after the addition of BAEE [22]. All assays were repeated at least 3 times. The kinetic parameters (Km , Vmax , kcat and kcat /Km ) were determined from the data of trypsin activity against a range of BAEE concentrations, based on the equation of Lineweaver-Burk. 2.7. Molecular docking studies Molecular docking studies of trypsin and putrescine were performed using AutoDock 4.0 software package [23]. The crystal structure of trypsin was downloaded from the Protein Data Bank (PDB) (trypsin accession number: 2PTN). The geometry of putrescine was modeled by HyperChem program [24] and minimized by the molecular mechanics method via MM+ force field (Fig. 1). Trypsin was considered as rigid and putrescine as fully flexible during docking. During molecular calculation studies, all water molecules were removed from the original Protein Data Bank file. Furthermore, Kollman united atom type charges, essential hydro-

L. Momeni et al. / International Journal of Biological Macromolecules 94 (2017) 145–153

147

6000 1 0 mM

2 mM 3 mM

0 mM

0.6

2000

∆G° (J/mol)

Fu

1 mM

4000

0.8

1 mM 2 mM

0.4

3 mM 4 mM

0.2

4 mM

0 314

316

318

320

322

324

326

328

-2000

0

-4000 303

313

323

333

343

T (K)

-6000

T (K) Fig. 2. Thermal denaturation curves of trypsin in the presence of different concentrations of putrescine (, 0 mM; 䊏, 1 mM; , 2 mM; × , 3 mM, 䊉 4 mM) at pH 8.0.

gen atoms and salvation parameters were also added in the trypsin [25]. The Gasteiger partial charges were added to the atoms of ligand, and rotatable bonds were defined. The program AutoGrid was used to generate the grid maps. Each grid was centered at the crystal structure of the corresponding trypsin bound ligand. In addition, the grid box size of putrescine–trypsin system was 87 × 87 × 87 A◦ , with the grid spacing of 10 A◦ . For the ligand, random starting positions, random orientations and torsions were used. The quaternion, translation and torsion steps were taken from default values in AutoDock. The pseudo-Solis and Wets methods and the Lamarckian genetic algorithm (LGA) were applied to look for the best binding site of trypsin and putrescine [26]. Docking calculation parameters were assigned as follows: number of Lamarckian job = 100, maximum number of energy evaluation = 25 × 106 , initial population = 100, a cross-over rate of 0.80, mutation rate of 0.02, and elitism value = 1, with all other parameters being kept as their default value. Docking energy in the largest cluster was selected as the representative binding pose [27].

3. Results and discussion 3.1. Thermal stability assays of trypsin To show the reversible unfolding of trypsin in the absence and presence of different concentrations of putrescine, protein thermal folding experiments were carried out by using UV–vis spectrophotometer. The transition temperature (Tm ) of the enzyme was obtained after normalizing the absorbance of native and denatured molecules of trypsin. In fact, denaturation data were analyzed by supposing the two-state mechanism between the folded (native) and unfolded (denatured) states. The fraction of the unfolded protein, Fu , was calculated using Eq. (1) [28,29]: Fu =

Y − Yf Yu − Yf

(1)

In this equation, Y is the observed variable parameter at a certain putrescine concentration, and Yf and Yu are, respectively, the absorbance of the folded and unfolded states of trypsin under the conditions in which Y is determined. Denaturation curves of trypsin in the absence and presence of different concentration of putrescine are shown in Fig. 2 As can be seen, by increasing the concentration of putrescine, the curves were shifted to the higher temperature, showing the more stability of trypsin. According to the two-state mechanism, the difference in Gibbs free energy (G◦ ) between the

Fig. 3. The effect of different concentrations of putrescine (, 0 mM; 䊏, 1 mM; , 2 mM; 䊉, 3 mM, × 4 mM) on G◦ at different temperatures and pH 8.0. Table 1 Thermodynamic data of trypsin at Tm in the presence of different concentrations of putrescine. [putrescine] (mM)

Tm (K)

Sm ◦ (J mol−1 K−1 )

Hm ◦ (kJ mol−1 )

R2

0 1 2 3 4

318.0 319.6 320.8 321.2 323.0

441.4 450.5 459.7 468.9 509.1

140.4 144.0 147.5 150.6 164.4

0.998 0.999 0.999 0.999 0.999

a

a

The correlation coefficient.

folded and unfolded conformation of the enzyme was defined as the enzyme stability. G◦ could be calculated by Eq. (2) [29–31]: G◦ = −RTln

 F  u 1 − Fu

(2)

where G◦ is the difference in free energy between the folded and unfolded conformation, R is the gas constant (8.314 J K−1 mol−1 ), and T is the absolute temperature. G◦ , the standard Gibbs free energy, was a function of temperature of trypsin in the presence and absence of putrescine, as displayed in Fig. 3. These results could be used to determine Tm at which G◦ = 0. Our results suggested that by increasing putrescine concentrations, the Tm value of trypsin was increased from 318.0 K (for pure trypsin in the buffer) to 323.8 K (Table 1). The standard-state entropy of transfer, S◦ m , was obtained from the slope denaturation curves at Tm . The standard enthalpy was calculated from H◦ m = Tm S◦ m (thermodynamic parameters are listed in Table 1) [30,31]. As can be seen, Tm , H◦ m , S◦ m and stability of trypsin were increased with the addition of the concentration of putrescine. The results indicated that trypsin interacted favorably with putrescine and putrescine stabilized the folded structure of trypsin. Similar results have been reported in the interaction of chymotrypsin with putrescine [29]. 3.2. Fluorescence quenching of trypsin by putrescine Fluorescence spectroscopy is a very powerful and accurate tool for studying ligand-protein interactions and ligand-induced conformational changes around the binding site [32,33]. This technique provides rapid and sensitive analysis for determining the binding of different chemical compounds to proteins. Fluorescence is the photon emission which is due to the transfer of an electron from a higher energy orbital to a lower one [19]. Some amino acid residues of enzymes such as tyrosine (Tyr), tryptophan (Trp) and phenyl alanine are intrinsically fluorescent. The quenching of fluorescence refers to any process that decreases the fluorescence intensity of a protein. Phe has a low quantum yield and the fluorescence of Tyr is quenched if it is near a Trp residue, a carbonyl

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L. Momeni et al. / International Journal of Biological Macromolecules 94 (2017) 145–153 700 Fluorescence Intensity at 332 nm (a. u.)

A)

Fluorescence Intensity

600

500

400

650

B)

600

298 K

550

308 K

500 450 0

2

4

6

8

[putrescine] (mM) 300

200

100

0 mM

1 mM

2 mM

3 mM

4 mM

5 mM

6 mM

7 mM

0 290

310

330

350

370

390

410

430

450

Wavelenght (nm)

Fig. 4. (A) Fluorescence emission of trypsin in the absence and presence of different concentrations of putrescine (0–7 mM) at 298 K, and (B) Fluorescence maximum emission of trypsin in the presence of different concentrations of putrescine at two temperatures.

3.2.1. Analysis of binding properties A variety of molecular interactions can quench the quantum yield of fluorophore. These include excited-state reactions, molecular rearrangement, energy transfer, ground-state complex formation (static quenching) and collisional quenching (dynamic quenching) [38,39]. Fluorescence quenching processes are usually explained by two mechanisms: dynamic and/or static type. When the static suppression mechanism predominates, the interaction between the fluorophore and quencher occurs in the ground state, with the formation of a new species which is not luminescent. The value of static quenching constants is reduced with increasing the temperature since the stability of the complex is decreased at higher temperatures. On the other hand, when quenching is

1.5 308 K

1.4

298 K 1.3

F0/F

group, an amino group, and/or when it is ionized. Trypsin contains four tryptophan residues (Trp 50, Trp 141, Trp 215, and Trp 237) [15,34]. The indole group of Trp residue leads to the fluorescence signal of proteins containing this residue [32,35]. Thus, for fluorescence quenching measurements, the intrinsic fluorescence of tryptophan residue(s) in the protein was measured in the presence and absence of putrescine. The changes in the emission spectra from Trp are also related to the protein conformational transitions, subunit association apart from anion binding, or direct denaturation [36]. The influence of different concentrations of putrescine on trypsin fluorescence intensity at ␭ex = 280 nm, 298 and 308 K in the 50 mM Tris–HCl buffer (pH 8) is shown in Fig. 4. Trypsin emitted the strong fluorescence peaks of 332 nm, contributing to the presence of Trp residues (excitation wavelengths of 280 nm). As shown in Fig. 4, the fluorescence intensity was decreased with the addition of putrescine to trypsin solution (at 332 nm), thereby demonstrating that the interaction between putrescine and trypsin occurred. After the conjugation of putrescine to trypsin, the exposure of hydrophobic amino acids to the environment was prevented. As shown, the conformational changes reflected in the reduction of fluorescence intensity could result from the change of interactions between putrescine and all residues of trypsin which contributed to the increase of surface hydrophilicity [1]. Since, in hydrophilic environment (exposed to solvent), their quantum yields were decreased, low fluorescence intensity was observed. In contrast, in the hydrophobic environment (buried within the core of the protein), Trp had a high quantum yield and therefore, a high fluorescence intensity was observed. [37]. No obvious shift of the maximum emission wavelength of the trypsin with the interaction of putrescine was observed (Fig. 4).

1.2 1.1 1 0.9 0

1

2

3

4

5

6

7

8

1010 [Q] Fig. 5. Stern–Volmer plots for the quenching of trypsin by putrescine at two temperatures of 298 K (䊉) and 308 K (䊏), ␭ex = 280 nm, ␭em = 290–450 nm, and pH 8.0.

dynamic, the interaction between the quencher and the excited state of the fluorophore occurs through diffusive collisions during the lifetime of the fluorophore excite state, and the process has diffusion control. The value of biomolecular dynamic quenching constants is expected to be higher with increasing the temperature. To determine the binding behavior, the fluorescence quenching data obtained in the above experiments were analyzed by the Stern–Volmer Eq. (3) [18,32,40]: F0 = 1 + KSV [Q ] = 1 + kq 0 [Q ] F

(3)

where F0 and F are the steady-state fluorescence intensities of trypsin in the absence and presence of putrescine, respectively, 0 is the average lifetime of molecule in the absence of quencher (10−8 ), and kq is the biomolecular quenching rate constant [41]. KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of putrescine. As brought in Fig. 5 the plot of F0 /F vs. [Q] showed a good linear behavior within the relative concentration range, indicating single quenching mechanism, either dynamic or static. The Ksv values for the putrescine-trypsin complex were determined by the linear regression of a plot of F0 /F against the concentration of putrescine and recorded at two temperatures of 298 and 308 K, as shown in Table 2. The obtained data strongly indicated that putrescine could bind to trypsin to form the trypsin-putrescine system. The results in Table 2 indicate that the quenching constant Ksv was inversely correlated with temperature. Substantial fluorescence quenching of the trypsin by putrescine indicated the static fluorescence quenching. The static quenching was due to the for-

L. Momeni et al. / International Journal of Biological Macromolecules 94 (2017) 145–153 Table 2 Stern–Volmer quenching constants of trypsin-putrescine complexes at pH 8.0.

149

Table 4 Changes in the secondary structures of trypsin in the absence and presence of putrescine.

T (K)

Ksv (×108 M−1 )

Kq (×1016 M−1 s−1 )

R2

298 308

5.12 3.18

5.12 3.18

0.988 0.988

[putrescine] (mM)

% ␣-Helix

% ␤ Sheet

B-Turn

% Random coil

0 2 4

5.4 5.6 6.0

40.8 40.7 40.0

19.7 19.5 19.7

34.1 34.2 34.3

log (F0-F)/F

0 -0.2

308 K

-0.4

298 K

-0.6 -0.8 -1 -1.2 -1.4 -1.6 -9.2

-9

-8.8

-8.6

-8.4

-8.2

-8

-7.8

3.2.3. Analysis of thermodynamic parameters In general, the interaction between biological macromolecules and small molecules can be described by four types of interaction: hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions. The dependence of the binding constant on temperature indicates that a thermodynamic process is responsible for the formation of the complex [19]. The sign and magnitude of H◦ and S◦ are important for confirming the main force contributing to the binding reaction. To explain the interaction between putrescine and trypsin, the thermodynamic parameters were calculated from van’t Hoff equation (Eq. (5)) [43]:

log [Q] Fig. 6. The plot of log [(F0 − F)/F] vs. log [Q] at two temperatures of 298 K (䊏) and 308 K (䊉) for trypsin with putrescine, ␭ex = 280 nm, ␭em = 290–450 nm, and pH 8.0.

mation of a ground state complex, and the stability of the complex could be decreased as temperature was increased, so the quenching constant was inversely correlated with temperature [42]. Also, kq , the quenching rate constant, was greater than 2.0 × 1010 M−1 s−1 . Therefore, it again indicated that the fluorescence quenching of trypsin by putrescine was mainly controlled by a static quenching mechanism (Fig. 5) [19]

3.2.2. Analysis of binding properties Since we observed that the static mechanism contributed to the quenching of trypsin, an analysis of the binding constants of compounds to trypsin was performed. The binding constant (Ka ) and the number of binding sites (n) were calculated by using the modified Stern–Volmer Eq. (4) [19,32]: log

F0 − F = logKa + n log [Q ] F

(4)

Ka and n could be obtained from the values of intercept and slope from the plot of log F0 -F/F vs. log [Q] (Fig. 6). According to Table 3, the values of n were determined to be 1 at both temperatures, suggesting the presence of a single binding site for the putrescine in the trypsin molecule. The relatively high Ka values (∼106 M) obtained for the compounds under investigation suggested that there was a strong interaction between the complexes and trypsin, thereby showing that the static component of quenching must be significant. Decreasing the value of Ka was caused by the higher temperature, indicating a reduction in the stability of the trypsinputrescine complex. Therefore, the binding was an exothermic reaction that was in agreement with Ksv , as mentioned above. Hence, the results indicated the putrescine-trypsin complex was not easily formed with increasing the temperature, and the formed complex could be unsteady and dissociated with the rising temperature.

ln

K2 = K1

1

T1

−

1 T2

 H ◦

(5)

RT

and the free energy change (G◦ ) was then calculated from the thermodynamic Eqs. (6) and (7): G◦ = −RTlnKa

(6)

H ◦ − G◦ S ◦ = T

(7)

where H◦ and S◦ are enthalpy and entropy changes of binding, respectively. The thermodynamic parameters presented in Table 3 G◦ are related to the spontaneity of reaction, while H◦ and S◦ are the main quantities used to assess the strength of a bond. The negative values of G◦ showed the spontaneous nature of the interaction process. Hence, according to the previous studies, the sign and magnitude of thermodynamic parameters could be associated with various individual kinds of interactions that might take place during protein association process. Negative H◦ and S◦ values indicate the presence of hydrogen bonds and/or van der Waals forces; positive H◦ and S◦ values show the presence of hydrophobic interactions and negative H◦ and positive S◦ values suggest the presence of electrostatic interactions [43–46]. As shown in Table 3, the negative values of S◦ and H◦ indicated that the van der Waals force and hydrogen bonding force were the main binding forces. Moreover, the result also suggested that the binding of putrescine to trypsin was enthalpy-driven, and the favorable enthalpy change (H◦ < 0) was offset partly by unfavorable entropy change (S◦ < 0). 3.3. Circular dichroism studies of putrescine/trypsin CD spectroscopy is a convenient technique for detecting changes in the structure of a protein when interacting with ligands. Far UVCD (200–260 nm) and the near UV-CD (260–320 nm) spectra could be used to monitor secondary and tertiary conformation changes of proteins upon interaction with the putrescine, respectively. Far-UV CD spectra reflected peptide bond absorption and characterized the secondary structure of a protein. Fig. 7A shows Far-UV CD spectra of trypsin-putrescine complexes. The far UV-CD of trypsin showed a strong negative band at about 208 nm that corresponded to the

Table 3 Binding and Thermodynamic parameters of trypsin-putrescine interaction at pH 8.0 and two different temperatures. T (K)

Ka (×106 M−1 )

n

R2

H◦ (kJ mol−1 )

G◦ (kJ mol−1 )

S◦ (J mol−1 K−1 )

298 308

3.1 2.5

0.86 0.85

0.9977 0.9994

−16.79 − 16.79

−37.04 −37.72

−67.96 −67.96

150

L. Momeni et al. / International Journal of Biological Macromolecules 94 (2017) 145–153 Table 5 The kinetic values of trypsin at various concentrations of putrescine.

10

A)

[Ɵ]×10-3 (deg cm2 mol-1)

5 0 -5 -10

0 mM

-15

2 mM

-20

4 mM

-25

[putrescine] (mM)

Km (mM)

kcat × 106 (s−1 ) Vmax (mM s−1)

kcat /Km × 106 (mM−1 s−1 )

0 1 3 5

0.9 1.1 1.2 1.5

3.1 4.1 4.6 5.8

3.4 3.7 3.8 3.9

3.3 4.3 4.9 6.1

3.4. UV absorption spectroscopy

-30 200

210

220

230

240

250

260

Wavelength (nm)

2

B)

[Ɵ] MR (deg cm2 mol-1)

0 -2 -4

0 mM

-6

2 mM 4 mM

-8 -10 -12 260

270

280

290

300

310

320

Wavelenght (nm)

Fig. 7. (A): Far-UV CD and (B): Near-UV CD spectra of trypsin in the absence and presence of putrescine (dotted line: 2 mM and dashed line: 4 mM) in the 50 mM Tris–HCl buffer and at pH 8. The Y-axis is the mean-residue ellipticity with the unit of degree cm2 /mol.

mostly ␣-Helix structure of trypsin in the crystalline structure of trypsin and originated from the n-␲* transitions changed obviously (Fig. 7A). The addition of putrescine slightly changed the intensity of the negative peak, revealing the slight alternations of trypsin secondary structure. The fraction contents of different secondary structures of trypsin in the presence and absence of putrescine were calculated using the CDNN program; the detailed fraction changes are listed in Table 4. There was an increasing tendency for the ␣-Helix (from 5.40% to 5.96%) and a deceasing one for the ␤sheet (from 40.80% to 40.04%) with the addition of putrescine. The results suggested that putrescine could make the secondary structure change. The slight increase of ␣-Helix content indicated that the binding of trypsin and putrescine induced further H-bonding formation. According to founding of Chanphai et al., polyamines bind via van der Waals and H-bonding contacts with trypsin and other proteins forming more stable conjugates [45,46]. The stability of the tertiary structure of trypsin upon interaction with putrescine was also monitored by near UV-CD to obtain complementary information about the structural changes mechanism of proteins. Fig. 7B demonstrates tertiary structural changes of trypsin after the titration of raising the concentration of putrescine. When putrescine was added into the solution, the intensity of trypsin spectra was decreased; this reduction was pronounced for putrescine/trypsin interaction. This was because of the intramolecular vibrations that the loss of asymmetry in the environment of amino acid residues occurred (Fig. 7B) [15]. If the aromatic residues become far from each other, the band intensity can be decreased. These results confirmed that the tertiary structure of trypsin was changed with putrescine, which was consistent with the results of absorption and fluorescence spectroscopy.

The change of hydrophobic groups induced by putrescine was also investigated by UV–vis absorption spectra [1]. The results obtained from UV–vis absorption spectra are usually in agreement with CD measurements, which could be regarded as a simple and excellent tool to study the interaction of enzyme with ligand and determine the conformational transitions in enzyme. These transitions are presented by the alterations in the wavelength of absorption and the strength of their absorbance [25]. Furthermore, the effect of different concentrations of putrescine on the absorption spectrum of trypsin was investigated. The absorption spectra of trypsin-putrescine combinations are shown in Fig. 8. Trypsin had a strong absorption peak at 216 nm due to a transition from ␲ → ␲* of the typical polypeptide backbone structure to C O of trypsin, reflecting the change of the secondary structure of biomacromolecule. Also, trypsin had a weak one at 280 nm due to the absorption of aromatic amino acids (Phe, Trp and Tyr), showing the alteration of the microenvironment of the chromophore [47,48]. It could be seen that by the addition of putrescine, the absorption intensity of trypsin was changed and a slight hyperchromic effect was observed in the maximum absorption peaks of trypsin at 216 and 280 nm, but the peak position of absorption spectra did not have any blue or red shift. The evidence suggested that there was an interaction between putrescine and trypsin. The changes in peak at 216 nm were related to the alterations in the secondary structures of trypsin. Also, the results were could prove that the binding between trypsin and putrescine decreased the hydrophobicity of the microenvironment of the aromatic amino acids residues in the internal hydrophobic region because of the formation of the ground state complex. The results also indicated that the probable quenching mechanism of the fluorescence of trypsin by putrescine was a static one; this is because dynamic quenching is largely due to collision and the energy transfer from trypsin to quencher. These results were consistent with the fluorescence results [41,49]. 3.5. The effects of putrescine on the trypsin activity According to the fluorescence, UV–vis and CD spectral results, it was found that putrescine not only affected the secondary structure of trypsin, but also partly influenced its tertiary structure. In order to explore the functional effects of putrescine on trypsin, at first, trypsin activity was assayed in the presence and absence of different concentrations of putrescine (0–5 mM) and substrate (BAEE), at pH 8.0 and the temperature of 37 ◦ C. The average velocity of reaction was determined by the generation of N␣ -benzoyl-l-arginine per seconds, as quantified via optical absorption at 253 nm. As illustrated in Fig. 9 and Table 5, addition of a higher concentration of putrescine to enzyme solution resulted in a gradual increase in the trypsin activity. The change in enzyme conformation led to the increase in the activity of trypsin. Overall, the binding of putrescine could change the micro-environment of the catalytic triad for the intruding molecules in their vicinity. Consequently, putrescine could influence the enzyme activity. It is possible that the binding of putrescine to trypsin would lead to the further exposure of the catalytic activity center [15]. With the addition of the putrescine, the

L. Momeni et al. / International Journal of Biological Macromolecules 94 (2017) 145–153

3

151

0.25

Absorbance

0.2

2.5

Absorbance

2

0.15 0.1 0.05 0

1.5

260

270

280

290

300

310

Wavelength (nm) 1

0.5

0 mM

1 mM

2 mM

3 mM

4 mM

0 200

220

240

260

280

300

Wavelength (nm) Fig. 8. UV–vis absorption spectra of trypsin in the absence and presence of putrescine at pH 8 and 310 K. The inset shows the increase in absorbance around 278 nm with increasing putrescine (0–4 mM).

2.5

1/V (S.mM-1)

2

1.5 0 mM

1

1 mM 3 mM

0.5

-1.5

0 -0.5

5 mM

0.5

1.5

2.5

3.5

1/[S]

4.5

5.5

6.5

7.5

8.5

(mM-1)

Fig. 9. Line weaver-Burk plot of trypsin at various putrescine concentrations (, 0 mM; 䊏, 1 mM; 䊉, 3 mM; , 5 mM) at 310 K, and pH 8.0. Table 6 Docking results for the putrescine – Trypsin system. Lowest Binding Energy (kcal/mol)

−4.43

Estimated Inhibition Constant, Ki

567.44

Final Intermolecular Energy (kcal/mol)

−5.92

H-bond (kcal/mol)

−2.17

affinity of the trypsin for BAEE was reduced (Km was increased). The term kcat /Km refers to the catalytic efficiency and a high value shows that the limiting factor for the overall reaction is the frequency of collisions of trypsin with BAEE molecules. Review of the literature showed that change in kcat could generally be explained in terms of cosolute-induced variations of the catalytic rate constant and the enzyme concentration. The former effect might be derived from minor changes in conformation, hydration and dynamics in the enzyme active site (by change in hydrogen bonds), as well as change in the stabilization of the enzyme-product complex [50]. These results proposed that by the binding of putrescine to trypsin, the high affinity sites like cationic lysine and arginine were exposed on the surface of trypsin, thereby decreasing the concentration of BAEE in the active site of trypsin. According to the results, Tm of trypsin and the enzyme activity were increased with the addition of putrescine concentrations. As a result, putrescine could be considered as an activator and stabilizer for trypsin. Kumar and Venkatesu

Electrostatic Energy (kcal/mol)

−3.75

Interaction bonds

Hydrogen-Bonding

Hydrophobic- Bonding

Glu77

Gly23 Ala24 Thr21

Asp71

have also suggested that polyamines are good enhancers for the native conformation of chymotrypsin [51]. 3.6. Molecular docking studies The results obtained multi-spectroscopic techniques were accompanied by docking studies in which the putrescine molecule was docked to trypsin to analyze the favorite binding sites on this enzyme. The final Intermolecular energy, hydrogen bond, total internal energy, binding energies and amino acid interactions in the docked complexes of Polyamine (putrescine) with protein trypsin are shown in Table 6; also, the interaction modes and H bond and hydrophobic interactions of the ligand Polyamines (putrescine) and trypsin are depicted in Figs. 10 and 11. As shown in Table 6, the negative value of the estimated free energy of binding revealed the spontaneity of the binding of trypsin with putrescine. As shown in Fig. 10, there were two hydrogen bonds (Glu 77,

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4. Conclusion

Fig. 10. H-bond (Asp 71, Glu 77) and hydrophobic (Gly 23, Ala 24, Thr 21) analysis of docking poses by Ligplot plus tool for putrescine ligand in Trypsin.

In the present study, the interaction between trypsin and putrescine was investigated for the first time by steady state thermal stability, intrinsic fluorescence, UV–vis spectroscopy, far and near-UV CD and kinetic techniques, as well as molecular docking. The results showed that there were binding interactions between putrescine and trypsin. The secondary and tertiary structures of trypsin were affected upon putrescine binding. It was also found that putrescine quenched the fluorescence of trypsin by the static quenching mechanism at pH 8, the binding process was spontaneous, and the fluorescence quenching resulted from the formation of the ground state complex. The increase in the intensity of UV spectroscopy and the decrease in the intensity of intrinsic fluorescence established the combination of putrescine and trypsin, which prevented the exposure of the hydrophobic amino acids to the environment and reduced the surface hydrophobicity. According to the thermodynamic parameters (H◦ , S◦ and G◦ ) and molecular docking, we could conclude that the hydrogen bonding and Van der Waals forces played a major role in stabilizing the complex. The change in conformation of trypsin was studied by CD and UV–vis spectroscopy in the presence of putrescine. Enzyme kinetics results and the thermal stability studies also demonstrated that putrescine increased the activity and the stability of trypsin. The docking study also indicated that putrescine was especially absorbed on the surface of the enzyme, leading to the increase of the thermal stability. The higher thermal stability of putrescinetrypsin complex might be relevant to the conformational change induced by the higher hydrogen bonds formation and the lower surface hydrophobicity after modification. The results also suggested that those poly-methylene skeletons of polyamine and its primary and secondary amines contributed to this effect. It seemed that these effects of polyamines were due their kosmotropic properties. The results from this study could be useful for developing the applications of putrescine and polyamines in the industry. References

Fig. 11. The superimposition of the docked putrescine into Trypsin.

Asp 71) in the binding interaction of putrescine with trypsin. In addition, there were three hydrophobic bonds (Gly 23, Ala 24 and Thr 21) between putrescine and trypsin. Many polar and nonpolar amino acid residues could take part in the binding of trypsin with putrescine; thus other binding forces including hydrophobic forces should not be excluded. Therefore, according to the results of quenching fluorescence, we could propose that the key mechanism of the binding reaction was related to hydrogen bonds and van der Waals forces. The docking studies further revealed the possibility of these binding interactions. The occurrence of hydrophobic association and hydrogen bonds and van der Waals interactions could change the native conformation of trypsin. It is, therefore, expected that putrescine would influence the enzyme activity.

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