Effect of Tb(III) on activity and stability of nattokinase

JOURNAL OF RARE EARTHS, Vol. 35, No. 5, May 2017, P. 510

Effect of Tb(III) on activity and stability of nattokinase MOU Xiaochao (牟小超)1, YANG Renjia (杨仁佳)2, ZHANG Wenlong (张文龙)1, YANG Binsheng (杨斌盛)1,* (1. Institute of Molecular Science, Key Laboratory of Chemical Biology of Molecular Engineering of Education Ministry, Shanxi University, Taiyuan 030006, China; 2. Blood Division, Shanxi Dayi Hospital, Taiyuan 030006, China) Received 8 September 2016; revised 1 December 2016

Abstract: Nattokinase, is an effective fibrinolytic enzyme with the potential for fighting cardiovascular disease. The aim of study was to investigate the interaction of Tb(III) with nattokinase and the impact of Tb(III) on the enzyme activity and protein stability. The binding of Tb(III) with nattokinase was studied by fluorescence spectrum in 100 mmol/L Tris-HCl (pH 8.0). It could be seen that the protein bound one Tb(III) with low affinity, and the binding constants K were 2.90×104 L/mol at 288 K. Although the activity of nattokinase determined by tetra-peptide substrate method at proper pH and temperature was not influenced for the binding of Tb(III), the transformation rate of substrate was increased to 113%. To better assess the stability of protease in the absence and presence of Tb(III), nattokinase was unfolded through continuous concentrations urea. Based on the model of structural element, the results showed that Tb(III) could not change the average structural element free energy <∆G0element (H2O)> of nattokinase by the measurement of enzyme activity, but it could improve the stability of the global protein by the fluorescence spectral measurement. Keywords: nattokinase; Tb(III); enzyme activity; protein stability; rare earths

Nattokinase (Subtilisin NAT or NK), one of the most considerable extracellular enzymes with strong fibrinolytic and thrombolytic activity, was first found in natto which is typical soybean food eaten in Japan[1]. Nattokinase has potential for fighting cardiovascular diseases including heart disease, high blood pressure and has also been used for curing other disease, such as Alzheimer’s disease, pain, vitreoretinal disorder, chronic fatigue syndrome, uterine fibroids[2,3]. Thus, NK is currently considered as an efficient, secure, economic, and preventative drug. As a number of subtilisin-like serine protease family, NK, encoded by aprN, was found code for a 29 residues signal peptide, a 77 residues propeptide and a 275 residues mature polypeptide, with a molecular mass of 27.7 kDa and an isoelectric point of 8.7[1,4]. NK not only could lyse fibrin directly or indirectly, but also showed amidolytic activity invested by several synthetic substrates, the most sensitive substrate for NK was the Suc-Ala-Ala-Pro-Phe-pNA for subtilisin[1]. So far, NK has been extensively studied not only for the insight into the mechanism of enzyme catalysis, but also for its significant applications in commercial field. The most important is to improve its activity and stability so as to broaden their utility in medical and commercial application. There are various methods to improve the activity and stability of NK at present. On the level of molecular biology, DNA family shuffling is one of the ways to improve the fibrinolytic activity of nattokinase[5].

Site-directed mutagenesis is another means to enhance activity and oxidative stability of NK[6,7]. During the NK purification process, the traditional protein expression and purification technology were changed and improved for better activity, but the results were less useful[8]. Beyond that, chemical modification has become an effective and popular approach to improve nattokinase activity and stability. NK was first well immobilized onto magnetic nanoparticles Fe3O4 in the presence of 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), and it turned out that it had much higher thrombolytic activity, even higher than the pure NK[9]. Immobilization of nattokinase upon polyhydroxybutyrate (PHB) nanoparticles resulted in a 20% increase in the enzyme activity[10]. The adsorption capacity of nanosilver (AgNPs) on nattokinase enhanced heat stability and anticoagulant effect of NK[11]. The effect of various metal ions, such as Ag(I), K(I), Na(I), Ba(II), Ca(II), Cd(II), Co(II), Cu(II), Hg(II), Mg(II), Mn(II), Ni(II), Zn(II), Fe(II), Fe(III) and Al(III), had been tested on enzyme activity with a final concentrations from 1 to 10 mmol/L[12–17]. It is not difficult to find that the metal ions and NK were added not at proper chemical stoichiometry in the experiment. So some new problems have arisen: whether and how these metal ions combined with NK, and how these metal ions impact the activity and stability of NK at the molecular level? The first X-ray diffraction analysis of nattokinase revealed the protease crystal structure with two Ca(II),

Foundation item: Project supported by the National Natural Science Foundation of China (21571117) and the PhD Programs Foundation of the Ministry of Education of China (20131401110011) * Corresponding author: YANG Binsheng (E-mail: [email protected]; Tel.: +86-351-7016358) DOI: 10.1016/S1002-0721(17)60941-4

MOU Xiaochao et al., Effect of Tb(III) on activity and stability of nattokinase

which were important for activity and stability of serine proteinase[18–21]. While, deep studies about Ca(II) having impact on NK stability and activity, to date, had remained undetermined. Tb(III), as a fluorescence probe, due to the similarity to Ca(II) in ions radii, charge character, and coordination chemistry, was well known to replace Ca(II) and had been used to study protein structural characterization in many proteins[22–29]. Moreover, the application of rare earth element in medical science has hundred years of history at aboard, due to its broad pharmacological properties, low toxicity and good clinical curative effect, a shining example of this is the anticoagulant therapy[30–32]. In the present study, nattokinase was produced and purified successfully. Then, the interaction of nattokinase with terbium ion and the function of Tb(III) in the enzyme were analyzed in detail. Tb(III) fluorescence probe was first applied to characterize the binding of NK with Tb(III). Further, the effect of Tb(III) on activity and stability of NK was investigated for the first time.

1 Materials and methods 1.1 Materials Synthetic substrate N-Succinyl-L-Ala-L-Ala-L-Pro-LPhe-p-nitroanilide (Suc-AAPF-pNA), urea, HCl, Tris were obtained from Sigma-Aldrich, USA. Terbium oxide (Tb4O7) was 99.99%, purchased from Rare Earth Research Institute of Hunan, China. All the chemicals were analytical grade reagents. The UV-Vis spectra were conducted by UV-Visible spectrophotometer (Varian Cary 50 BIO, Agilent). The fluorescence spectra were recorded on a fluorescence spectrophotometer (FluoroMax-4, HORIBA). All pH measurements were measured with a pH meter (Metter Toledo, Switzerland). 1.2 Methods 1.2.1 Production and purification of nattokinase For the production of nattokinase, the stain of Bacillus subtilis natto YBNK-1 was previously isolated from health care products. In the experiment, nattokinase was produced by the liquid state fermentation according to the previous report[33]. After the purification procedures, the activity of purified enzyme was detected, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to determine the enzyme’s purity and the molecular mass. 1.2.2 Terbium stock solution The terbium stock solution was prepared[25] by dissolving Tb4O7 with a small volume of concentrated hydrochloric acid (HCl) and then diluted with a specified volume of deionized water. The terbium solution was standardized by titration with standard ethylene diamine

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tetraacetic acid (EDTA) solution in hexamethylene tetramine buffer at pH 5.5. Titration endpoint was observed with a color change from pink to yellow by xylenol orange as an indicator. 1.2.3 Steady-state fluorescence studies For the measurement of NK interacting with Tb(III), NK was titrated with Tb(III) solution in 100 mmol/L Tris-HCl (pH 8.0). Allowing for equilibrium time, spectra were recorded at 2-min intervals after the addition of Tb(III). The experiments with the same conditions except the temperature were done to obtain the thermodynamic data for the combination of NK with Tb(III). The excitation wavelength of the fluorescence spectra was set at 290 nm and the slit widths of excitation and emission were both 5 nm. To correct dilution errors after each titration, the fluorescence intensity at the maximum emission peak needs to deduct the dilution effect. Using Tb3+ as fluorescence probe, we further explored the Ca(II)-NK binding properties from the competition between Ca(II) and Tb(III). In this study, Tb(III) and NK solution were mixed with the concentration rate of 1:1. Then the high concentration of Ca(II) was added to the NK-Tb to monitor the competition between Tb(III) and Ca(II). The reaction was monitored using aromatic residue sensitized Tb(III) fluorescence spectra, recorded between 500–560 nm with excitation at 290 nm, and the slit width of excitation and emission were both at 10 nm. 1.2.4 Enzymatic activity assays The amidolytic activity of nattokinase was measured using the chromogenic substrate Suc-AAPF-pNA as previously reported[6]. Firstly, the protein sample was incubated in the 100 mmol/L Tris-HCl buffer (pH 8.0) at 303 K. Ten minutes later, the substrate Suc-AAPF-pNA was added into the protein samples. The absorbance of p-nitroaniline (pNA) produced by the hydrolysis of Suc-AAPF-pNA during the incubation was recorded on a UV-Vis spectrophotometer. 1.2.5 Effects of pH and temperature on enzyme activity The effect of pH on enzyme activity was determined at pH 5.0–10.0 (pH 5.0–7.0 using 100 mmol/L citrate buffer, pH 7.4–10.0 using 100 mmol/L Tris-HCl buffer). The optimal temperature on enzyme activity was measured at different temperatures ranging from 25 to 70 ºC. The residual activity of NK was measured and the maximum activity was defined as 100%. 1.2.6 Enzyme-catalyzed kinetics In order to introduce the effect of Tb(III) on nattokinase activity, Tb(III) solution was added into the protein sample (NK and Tb(III) in the stoichiometry of 1:1). The enzyme-catalyzed kinetic measurement was measured at the optimum pH and the optimum temperature as described above. Reactions were initiated by the addition of the Suc-AAPF-pNA. Dynamic data were acquired by measuring the change of absorbance at 390 nm on kinetic scan mode of UV spectrophotometer with a 30 s lag. To

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ensure that the results were reproducible, the kinetic experiment was performed three times. The activity of the control without any additive (Tb solution) was defined as 100%. 1.2.7 Chemical unfolding experiments Denaturation experiment of protein was measured in the presence and absence of Tb(III) using urea, to make sure the role of the Tb(III) that played on the stability of nattokinase. Urea stock solution was prepared by dissolving solid denaturant in distilled water. In the chemical unfolding experiments detected by enzyme activity, NK and Tb-NK were induced into unfolding by urea at various concentrations at 4 ºC overnight. The substrate Suc-AAPF-pNA was added into the unfolding protein sample in the same degree to measure the enzyme activity. Urea-induced unfolding of the NK and Tb-NK was also monitored on a fluorescence spectrophotometer in 100 mmol/L Tris-HCl (pH 8.0). The protein solution was equilibrated with different concentrations of urea. In each urea denaturation experiment, every sample was added with urea from 0 to 10 mol/L at a protein concentration of 6 μmol/L. All the samples were induced for overnight before emission spectrum measured. The fluorescence spectra were measured by exciting the protein at 295 nm, and recorded in the range from 300 to 500 nm. The excitation and emission slits were both set at 5 nm. 1.2.8 Analysis of denaturation data Denaturation data were analyzed by the new model proposed by our group to measure the stability of protein and enzyme[34–36]. Based on this model, the values of the free energy of the average structural element <∆G0element(H2O)>, m, D1/2 were obtained to compare the stability of protein.

JOURNAL OF RARE EARTHS, Vol. 35, No. 5, May 2017

by SDS-PAGE (Fig. 1). SDS-PAGE exhibited a single band with a molecular weight approximately 28 kDa based upon the migration of molecular weight markers, which is in perfect accordance with previous report by Fujita et al.[12]. The enzyme activity was measured immediately after purification. The enzyme activity assay showed that the nattokinase was relative stable (Table 2) after long-term cryopreservation treatment compared with the new nattokinase. 2.2 Binding of Tb(III)/Ca(II) to NK The sensitization and enhancement of Tb(III) fluorescence via aromatic chromophore to Tb ions non-radiative energy transfer processes, will be possible to appear when the complex is irradiated in the near-ultraviolet absorption region of the aromatic moiety. The four characteristic fluorescence peak of Tb(III) in the presence of protein at 490, 545, 590, 623 nm, corresponding to the transition from 5D4 to 7F6, 7F5, 7F4, 7F3, respectively, is enhanced greatly[24,25,37]. In this paper, fluorescence spectra were used to determine the binding of Tb(III) to NK. Fig. 2(a) shows the Tb(III) fluorescence titration spectra for NK in 100 mmol/L Tris-HCl (pH 8.0). In the presence of NK, the fluorescence intensity of Tb(III) at 490, 545 nm were increased greatly, while the fluorescence of Tb(III) at 590 and 630 nm were neglected due to the limitation of the instrument. The sensitization of Tb(III) at 490 and 545 nm means that the binding of NK to Tb(III) for the energy-transfer from tryptophan residues

2 Results and discussion 2.1 Purification of nattokinase from Bacillus subtilis natto The purified enzyme was homogenous and monomeric

Fig. 1 SDS-PAGE analysis of purified nattokinase (Lane Marker: standard molecular protein; Lane 1: purified enzyme)

Fig. 2 (a) Sensitization fluorescence spectra of NK under different concentrations of Tb(III) (Inset: titration curve for the addition of Tb(III) to the proteins by measuring the fluorescence intensity of Tb(III) at 545 nm in 100 mmol/L Tris-HCl (pH 8.0)); (b) The scatter diagram of lg((Fi–F0)/(F∞–Fi)) versus lg[Tb] and the fitting figures at different temperatures

MOU Xiaochao et al., Effect of Tb(III) on activity and stability of nattokinase

to the binding Tb(III). Fig. 2(a) insert is the plot of fluorescence intensity of Tb(III) at 545 nm versus different molar ratios of Tb(III) to NK. Furthermore, the binding constant K, the binding site n of NK with Tb(III) obtained at different temperatures by double-logarithm regression equation, are shown in Fig. 2(b). Carries on the data fitting to the experimental findings, the slope of the straight line represents the number binding site n, and the intercept represents lgK. The specific data are shown in Table 1. NK titration with Tb(III) indicated that a NK-Tb complex can be formed with 1:1 stoichiometry with an affinity of 2.90×104 L/mol, 0.85×104 L/mol, 0.32×104 L/mol at 288, 295, 302 K, respectively. Moreover, to identify whether the Tb(III) replaced the binding sites of Ca(II) in NK, we studied the interaction of NK-Tb(III) with Ca(II) by ionic competition. NK-Tb complex was titrated with Ca(II) solution. The addition of Ca(II) made the fluorescence at 545 nm of Tb-NK quenched 42% (shown in Fig. 3(a)), which indicated that there is a competition of binding sites in NK between Tb(III) and Ca(II) or the transformation from NK-Tb to Ca-NK. The relative binding constant of Ca-NK can be determined from the florescence quenching curves in Fig. 3(b) using Eq. (1). The relative binding constant Ka of Ca-NK was calculated to be (1.11±0.04)×102 L/mol. K Ca-P [Tb]f [Ca-P] [Ca]f = (1) K Tb-P [Tb-P] 2.3 Effects of pH and temperature on the activity of NK

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The effects of pH and temperature on the activity of nattokinase were measured by tetra-peptide substrate method. The catalytic reaction of substrate with NK for enzyme activity determination is revealed in Scheme 1 that the amide bound of Suc-AAPF-pNA was broken down by NK to generate pNA, accompanied with the change in absorbance values in UV-Vis spectrum shown in Fig. 4(a). As shown in Fig. 4(b), the enzyme had certain activity across a broad pH range from 5 to 10. It can remain over 80% of activity over a pH range of 6 to 10. The optimal pH of NK was 8.0. According to experimental results, NK was observed to have a thermal stability range of 20–50 ºC and the optimum temperature was 37 ºC. The substrate was stable under the experimental conditions (data not shown). 2.4 Effect of Tb(III) on the activity of NK To determine the difference of the activity of NK combining with Tb(III) before and after, enzyme activity was detected at proper pH and temperature. The initial reaction rate and the reaction rate constants were obTable 1 Binding constants and the numbers of binding site for NK-Tb at different temperatures Temperature/

Binding constant

Number of

Linear

K

K/104 (L/mol)

binding site n

coefficient R

288

2.90 ± 0.15

1.11 ± 0.03

0.9971

295

0.85 ± 0.07

1.00 ± 0.02

0.9998

302

0.32 ± 0.08

0.92 ± 0.02

0.9987

Fig. 3 (a) Fluorescence spectra of Tb(III) binding to NK in the presence of Ca(II) ((1) NK-Tb, (2–14): NK-Tb with the different concentrations of Ca(II) from 0.09 to 5 mmol/L, (14) NK alone); (b) Fluorescence quenching curve for the addition of Ca(II) to NK-Tb in 100 mmol/L Tris-HCl (pH 8.0) (Insert is the plot of [Tb3+][Ca-P]/[Tb-P] versus [Ca2+])

Scheme 1 Catalytic reaction of Suc-AAPF-pNA with nattokinase

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Fig. 4 (a) UV-Vis absorption spectrum of Suc-AAPF-pNA before (line 1) and after (line 2) hydrolysis by NK (The absorbance value in 310 nm of Suc-AAPF-pNA decreased gradually for the hydrolysis process along with the increase in 390 nm of pNA, in 100 mmol/L Tris-HCl (pH 8.0)); (b) Effect of pH on the activity of NK and (c) effect of temperature on the activity of NK (the maximal activity was taken as 100%)

tained from kinetics assay. Fig. 5 shows the representative kinetic traces of NK and NK-Tb with Suc-AAPFpNA, respectively. The two reactions were satisfactorily fit to a single exponential function. And the initial reaction rates were 0.0028±0.0001 min–1, 0.0029±0.0001 min–1 for NK and NK-Tb, respectively. According to the initial reaction rate of enzymatic reaction, the measurement of enzyme activity demonstrated that Tb(III) had no influence on the activity of NK. However, the binding of Tb(III) makes the transformation rate be increased to 113%.

2.5 Effect of Tb(III) on the stability of NK measured by enzyme activity Enzyme activity can be regarded as the most sensitive probe to study the changes in the enzyme conformation during various treatments as it reflects subtle readjustments at the active site, allowing very small conformational variations of an enzyme structure to be detected[38]. To make clear the effect of Tb(III) on the stability of NK, urea, an well-known chemical denaturant, was chosen to analyze the unfolding behavior of NK. As shown in Fig. 6, increasing urea concentrations caused the residual activity of NK gradually decreased. No significant effect of denaturant on enzymatic activity of NK was observed up to about 1.0 mol/L urea. However, between 3.0 and 6.0 mol/L urea, a steep decrease in enzyme activity (from 100% to about 1%) and a complete loss of enzymatic activity above 7.0 mol/L urea were observed. A similar denaturation curve of NK-Tb was also obtained under the same experimental conditions. Urea-induced denatu-

Fig. 5 Kinetics trace of NK (1), NK (new) (2), and Tb-NK (3) with Suc-AAPF-pNA in 100 mmol/L Tris-HCl buffer (pH 8.0, 310 K), monitored by measurement of the change of absorbance value of pNA at 390 nm (The protein concentration was 40 nmol/L and the substrate was 6 μmol/L. The curves were obtained after fitting to a single exponential term) Table 2 Initial reaction rate, relative enzyme activity, and substrate transformation rate for NK sample with Suc-AAPF-pNA Protein

Substrate

Initial reaction rate/

Relative

transformation rate/%

min–1

activity/% 100±15

NK

100±11

0.0028±0.0001

NK-Tb

113±13

0.0029±0.0001

100±24

NK(new)

102 ± 23

0.0030 ± 0.0002

100 ± 22

Fig. 6 Changes in residual enzyme activity of NK (circle) and NK-Tb (triangle) on treatment with increasing concentrations of urea. NK, in 100 mmol/L Tris-HCl buffer (pH 8.0), was incubated with the desired concentrations of chemical denaturant for 12 h at 4 ºC (The data in the figure were expressed in terms of residual activity using the activity of the native enzyme as reference (100%))

MOU Xiaochao et al., Effect of Tb(III) on activity and stability of nattokinase

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ration of NK-Tb and NK were found to be a typical two-state (N→U) process. Based on the model of structure element, the thermodynamic parameters for unfolding of NK and NK-Tb were obtained using a two-state mechanism and are complied in Table 3. A comparison of the thermodynamics parameter of NK and NK-Tb showed that the binding of Tb to NK did not change the nattokinase unfolding behavior, which suggested that Tb(III) had no significant influence on the stability of NK by the detection of enzyme activity. The binding site of Tb(III) at NK probably was not at the active center. This finding was consistent with the previous reports[18]. 2.6 Effect of Tb(III) on the stability of NK measured by fluorescence spectra The changes of the spectral parameters of tryptophan (Trp) fluorescence emission are dependent on the conformational changes, electronic and dynamic properties of the chromophore microenvironment, hence, the intrinsic fluorescence of Trp residues was considered as a probe to obtain information on the structure properties of protein[39]. There are three Trp residues in NK, so the change of Trp microenvironment can be assessed to reflect the global change in protein structure. Fig. 7(a) shows the steady-state fluorescence emission spectra for NK by exciting the protein at 295 nm in the presence of increasing concentrations of urea. It is notable that the fluorescence emission intensity increased, accompanied by a redshift (from 353 to 361 nm) in emission maximum with increasing concentrations of urea, which revealed that more tryptophan residues in protein is exposed to solvent as a result of denaturation. The similar results were found in NK-Tb in the presence of urea (data no shown). To best evaluate the shape of the transitions of proteins, the values of emission intensity at 345 and 370 nm (I370 nm/I345 nm) obtained from selected fluorescence spectrum of NK and NK-Tb upon excitation at 295 nm, were plotted against denaturant concentrations. A sigmoidal dependence of fluorescence intensity ratio on urea concentrations was observed. As evident from Fig. 7(b), urea-induced denaturation of NK and NK-Tb was found to be a two-state, single-step process with no detectable intermediate state(s). The transition of NK started at 2.0 mol/L and slopes off at 7.0 mol/L urea with a midpoint occurring at 4.6 mol/L urea. While, the unfolding curve of NK-Tb showed difference in unfolding process compared to NK, as it started at more later around 3.0 mol/L urea, and completed at 8.0 mol/L with a midpoint about 5.1 mol/L urea. The unfolding curve of NK compared with the NK-Tb, was indicative of Tb(III) playing a vital role in increasing the stability of protein against the increasing the urea concentrations. The data in Table 3 showed that the stability of nattokinase was increased as the binding of Tb(III). Maybe the electrostatic interaction

  Fig. 7 Fluorescence spectrum of NK-Tb in the presence of different concentrations of urea for the curves from bottom to top shown by arrow corresponding to increasing urea concentrations (Unfolding of NK-Tb was induced by urea in 100 mmol/L Tris-HCl buffer (pH 8.0, 298 K), as followed by intrinsic fluorescence measurements upon excitation at 295 nm (a) and unfolding transitions of denaturation of NK (circle) and NK-Tb (triangle), was monitored by fluorescence ratio at I370 nm/I345 nm after exciting at 295 nm (b)) Table 3 Thermodynamic parameters of NK, Tb-NK from urea-induced unfolding by spectroscopy and enzyme activity detection Test Spectroscopy Enzyme activity

Protein

<∆G0element(H2O)> / (kJ/mol)

–m/ 2

[D]1/2/ –1

(kJ/mol /L ) (mol/L)

NK

12.32 ± 0.25

2.67 ± 0.05

4.58

NK-Tb

13.50 ± 0.78

2.68 ± 0.14

5.11

NK

13.26 ± 0.54

2.73 ± 0.12

4.71

NK-Tb

13.72 ± 0.69

2.72 ± 0.15

4.98

of Tb(III) and NK made the structure of protein more compact and led to stabilization of NK during protein unfolding.

3 Conclusions In summary, nattokinase had one Tb(III) binding site with the association constant values 2.90×104 L/mol at 288

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K. Enzymic catalytic reaction by kinetic spectrophotometric determination, displayed that enzyme activity of NK was not changed in presence of Tb(III), but the transformation rate of substrate was increased. Chemical denaturation studies by enzyme activity assay suggested that the binding of Tb(III) brought about no change in the average structural element free energy of NK. However, there was certain enhancement in stability of the global protein upon binding of Tb(III) analyzed by spectrofluorometry through various concentrations of urea.

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