# Interaction of Hyperoside with Human Serum Albumin and Effect of Glucose on the Binding

Authors: Jie Yang; Lingling Qu; Wenyue Yang; Yun Huang; Ning Jiao; Wenhong Zhan; Ding Zhao; Lijian Cui
Journal: Journal of Spectroscopy
 (2014)

---

## Abstract

The interaction of hyperoside (Hyp) with human serum albumin (HSA) and effect of glucose on the binding were studied in simulating physiological condition (pH 7.40). The results suggested that Hyp quenched the endogenous fluorescence of HSA via a static quenching process with the distance of 1.95 nm between Hyp and HSA. Hydrophobic forces played a major role in stabilizing the Hyp-HSA complex. Through synchronous fluorescence monitoring of conformation of HSA, we found that the binding to Hyp can change the microenvironment around tryptophan (Trp) residues. Increasing in glucose concentration over a range from 0 to 9 mM decreased the binding ability of HSA to Hyp, implying that increasing in glucose concentration would increase the concentration of free Hyp.


---

## Body

## 1. Introduction

Hyperoside (Hyp) (Figure1), named 2-(3,4-dihydroxyphenyl)-3-[(3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4H-chromene-4,5,7-triol, is a flavonol compound isolated from the plants of Hypericaceae and Rosaceae. Hyp has a variety of physiological activities, including antioxidant, anti-inflammatory, and antiviral and so on [1–4]. In addition, Hyp plays a protective role against myocardial ischemia and cerebral ischemia injury through fighting free radicals, reducing cell apoptosis, the release of lactate dehydrogenase, and calcium inflow [5–7]. In recent years, a significant discovery has been made through the studies on the inhibitory action of Hyp on eye aldose reductase (AR) which in mammals catalyzes the conversion of glucose to sorbitol that is one of the leading causes of diabetes complications such as cataract and neurological diseases. AR inhibitory activities of 2 μM Hyp were from 57.81% to 54.41% with the 50% inhibition concentration (IC50) 1.85±0.06 μM, which may be good for the prevention of diabetes complication [8].Figure 1
The structure of hyperoside (Hyp).Little attention has been paid on Hyp binding with human serum albumin (HSA) and the effect of glucose on Hyp-HSA binding even though the interaction of Hyp with bovine serum albumin was reported [9]. HSA is the most abundant plasma protein in humans and acts as a transport protein and plasma carrier by nonspecific binding. As one of the most extensively studied model protein, HSA has high affinity to many endogenous and exogenous compounds, serving in transporting drugs or other organic molecules to their targets [10, 11].The concentration of blood glucose is the amount of glucose present in the blood of a human or animal. Normally in human, the body maintains the blood glucose level at 3.9–6.1 mM (fasting plasma glucose) and 3.9–7.8 mM (postprandial blood glucose), above or below which may be an indicator of a medical condition [12]. Our previous research showed that blood glucose affected the binding between some flavonol compounds and BSA [13]. It is very informative to investigate on the binding of Hyp to HSA in the presence and absence of glucose.In order to further explore whether the interactions between Hyp and HSA occurred and the influence of glucose on them, we primarily focus on investigating the effect of Hyp on the fluorescence spectrum of HSA, the fluorescence quenching mechanism, the binding constant, the number of binding sites, the binding mode, and the effect of different glucose concentrations including 0, 3, 6, or 9 mM on the binding. These parameters are expected to provide more information about the interaction between Hyp and HSA. The studies on intermolecular interaction of Hyp with HSA and the influence of glucose on the binding will contribute to the understanding of the functions and the metabolic processes of Hyp. Finally, the results may provide an important theoretical support for the use of Hyp in the treatment of diabetic cataract.


## 2. Materials and Methods

### 2.1. Materials and Apparatus

HSA purchased from Sigma (New York, USA) (lyophilized powder) was used without further purification and the powder (a molecular mass 66000 Da) was 98-99% pure (agarose gel electrophoresis), which was prepared to the experimental concentration according to weight and the molecular mass. Tris (no less than 99.5% pure) was also from Sigma. Hyp (no less than 98% pure) was bought from national institute for the control of pharmaceutical and biological products (Beijing, China). All other chemicals were of analytical reagent grade.Fluorescence measurements were performed with a Perkin-Elmer spectrofluorometer Model LS-55 equipped with a 150 W Xenon lamp and a thermostat bath. The widths of both the excitation slit and emission slit were set at 5.0 nm with a nominal resolution of 0.5 nm. The absorption spectra were measured on a Shimadzu UV-2450 UV-vis spectrophotometer. The pH measurements were carried out on a PHS-3C digital pH meter. Titrations were done manually by using trace syringes. A quartz cell of 1.00 cm path length was used for the measurements.


### 2.2. Procedures

1 × 10−5 M and 3 × 10−3 M Hyp stock solutions were prepared in methanol and 1 × 10−5 M HSA stock solution in Tris-HCl buffer. The stock solution was diluted with 5 × 10−2 M Tris-HCl buffer solution (pH 7.4) to some lower concentration containing 5 × 10−2 M NaCl for actual use.Three mL HSA (1 × 10−6 M) was titrated with 0, 1, 2, 3, 4, 5, 6, or 7 μL Hyp (3 × 10−3 M), respectively, and a certain amount of Tris-HCl buffer solution to give the final volume of 3.01 mL. Another solution containing HSA in the absence and presence of glucose (3, 6, or 9 mM) was prepared similarly. The final concentration of Hyp varied from 0 to 7 × 10−6 M at an increment of 1 × 10−6 M. The resultant mixture was subsequently ultrasonicated for 1 min and incubated for 5 min at 298, 304, or 310 K.Fluorescence spectra were recorded at 298 and 310 K in the range from 300 to 420 nm at excitation wavelength 280 nm. The synchronous fluorescence spectra were recorded whenΔλ=60nm. The three-dimensional fluorescence spectra were performed under the following conditions: emission wavelengths over a range of 280–420 nm, excitation at 260 nm, scanning number 13, and increment 5 nm. The UV-vis absorption spectrum of Hyp (HSA or Hyp-HSA) was recorded in the range from 300 to 420 nm using UV-2450.


### 2.3. Correction of Fluorescence Intensity

Stern-Volmer equation [14, 15] was used to deduce HSA quenching mechanism caused by Hyp; a correction for the inner filter effect [16, 17] was made using (1). Consider
(1)Fc=Fa×e(A1+A2)/2,
where Fc and Fa are the fluorescence intensity corrected and observed, respectively. A1 and A2 are the absorption of Hyp solution at excitation and emission wavelength, respectively. All intensities of fluorescence used were corrected in this paper.


### 2.4. Determination of Free HSA Concentration

Binding ratio of HSA (HSAbr) was calculated by (2).The intrinsic fluorescence is mainly manifested by emission of Trp residues in molecule. WhenΔλ=60nm, synchronous fluorescence intensities (F′) of HSA in the range from 0 to 1 × 10−6 M were recorded, which just show the fluorescence of Trp residues. The calibration curve was constructed, and a linear regression analysis between the relationship of [HSA]free×107 (HSA without Hyp) (x) and F′(y) is plotted. The [HSA]free were obtained through the plot. Consider
(2)HSAbr=[HSA]bound[HSA]total,[HSA]bound=[HSA]total-[HSA]free.Then synchronous fluorescence intensities after Hyp being added were brought into the calibration curve to get the[HSA]free. Thus, HSAbr can be evaluated from (2).


### 2.5. Calculation ofKA and n

Binding constant and the number of binding sites were calculated by (3).When Hyp binds independently to a set of equivalent sites on a HSA, binding constant (KA) and the number of binding sites (n) are given by the double logarithm regression equation [15, 18]:
(3)log⁡[F0-FF]=log⁡KA+nlog⁡[Hyp].


### 2.6. Calculation of Distance between HSA and Hyp

Distance between HSA and Hyp was obtained by the following equations [13]:
(4)EC=R06(R06+r6),(5)EC=E′HASbr,(6)E′=1-FF0,
where EC is the corrected energy transfer efficiency and E′ the measured energy transfer efficiency. Consider
(7)R06=8.8×10-25K2N-4ΦJ(8)J=(∑Fλελλ4Δλ)∑FλΔλ.F is the fluorescence intensity of HSA when CHSA:CHyp=1:1, F0 is the fluorescence intensity without Hyp, and r is the binding distance between HSA and Hyp. R0 is the critical distance with K2 (the spatial orientation factor of the dipole) = 2/3, N (the refractive index of the medium) = 1.34, and Φ (the fluorescence quantum yield of HSA) = 0.12, when the transfer efficiency is 50%. J is the overlap integral of the fluorescence emission spectrum of HSA and the absorption spectrum of Hyp. Fλ is the fluorescence intensity of HSA at wavelength λ, ελ is the molar absorptivity of Hyp at wavelength λ, and Δλ is wavelength span.


### 2.7. Binding Forces

The interaction forces between Hyp and HSA were estimated by the relationships between the thermodynamic parameters; according to the enthalpy change(ΔH°) and entropy change (ΔS°) before and after reaction, the main interaction forces can be estimated. Based on the binding constants at different temperatures, the free energy change (ΔG°) can be estimated [14, 15]. Consider
(9)ln⁡K2K1=(1T1-1T2)ΔH°RΔG°=-RTln⁡KΔG°=ΔH°-TΔS°,
where K is the binding constant at the corresponding experimental temperature T and R is the gas constant.


## 2.1. Materials and Apparatus

HSA purchased from Sigma (New York, USA) (lyophilized powder) was used without further purification and the powder (a molecular mass 66000 Da) was 98-99% pure (agarose gel electrophoresis), which was prepared to the experimental concentration according to weight and the molecular mass. Tris (no less than 99.5% pure) was also from Sigma. Hyp (no less than 98% pure) was bought from national institute for the control of pharmaceutical and biological products (Beijing, China). All other chemicals were of analytical reagent grade.Fluorescence measurements were performed with a Perkin-Elmer spectrofluorometer Model LS-55 equipped with a 150 W Xenon lamp and a thermostat bath. The widths of both the excitation slit and emission slit were set at 5.0 nm with a nominal resolution of 0.5 nm. The absorption spectra were measured on a Shimadzu UV-2450 UV-vis spectrophotometer. The pH measurements were carried out on a PHS-3C digital pH meter. Titrations were done manually by using trace syringes. A quartz cell of 1.00 cm path length was used for the measurements.


## 2.2. Procedures

1 × 10−5 M and 3 × 10−3 M Hyp stock solutions were prepared in methanol and 1 × 10−5 M HSA stock solution in Tris-HCl buffer. The stock solution was diluted with 5 × 10−2 M Tris-HCl buffer solution (pH 7.4) to some lower concentration containing 5 × 10−2 M NaCl for actual use.Three mL HSA (1 × 10−6 M) was titrated with 0, 1, 2, 3, 4, 5, 6, or 7 μL Hyp (3 × 10−3 M), respectively, and a certain amount of Tris-HCl buffer solution to give the final volume of 3.01 mL. Another solution containing HSA in the absence and presence of glucose (3, 6, or 9 mM) was prepared similarly. The final concentration of Hyp varied from 0 to 7 × 10−6 M at an increment of 1 × 10−6 M. The resultant mixture was subsequently ultrasonicated for 1 min and incubated for 5 min at 298, 304, or 310 K.Fluorescence spectra were recorded at 298 and 310 K in the range from 300 to 420 nm at excitation wavelength 280 nm. The synchronous fluorescence spectra were recorded whenΔλ=60nm. The three-dimensional fluorescence spectra were performed under the following conditions: emission wavelengths over a range of 280–420 nm, excitation at 260 nm, scanning number 13, and increment 5 nm. The UV-vis absorption spectrum of Hyp (HSA or Hyp-HSA) was recorded in the range from 300 to 420 nm using UV-2450.


## 2.3. Correction of Fluorescence Intensity

Stern-Volmer equation [14, 15] was used to deduce HSA quenching mechanism caused by Hyp; a correction for the inner filter effect [16, 17] was made using (1). Consider
(1)Fc=Fa×e(A1+A2)/2,
where Fc and Fa are the fluorescence intensity corrected and observed, respectively. A1 and A2 are the absorption of Hyp solution at excitation and emission wavelength, respectively. All intensities of fluorescence used were corrected in this paper.


## 2.4. Determination of Free HSA Concentration

Binding ratio of HSA (HSAbr) was calculated by (2).The intrinsic fluorescence is mainly manifested by emission of Trp residues in molecule. WhenΔλ=60nm, synchronous fluorescence intensities (F′) of HSA in the range from 0 to 1 × 10−6 M were recorded, which just show the fluorescence of Trp residues. The calibration curve was constructed, and a linear regression analysis between the relationship of [HSA]free×107 (HSA without Hyp) (x) and F′(y) is plotted. The [HSA]free were obtained through the plot. Consider
(2)HSAbr=[HSA]bound[HSA]total,[HSA]bound=[HSA]total-[HSA]free.Then synchronous fluorescence intensities after Hyp being added were brought into the calibration curve to get the[HSA]free. Thus, HSAbr can be evaluated from (2).


## 2.5. Calculation ofKA and n

Binding constant and the number of binding sites were calculated by (3).When Hyp binds independently to a set of equivalent sites on a HSA, binding constant (KA) and the number of binding sites (n) are given by the double logarithm regression equation [15, 18]:
(3)log⁡[F0-FF]=log⁡KA+nlog⁡[Hyp].


## 2.6. Calculation of Distance between HSA and Hyp

Distance between HSA and Hyp was obtained by the following equations [13]:
(4)EC=R06(R06+r6),(5)EC=E′HASbr,(6)E′=1-FF0,
where EC is the corrected energy transfer efficiency and E′ the measured energy transfer efficiency. Consider
(7)R06=8.8×10-25K2N-4ΦJ(8)J=(∑Fλελλ4Δλ)∑FλΔλ.F is the fluorescence intensity of HSA when CHSA:CHyp=1:1, F0 is the fluorescence intensity without Hyp, and r is the binding distance between HSA and Hyp. R0 is the critical distance with K2 (the spatial orientation factor of the dipole) = 2/3, N (the refractive index of the medium) = 1.34, and Φ (the fluorescence quantum yield of HSA) = 0.12, when the transfer efficiency is 50%. J is the overlap integral of the fluorescence emission spectrum of HSA and the absorption spectrum of Hyp. Fλ is the fluorescence intensity of HSA at wavelength λ, ελ is the molar absorptivity of Hyp at wavelength λ, and Δλ is wavelength span.


## 2.7. Binding Forces

The interaction forces between Hyp and HSA were estimated by the relationships between the thermodynamic parameters; according to the enthalpy change(ΔH°) and entropy change (ΔS°) before and after reaction, the main interaction forces can be estimated. Based on the binding constants at different temperatures, the free energy change (ΔG°) can be estimated [14, 15]. Consider
(9)ln⁡K2K1=(1T1-1T2)ΔH°RΔG°=-RTln⁡KΔG°=ΔH°-TΔS°,
where K is the binding constant at the corresponding experimental temperature T and R is the gas constant.


## 3. Results and Discussion

### 3.1. Effect of Hyp on Fluorescence Spectrum of HSA

The study shows that the peak shape and intensity of HSA fluorescence emission were very stable and highly repeatable in Tris-HCl buffer solution and not affected by a small amount of methanol.At excitation wavelength 280 nm, scanning fluorescence spectra showed that HSA had a strong fluorescence emission peak at 349 nm and Hyp did not. Because Hyp has weak UV absorption at 280 and 349 nm, (1) was used to correct for the inner filter effects caused by the absorption of Hyp.HSA emission spectrum is the reflection of the kind, structure, and microenvironment of fluorescence chromophore, mainly including Trp and Tyr residues [19], through which drug-protein complex is usually studied. The maximum emission wavelength of HSA, used to judge whether the protein conformation is changed, is closely related to the microenvironment of aromatic residues [20–22]. The fluorescence quenching spectra of HSA with different concentrations of Hyp are shown in Figure 2. The fluorescence intensities of HSA at 349 nm decreased regularly with the increase of Hyp concentration and the maximum hypsochromic shift was 5 nm (λmax⁡ from 349 to 344 nm) at 310 K, suggesting that the complex of Hyp-HSA was formed, which is responsible for the quenching of HSA fluorescence.Figure 2
The fluorescence quenching spectra of HSA at different concentration of Hyp (T=298 K, pH = 7.4 and λex=280 nm); from curve 1 to 8, CHSA=1.0×10-6 M, CHyp=0, 1, 2, 3, 4, 5, 6, and 7 × 10−6 M, respectively.Dynamic quenching, static quenching, and nonradiative energy transfer are three major causes of intrinsic fluorescence quenching [15]. The possible quenching mechanism can be interpreted by the fluorescence quenching spectra and the Stern-Volmer curves of Hyp with HSA. In order to confirm the view, the procedure of the fluorescence quenching was first assumed to be dynamic quenching.TheKSV values increase with an increase in temperature for dynamic quenching, for which an increase in temperature is favorable, and the reverse effect would be observed for static quenching [15, 23]. If the quenching type includes both static and dynamic, the Stern-Volmer plot is an upward curvature [24].Figure3 shows the Stern-Volmer plots of the fluorescence quenching of HSA by Hyp at different temperatures. The SV plots have good linearity with each regression coefficient above 0.99. The results (Table 1) show that within the detected concentrations the KSV is correlated with temperature and decrease from 1.73×105 to 9.75×104 L·mol−1 with the temperature increasing, which indicates that the probable quenching mechanism of Hyp-HSA is static quenching, rather than dynamic quenching [15, 25–27].Table 1
Quenching rate constant of the bimolecule (Kq), quenching constant (KSV), binding constant (KA), and the number of binding sites (n) of Hyp to HAS.
T (K)
Glucose concentration mM
Stern-Volmer equation
Double logarithm regression equation
K
q ×1012 L·mol−1 s−1
K
SV ×104 L·mol−1
R
a
SD
b
K
A ×105 L·mol−1
n
R
a
SD
b
298
0
17.3
17.3
0.992
0.0159
2.47
1.09
0.996
0.0108
310
0
9.75
9.75
0.993
0.0097
42.7
1.28
0.995
0.0123
3
16.2
1.19
0.997
0.0098
6
11.1
1.17
0.984
0.0130
9
3.23
1.10
0.992
0.0103
aThe correlation coefficient.
bThe standard deviation.Figure 3
Stern-Volmer plots of Hyp-HSA at different temperatures,C(Hyp) = 1, 2, 3, 4, 5, 6, 7 × 10−6 M.In general, for static quenching, the value ofKq is above 2.0×1010 L mol−1 s−1 [11]. Kq initiated by Hyp (17.3×1012 L·mol−1 s−1 (298 K) or 9.75 × 1012 (310 K)) is much greater than 2.0×1010 L·mol−1 s−1, which means that the quenching process is static.


### 3.2. Binding Ratio of HSA (HSAbr)

By the linear regression analysis between[HSA]free×107 (without Hyp) and F′ (Figure 4), (10) was obtained
(10)y=62.129x+7.6217(R2=0.9972),
where R is the correlation coefficient.Figure 4
Linear regression analysis between[HSA]free and F′ (F′ the Synchronous fluorescence intensity when Δλ=60 nm), C(HSA) = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 × 10−7 M.WhenΔλ=60nm, the synchronous fluorescence intensities after Hyp added were brought into (7). [HSA]free=0.96×10-6 M was got when CHSA=CHyp=1×10-6 M; thus, HSAbr=4% from (2).HSAbr was 7.9% when CHSA:CHyp=1:2.


### 3.3. Effect of Hyp on UV Absorption of HSA

The structural change and the complex formation between Hyp and HSA can also be explored by UV absorption measurement. For static quenching, due to forming nonfluorescent complexes with fluorophores in the ground state, the absorption spectra of HSA with quencher are different from those without quencher, while for dynamic quenching affecting the excitation state of fluorophores does not change the absorption spectra of HSA [15, 23–25, 28].The absorption spectra of HSA in the absence and presence of Hyp were recorded by subtracting the corresponding spectrum of Hyp free form in the buffer from that of Hyp-HSA system in Figure5. HSA has two main peaks (210 and 278 nm) in its absorption spectrum, which shows that the addition of Hyp made the absorption peak of HSA very small decrease at 210 nm and no significant changes at 278 nm when HSAbr was 4% (Figure 5(a)) and significant drop at 210 nm and 278 nm when HSAbr was 7.9% (Figure 5(b)). The absorption spectral changes of HSA in the presence of Hyp strongly suggest the complex formation between Hyp and HSA.The absorption spectra of HSA, Hyp, and Hyp-HSA system. a: [Hyp-HSA], b: [HSA], and c: [Hyp-HSA]—[Hyp], d: [Hyp].
(a)
C
(
HSA
)
=
C
(
Hyp
)
=
1.0
×
10
-
6 M
(b)
C
(
HSA
)
=
1.0
×
10
-
6 M; C(Hyp)=2.0×10-6 M


### 3.4. Binding Constant and the Number of Binding Sites

From Figure6, the double logarithm regression curves possessed good linearity and the slope significantly increased with temperature increasing.Figure 6
The double logarithm regression plots of Hyp-HSA at different temperatures,C(HSA)=1,2,3,4,5,6,7×10-6M.The values ofKA and n of Hyp binding to HSA increased with temperature increasing from 2.47×105 to 4.27×106 L·mol−1and from 1.09 to 1.28, respectively


### 3.5. Distance between HSA and Hyp

Figure7 shows the overlapping plot between absorption spectrum of Hyp and emission spectrum of HSA. The value of J is evaluated from (8), which was J=2.67×10-14 cm3·L·mol−1. Thus, we obtained that R0=3.00 nm from (7); E′=0.04 from (6).Figure 7
Overlap of fluorescence emission spectrum of HSA (curve 1) and UV absorption spectrum of Hyp (curve 2);C(Hyp)=C(HSA)=1×10-6 M.WhenCHSA=CHyp=1×10-6 mol L−1 and HSA binding ratio was calculated to be 4%, then, EC=0.93 was obtained from (5).The distance (r) was 1.95 nm from (4).


### 3.6. Binding Mode

The interaction forces between Hyp and HSA mainly involve hydrogen bonds, Van der Waals forces, electrostatic interactions and hydrophobic forces, and so forth [14, 15].From the experimental data shown in Table2, the thermodynamic parameters of Hyp binding to HSA were ΔH°>0 and ΔS°>0, which indicated that the predominant intermolecular forces to stabilize the complex of Hyp-HSA were hydrophobic forces. The negative ΔG° value was taken as evidence for the spontaneity of the binding of Hyp with HSA at standard conditions.Table 2
Thermodynamic parameters of the interaction between Hyp and HAS.
T/K
ΔH°/(kJ·mol−1)
ΔS°/(J·mol−1 K−1)
ΔG°/(kJ·mol−1)
298 K
36.70
226.37
-
30.76
304 K
36.70
227.39
-
29.75
310 K
36.70
227.61
-
29.28


### 3.7. Synchronous Fluorescence Spectra

The intrinsic fluorescence of HSA is manifested by emission of Trp and Tyr residues in molecule [19]. When Δλ=60nm, synchronous fluorescence spectra just showed the fluorescence of Trp residues, the maximum emission wavelength of which is related to its microenvironment [14, 15].The synchronous fluorescence spectra of HSA (Figure8) show that the fluorescence intensity of HSA at 285 nm decreased regularly and the maximum emission wavelength had weak bathochromic shift (from 285 to 287 nm) with a fixed amount of HSA and the increase of Hyp concentration, which represented that the conformation of HSA was somewhat changed leading to decreased polarity around the Trp residues.Figure 8
Synchronous fluorescence spectra of interaction between HSA and Hyp; from curve 1 to 8,C(HSA)=1×10-6 M, C(Hyp) = 0, 1, 2, 3, 4, 5, 6, and 7 × 10−6 M, respectively.


### 3.8. Influence of Glucose on Binding Constant, the Number of Binding Sites, and Conformation

In order to investigate the influence of glucose on the interaction of Hyp with HSA, the binding constants and the number of binding sites in presence and absence of different concentrations of glucose (3, 6, or 9 mM) at 310 K were compared. Glucose decreased binding constant and the number of binding sites of Hyp with HSA at 310 K (KA from 4.27 × 106 to 1.62 × 106, 1.11 × 106 and 3.23 × 105 L·mol−1; n from 1.28 to 1.19, 1.17, and 1.10, resp.) (Table 1). The double logarithm curves possessed good linearity and the slope significantly decreased with the increase of sugar level (Figure 9).Figure 9
The double logarithm regression plots of Hyp-HSA at different concentrations of glucose.The three-dimensional fluorescence spectra of Hyp-HSA in the absence and presence of glucose were shown in Figure10, respectively.Three-dimensional fluorescence spectra of HSA (a), Hyp-HSA without glucose (b), Hyp-HSA with 3 mM glucose (c), Hyp-HSA with 6 mM glucose (d), and Hyp-HSA with 9 mM glucose (e) at 310 K;C(HSA)=1×10-6 M, C(Hyp)=7×10-6 M.
(a)
(b)
(c)
(d)
(e)The excitation wavelength, emission wavelength and fluorescence intensity as axes in the fluorescence emission spectra, three-dimensional fluorescence technique is commonly used for the investigation of conformational changes of proteins interacting with drugs [29, 30]. It was reported that the three-dimensional fluorescence spectrum is an effective means to investigate the characteristic conformational change of protein [31–33].Two fluorescence peaks 1 and 2 were easily observed in three-dimensional fluorescence spectra. Peak 1 is the Rayleigh scattering peak(λex=λem). Peak 2 (λex<λem,Δλ>0) which reveals the spectral behavior of Trp and Tyr residues, of which the maximum emission wavelength and fluorescence intensity show a close relation to the polarity of their microenvironment, was the primary fluorescence peak we studied [34, 35].The fluorescence intensity of Hyp-HSA in the presence of glucose (at 433, 426, and 415, resp.) was weaker than that (446 nm) without glucose.By comparison, it could be seen that all the maximum excitation wavelengths were at 280 nm and the maximum excitation wavelengths were from 347 to 342 nm in the three-dimensional fluorescence spectra (Figures10(a), 10(b), 10(c), 10(d), and 10(e)) and the Stokes shift from 67 to 62 nm (Δλ between excitation wavelength and emission wavelength) changed which demonstrated that glucose had an effect on the polarity and hydrophilicity around Trp microenvironment [33].By decreasing the binding constant and the number of binding sites of Hyp with HSA, glucose could improve the plasma concentration of free Hyp, which may be help for the inhibitory action on eye aldose reductase and diabetic cataract.


## 3.1. Effect of Hyp on Fluorescence Spectrum of HSA

The study shows that the peak shape and intensity of HSA fluorescence emission were very stable and highly repeatable in Tris-HCl buffer solution and not affected by a small amount of methanol.At excitation wavelength 280 nm, scanning fluorescence spectra showed that HSA had a strong fluorescence emission peak at 349 nm and Hyp did not. Because Hyp has weak UV absorption at 280 and 349 nm, (1) was used to correct for the inner filter effects caused by the absorption of Hyp.HSA emission spectrum is the reflection of the kind, structure, and microenvironment of fluorescence chromophore, mainly including Trp and Tyr residues [19], through which drug-protein complex is usually studied. The maximum emission wavelength of HSA, used to judge whether the protein conformation is changed, is closely related to the microenvironment of aromatic residues [20–22]. The fluorescence quenching spectra of HSA with different concentrations of Hyp are shown in Figure 2. The fluorescence intensities of HSA at 349 nm decreased regularly with the increase of Hyp concentration and the maximum hypsochromic shift was 5 nm (λmax⁡ from 349 to 344 nm) at 310 K, suggesting that the complex of Hyp-HSA was formed, which is responsible for the quenching of HSA fluorescence.Figure 2
The fluorescence quenching spectra of HSA at different concentration of Hyp (T=298 K, pH = 7.4 and λex=280 nm); from curve 1 to 8, CHSA=1.0×10-6 M, CHyp=0, 1, 2, 3, 4, 5, 6, and 7 × 10−6 M, respectively.Dynamic quenching, static quenching, and nonradiative energy transfer are three major causes of intrinsic fluorescence quenching [15]. The possible quenching mechanism can be interpreted by the fluorescence quenching spectra and the Stern-Volmer curves of Hyp with HSA. In order to confirm the view, the procedure of the fluorescence quenching was first assumed to be dynamic quenching.TheKSV values increase with an increase in temperature for dynamic quenching, for which an increase in temperature is favorable, and the reverse effect would be observed for static quenching [15, 23]. If the quenching type includes both static and dynamic, the Stern-Volmer plot is an upward curvature [24].Figure3 shows the Stern-Volmer plots of the fluorescence quenching of HSA by Hyp at different temperatures. The SV plots have good linearity with each regression coefficient above 0.99. The results (Table 1) show that within the detected concentrations the KSV is correlated with temperature and decrease from 1.73×105 to 9.75×104 L·mol−1 with the temperature increasing, which indicates that the probable quenching mechanism of Hyp-HSA is static quenching, rather than dynamic quenching [15, 25–27].Table 1
Quenching rate constant of the bimolecule (Kq), quenching constant (KSV), binding constant (KA), and the number of binding sites (n) of Hyp to HAS.
T (K)
Glucose concentration mM
Stern-Volmer equation
Double logarithm regression equation
K
q ×1012 L·mol−1 s−1
K
SV ×104 L·mol−1
R
a
SD
b
K
A ×105 L·mol−1
n
R
a
SD
b
298
0
17.3
17.3
0.992
0.0159
2.47
1.09
0.996
0.0108
310
0
9.75
9.75
0.993
0.0097
42.7
1.28
0.995
0.0123
3
16.2
1.19
0.997
0.0098
6
11.1
1.17
0.984
0.0130
9
3.23
1.10
0.992
0.0103
aThe correlation coefficient.
bThe standard deviation.Figure 3
Stern-Volmer plots of Hyp-HSA at different temperatures,C(Hyp) = 1, 2, 3, 4, 5, 6, 7 × 10−6 M.In general, for static quenching, the value ofKq is above 2.0×1010 L mol−1 s−1 [11]. Kq initiated by Hyp (17.3×1012 L·mol−1 s−1 (298 K) or 9.75 × 1012 (310 K)) is much greater than 2.0×1010 L·mol−1 s−1, which means that the quenching process is static.


## 3.2. Binding Ratio of HSA (HSAbr)

By the linear regression analysis between[HSA]free×107 (without Hyp) and F′ (Figure 4), (10) was obtained
(10)y=62.129x+7.6217(R2=0.9972),
where R is the correlation coefficient.Figure 4
Linear regression analysis between[HSA]free and F′ (F′ the Synchronous fluorescence intensity when Δλ=60 nm), C(HSA) = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 × 10−7 M.WhenΔλ=60nm, the synchronous fluorescence intensities after Hyp added were brought into (7). [HSA]free=0.96×10-6 M was got when CHSA=CHyp=1×10-6 M; thus, HSAbr=4% from (2).HSAbr was 7.9% when CHSA:CHyp=1:2.


## 3.3. Effect of Hyp on UV Absorption of HSA

The structural change and the complex formation between Hyp and HSA can also be explored by UV absorption measurement. For static quenching, due to forming nonfluorescent complexes with fluorophores in the ground state, the absorption spectra of HSA with quencher are different from those without quencher, while for dynamic quenching affecting the excitation state of fluorophores does not change the absorption spectra of HSA [15, 23–25, 28].The absorption spectra of HSA in the absence and presence of Hyp were recorded by subtracting the corresponding spectrum of Hyp free form in the buffer from that of Hyp-HSA system in Figure5. HSA has two main peaks (210 and 278 nm) in its absorption spectrum, which shows that the addition of Hyp made the absorption peak of HSA very small decrease at 210 nm and no significant changes at 278 nm when HSAbr was 4% (Figure 5(a)) and significant drop at 210 nm and 278 nm when HSAbr was 7.9% (Figure 5(b)). The absorption spectral changes of HSA in the presence of Hyp strongly suggest the complex formation between Hyp and HSA.The absorption spectra of HSA, Hyp, and Hyp-HSA system. a: [Hyp-HSA], b: [HSA], and c: [Hyp-HSA]—[Hyp], d: [Hyp].
(a)
C
(
HSA
)
=
C
(
Hyp
)
=
1.0
×
10
-
6 M
(b)
C
(
HSA
)
=
1.0
×
10
-
6 M; C(Hyp)=2.0×10-6 M


## 3.4. Binding Constant and the Number of Binding Sites

From Figure6, the double logarithm regression curves possessed good linearity and the slope significantly increased with temperature increasing.Figure 6
The double logarithm regression plots of Hyp-HSA at different temperatures,C(HSA)=1,2,3,4,5,6,7×10-6M.The values ofKA and n of Hyp binding to HSA increased with temperature increasing from 2.47×105 to 4.27×106 L·mol−1and from 1.09 to 1.28, respectively


## 3.5. Distance between HSA and Hyp

Figure7 shows the overlapping plot between absorption spectrum of Hyp and emission spectrum of HSA. The value of J is evaluated from (8), which was J=2.67×10-14 cm3·L·mol−1. Thus, we obtained that R0=3.00 nm from (7); E′=0.04 from (6).Figure 7
Overlap of fluorescence emission spectrum of HSA (curve 1) and UV absorption spectrum of Hyp (curve 2);C(Hyp)=C(HSA)=1×10-6 M.WhenCHSA=CHyp=1×10-6 mol L−1 and HSA binding ratio was calculated to be 4%, then, EC=0.93 was obtained from (5).The distance (r) was 1.95 nm from (4).


## 3.6. Binding Mode

The interaction forces between Hyp and HSA mainly involve hydrogen bonds, Van der Waals forces, electrostatic interactions and hydrophobic forces, and so forth [14, 15].From the experimental data shown in Table2, the thermodynamic parameters of Hyp binding to HSA were ΔH°>0 and ΔS°>0, which indicated that the predominant intermolecular forces to stabilize the complex of Hyp-HSA were hydrophobic forces. The negative ΔG° value was taken as evidence for the spontaneity of the binding of Hyp with HSA at standard conditions.Table 2
Thermodynamic parameters of the interaction between Hyp and HAS.
T/K
ΔH°/(kJ·mol−1)
ΔS°/(J·mol−1 K−1)
ΔG°/(kJ·mol−1)
298 K
36.70
226.37
-
30.76
304 K
36.70
227.39
-
29.75
310 K
36.70
227.61
-
29.28


## 3.7. Synchronous Fluorescence Spectra

The intrinsic fluorescence of HSA is manifested by emission of Trp and Tyr residues in molecule [19]. When Δλ=60nm, synchronous fluorescence spectra just showed the fluorescence of Trp residues, the maximum emission wavelength of which is related to its microenvironment [14, 15].The synchronous fluorescence spectra of HSA (Figure8) show that the fluorescence intensity of HSA at 285 nm decreased regularly and the maximum emission wavelength had weak bathochromic shift (from 285 to 287 nm) with a fixed amount of HSA and the increase of Hyp concentration, which represented that the conformation of HSA was somewhat changed leading to decreased polarity around the Trp residues.Figure 8
Synchronous fluorescence spectra of interaction between HSA and Hyp; from curve 1 to 8,C(HSA)=1×10-6 M, C(Hyp) = 0, 1, 2, 3, 4, 5, 6, and 7 × 10−6 M, respectively.


## 3.8. Influence of Glucose on Binding Constant, the Number of Binding Sites, and Conformation

In order to investigate the influence of glucose on the interaction of Hyp with HSA, the binding constants and the number of binding sites in presence and absence of different concentrations of glucose (3, 6, or 9 mM) at 310 K were compared. Glucose decreased binding constant and the number of binding sites of Hyp with HSA at 310 K (KA from 4.27 × 106 to 1.62 × 106, 1.11 × 106 and 3.23 × 105 L·mol−1; n from 1.28 to 1.19, 1.17, and 1.10, resp.) (Table 1). The double logarithm curves possessed good linearity and the slope significantly decreased with the increase of sugar level (Figure 9).Figure 9
The double logarithm regression plots of Hyp-HSA at different concentrations of glucose.The three-dimensional fluorescence spectra of Hyp-HSA in the absence and presence of glucose were shown in Figure10, respectively.Three-dimensional fluorescence spectra of HSA (a), Hyp-HSA without glucose (b), Hyp-HSA with 3 mM glucose (c), Hyp-HSA with 6 mM glucose (d), and Hyp-HSA with 9 mM glucose (e) at 310 K;C(HSA)=1×10-6 M, C(Hyp)=7×10-6 M.
(a)
(b)
(c)
(d)
(e)The excitation wavelength, emission wavelength and fluorescence intensity as axes in the fluorescence emission spectra, three-dimensional fluorescence technique is commonly used for the investigation of conformational changes of proteins interacting with drugs [29, 30]. It was reported that the three-dimensional fluorescence spectrum is an effective means to investigate the characteristic conformational change of protein [31–33].Two fluorescence peaks 1 and 2 were easily observed in three-dimensional fluorescence spectra. Peak 1 is the Rayleigh scattering peak(λex=λem). Peak 2 (λex<λem,Δλ>0) which reveals the spectral behavior of Trp and Tyr residues, of which the maximum emission wavelength and fluorescence intensity show a close relation to the polarity of their microenvironment, was the primary fluorescence peak we studied [34, 35].The fluorescence intensity of Hyp-HSA in the presence of glucose (at 433, 426, and 415, resp.) was weaker than that (446 nm) without glucose.By comparison, it could be seen that all the maximum excitation wavelengths were at 280 nm and the maximum excitation wavelengths were from 347 to 342 nm in the three-dimensional fluorescence spectra (Figures10(a), 10(b), 10(c), 10(d), and 10(e)) and the Stokes shift from 67 to 62 nm (Δλ between excitation wavelength and emission wavelength) changed which demonstrated that glucose had an effect on the polarity and hydrophilicity around Trp microenvironment [33].By decreasing the binding constant and the number of binding sites of Hyp with HSA, glucose could improve the plasma concentration of free Hyp, which may be help for the inhibitory action on eye aldose reductase and diabetic cataract.


## 4. Conclusions

This paper presents the interaction of Hyp with HSA and the effect of glucose on the binding by equilibrium UV-vis absorption, fluorescence emission, synchronous fluorescence, and three-dimensional fluorescence spectra.It was shown that Hyp quenched the endogenous fluorescence of HSA via a static quenching procedure. The distance between Hyp and HAS was 1.95 nm. Hydrophobic forces played a major role in stabilizing the complex of Hyp-HSA. The conformation and microenvironment of HSA were changed after Hyp binding to HSA. By decreasingKA and n of Hyp to HSA, glucose can elevate blood drug level of Hyp, from which we can deduce that Hyp may have stronger biological activity in diabetics than that in normal people. The binding constants decreased with sugar concentration increasing, which indicate concentration of free Hyp rising that is advantageous to prevent of diabetic cataract.



---

*Source: 386586-2014-07-23.xml*