3.2. Conformational changes of BSA

3.2. Conformational changes of BSA investigated by UV–vis
UV–vis absorption spectroscopy is a simple but effective method,
which is often applied to explore the structural changes of protein and ligand–protein complex formation. BSA has two main
absorption bands (Fig. 3). The strong absorption band at around
220 nm is mainly due to the peptide bond p ? p⁄ electronic
transitions of the peptide backbone (Scopes, 1974). The weak
absorption band around 280 nm is mainly attributed to the absorption
of tryptophan and tyrosine (Pace et al., 1995). The absorption
spectrum of BSA shows a hypochromic effect around 220 nm with
concomitant bathochromic shift as the concentration of chrysoidine
increased. The reason for this phenomenon is that the energy
required for p ? p⁄ transition decreased when the polar of the BSA
solution increased with increasing added chrysoidine concentration.
No detectable change was observed in BSA absorption spectra
at around 280 nm in the presence of chrysoidine. The results indicate
that the binding of chrysoidine to BSA does not drastically
change the conformation of BSA.
3.3. Fluorescence quenching of BSA by chrysoidine
Three aromatic amino acid residues (tryptophan, tyrosine, and
phenylalanine) contribute to the intrinsic fluorescence of BSA.
When the excitation wavelength is set to 280 nm, the emission
fluorescence from BSA is dominated by tryptophan and tyrosine,
while when excitation is selected at 295 nm, it minimizes the
emission from tyrosine residues and is known to selectively excite
tryptophan residues (Prasad et al., 1986). As shown in
Fig. 4a, when the BSA was excited at 280 nm, the chrysoidine
quenched the fluorescence emission spectrum of BSA. BSA contains
2 tryptophan residues: Trp-134 in domain I and Trp-213
in domain II. Trp-213 is located in a hydrophobic binding pocket,
whereas Trp-134 is more exposed to solvent and it is located on
the surface of molecule (Mallick et al., 2005). The fluorescence
properties of tryptophan residues are exceptionally sensitive to
the local environment, making it an ideal choice for the detection
of conformational alterations in proteins (Lynch and
Dawson, 2008). Fig. 4b shows the fluorescence quenching of
BSA by chrysoidine when excitation was set to 295 nm. The
maximum emission wavelength of BSA had no significant shift
with the addition of chrysoidine, which suggested that chrysoidine
had a slight impact on the conformation of the protein
(Berger et al., 1998).
Synchronous fluorescence spectroscopy is a useful method for
the evaluation of protein conformational changes, which gives
the information about the microenvironments around the fluorophore
functional groups by simultaneous scanning of the excitation
and emission monochromators with a constant wavelength
interval. When the wavelength interval (Dk) is set to 60 nm or
15 nm, synchronous fluorescence spectra give the characteristic
information of tryptophan residues or tyrosine residues, respectively
(Liu and Liu, 2012). As seen in Fig. 4c and d, the intensity
of the synchronous fluorescence of BSA at Dk = 60 nm and
Dk = 15 nm progressively reduced with gradual addition of chrysoidine.
No obvious shift in the emission maximum upon quenching
was observed, indicating that the interaction of chrysoidine
with BSA does not perturb the polarity around these residues
significantly.
3.4. Fluorescence quenching mechanism
The mechanisms of fluorescence quenching are usually classi-
fied into dynamic quenching and static quenching (Xie et al.,
2010). During dynamic quenching, the quencher interacts with
the excited state of the fluorophore leading to a change of the fluorescence
lifetime. By contrast, during the static quenching, the formation
of ground-state complex between the fluorophores and the
quencher inhibits the excited state formation of the fluorophore,
without affecting the fluorescence lifetime (van de Weert and
Stella, 2011). Consequently, the most definitive method to distinguish
static and dynamic quenching is to measure the fluorescence
lifetimes in the presence and absence of quencher (Gauthier et al.,
1986). The data obtained from fluorescence lifetime experiments
were found to fit well to a single-exponential decay with a v2
values approached 1.00. The lifetime (s) of BSA was decreased from
6.20 to 5.08 ns with the increasing concentration of chrysoidine
(Table 2), indicating that the quenching mechanism was a dynamic
quenching. For the dynamic quenching, it often involves collisional
quenching, photoinduced electron transfer (PET), radiative energy
transfer and Förster resonance energy transfer (FRET), which do
not require a direct contact of the quencher with fluorophore. In
collisional quenching, higher temperatures result in faster diffusion,
and hence the quenching constants will increase with the
temperature. The fluorescence quenching data of chrysoidine-BSA
binding at different temperatures are analyzed by the Stern–Volmer
equation:
F0=F ¼ 1 þ KSV½Q ð2Þ
where F0 and F are the steady-state fluorescence intensities in
the absence and presence of quencher, respectively; Ksv is the
Stern–Volmer quenching constant; and [Q] is the concentration
of the quencher (chrysoidine). The Stern–Volmer plots are shown
in Fig. 5 and the Ksv values calculated from the Stern–Volmer equation
are presented in Table 3. The results show the Ksv values decrease
with increasing temperature, indicating that the
quenching is not initiated from collisions. Since the interactions
of chrysoidine with BSA induce no discernible change in the protein
(results obtained from UV–vis spectra experiments), and the
fluorescence emission spectrum of BSA overlaps with the UV
absorption spectrum of chrysoidine (see Fig. 6), we inferred that
the quenching of the BSA fluorescence caused by chrysoidine was
attributable to FRET. The reason why the fluorescence of BSA is
quenched to a greater extent at higher temperatures is due to
the chrysoidine-BSA diffusion during the excited-state lifetime.
Lakowicz also observed this phenomenon in the study of FRET
between TMA and TU2D (Lakowicz et al., 1994).
According to the Förster’s non-radiative energy transfer theory,
the parameters related to energy transfer can be calculated based
on the equation as follows:
where E is the efficiency of energy transfer; s0 and s are the relative
lifetimes of the donor, in the absence (s0) and presence (s) of
acceptor; r is the distance between the acceptor and the donor, R0
is the distance at which transfer efficiency equals 50%, which can
be calculated using the following equation:
R6
0 ¼ 8:79  1025K2
n4/J ð4Þ
where K2 is the spatial orientation factor of the dipole, n is the
refractive index of the medium, U is the fluorescence quantum yield
of the donor, and J is the overlap integral of the fluorescence emission
spectrum of the donor and the absorption spectrum of the
acceptor:
J ¼
R 1
0 FðkÞeðkÞk4
dk
R 1
0 FðkÞdk ð5Þ
where F(k) is the fluorescence intensity of the fluorescence donor at
wavelength k, and e(k) is the molar absorption coefficient of the
acceptor at wavelength k. It has been reported for BSA, that
K2 = 2/3, U = 0.15 and n = 1.36 (Deepa and Mishra, 2005; Kandagal
et al., 2006). Thus according to the above equations it is calculated
that J = 3.78  1014 cm3 L mol1
, R0 = 3.21 nm. Values of the r and
E corresponding to the different concentration of chrysoidine are
summarized in Table 4. With the concentration of chrysoidine
increasing, the Förster distance (r) reduces from 5.76 to 4.05, and
accompanied with the increase of transfer efficiency.
According to Förster’s theory, the distance between the acceptor
and donor should be smaller than 10 nm. r, the distance between
chrysoidine and Trp residues of BSA, is in the range of 4–6 nm
and 0.5R0 < r < 2R0, indicating the results are in accordance with
the conditions of FRET and the energy transfer from BSA to chrysoidine
occurs with high probability.
3.5. Conformational changes of BSA monitored by circular dichroism
spectroscopy
To gain a better understanding of the effects of chrysoidine on
the conformational change of BSA, CD spectroscopy were employed
in the present study. Far UV-CD studies revealed that there
were no significant changes in the ellipticity values at 208 or
222 nm, which is characteristic of an a-helical structure of the protein
(Xu et al., 2012; Zhao et al., 2009) (Fig. S1, Supplementary
material). Similarly, the content of secondary structural elements
in BSA was nearly unchanged with increasing the concentration
of chrysoidine (Table 5) indicated that the binding of chrysoidine
to BSA does not lead to an apparent alteration of the secondary
structure of BSA, which is consistent with the results from
UV–vis spectroscopy studies.
3.6. Molecular docking
To predict the binding sites of chrysoidine on BSA, molecular
docking simulations were run with Autodock program (version
4.2), which is available free of charge from The Scripps Research
Institute (http://autodock.scripps.edu/) (Morris et al., 1998). The
structure of chrysoidine was optimization using Gaussian 03 package.
During the docking simulation, 10 conformations were obtained
(Table S1, Supplementary material), and the lowest energy
solution structure of chrysoidine–BSA are shown in Fig. 7. Chrysoidine
binds to BSA at a cavity close to Sudlow site I in domain IIA,
where small organic compounds, including many drugs, were
found to bind to serum albumin (Perry et al., 2006; Saquib et al.,
2011). The distance between tryptophan (the donor) and chrysoidine
(the acceptor) obtained from the docking simulation is less
than 10 nm, which satisfied the condition of FRET (Gadella, 2009;Lakowicz, 2006) and verified the conclusion obtained from fluorescence
quenching analysis. Calculation of the binding energy from
docking simulations revealed that van der Waals and hydrogen
bonding interactions (chrysoidine with Arg 256) play a major role
in the binding of chrysoidine to BSA, in agreement wi

ผลลัพธ์ (ไทย) 1:

3.2. การเปลี่ยนแปลงของบีเอสเอในการสอบสวน โดย UV – vis conformationalกดูดซึม UV – vis เป็นวิธีง่าย ๆ แต่มีประสิทธิภาพซึ่งมักจะใช้การเปลี่ยนแปลงโครงสร้างของโปรตีนและลิแกนด์ – โปรตีนก่อซับซ้อน บีเอสเอมีสองหลักวงดูดซึม (Fig. 3) แถบดูดซับแรงที่รอบ220 nm เป็นส่วนใหญ่เนื่องจากเพปไทด์ p p⁄ อิเล็กทรอนิกส์ช่วงการเปลี่ยนภาพของแกนหลักของเพปไทด์ (ขอบเขต 1974) อ่อนแอดูดซึมวงประมาณ 280 nm เป็นส่วนใหญ่เกิดจากการดูดซึมทริปโตเฟนและ tyrosine (ก้าวและ al., 1995) การดูดซึมสเปกตรัมของบีเอสเอแสดงผล hypochromic ประมาณ 220 nm มีbathochromic มั่นใจกะเป็นความเข้มข้นของ chrysoidineเพิ่มขึ้น สาเหตุของปรากฏการณ์นี้คือพลังงานต้องการ p เปลี่ยน p⁄ ลดลงเมื่อขั้วโลกบีเอสเอโซลูชันเพิ่มขึ้นกับความเข้มข้น chrysoidine เพิ่มมากขึ้นไม่เปลี่ยนแปลงสามารถถูกตรวจสอบในแรมสเป็คตราการดูดซึมของบีเอสเอที่ประมาณ 280 nm ในต่อหน้าของ chrysoidine ระบุผลลัพธ์ที่รวมของบีเอสเอ chrysoidine ไม่ไม่อย่างรวดเร็วเปลี่ยน conformation ของบีเอสเอ3.3. fluorescence ชุบของบีเอสเอ โดย chrysoidineตกค้างสามหอมกรดอะมิโน (tyrosine ทริปโตเฟน และphenylalanine) ช่วยการ fluorescence intrinsic ของบีเอสเอเมื่อการตั้งค่าความยาวคลื่นในการกระตุ้นเป็น 280 nm การปล่อยก๊าซfluorescence จากบีเอสเอที่ถูกครอบงำ ด้วยทริปโตเฟนและ tyrosineในขณะที่เมื่อเลือกในการกระตุ้นที่ 295 nm ลดการemission from tyrosine residues and is known to selectively excitetryptophan residues (Prasad et al., 1986). As shown inFig. 4a, when the BSA was excited at 280 nm, the chrysoidinequenched the fluorescence emission spectrum of BSA. BSA contains2 tryptophan residues: Trp-134 in domain I and Trp-213in domain II. Trp-213 is located in a hydrophobic binding pocket,whereas Trp-134 is more exposed to solvent and it is located onthe surface of molecule (Mallick et al., 2005). The fluorescenceproperties of tryptophan residues are exceptionally sensitive tothe local environment, making it an ideal choice for the detectionof conformational alterations in proteins (Lynch andDawson, 2008). Fig. 4b shows the fluorescence quenching ofBSA by chrysoidine when excitation was set to 295 nm. Themaximum emission wavelength of BSA had no significant shiftwith the addition of chrysoidine, which suggested that chrysoidinehad a slight impact on the conformation of the protein(Berger et al., 1998).Synchronous fluorescence spectroscopy is a useful method forthe evaluation of protein conformational changes, which givesthe information about the microenvironments around the fluorophorefunctional groups by simultaneous scanning of the excitationand emission monochromators with a constant wavelengthinterval. When the wavelength interval (Dk) is set to 60 nm or15 nm, synchronous fluorescence spectra give the characteristicinformation of tryptophan residues or tyrosine residues, respectively(Liu and Liu, 2012). As seen in Fig. 4c and d, the intensityof the synchronous fluorescence of BSA at Dk = 60 nm andDk = 15 nm progressively reduced with gradual addition of chrysoidine.No obvious shift in the emission maximum upon quenchingwas observed, indicating that the interaction of chrysoidinewith BSA does not perturb the polarity around these residuessignificantly.3.4. Fluorescence quenching mechanismThe mechanisms of fluorescence quenching are usually classi-fied into dynamic quenching and static quenching (Xie et al.,2010). During dynamic quenching, the quencher interacts withthe excited state of the fluorophore leading to a change of the fluorescencelifetime. By contrast, during the static quenching, the formationof ground-state complex between the fluorophores and thequencher inhibits the excited state formation of the fluorophore,without affecting the fluorescence lifetime (van de Weert andStella, 2011). Consequently, the most definitive method to distinguishstatic and dynamic quenching is to measure the fluorescencelifetimes in the presence and absence of quencher (Gauthier et al.,1986). The data obtained from fluorescence lifetime experimentswere found to fit well to a single-exponential decay with a v2values approached 1.00. The lifetime (s) of BSA was decreased from6.20 to 5.08 ns with the increasing concentration of chrysoidine(Table 2), indicating that the quenching mechanism was a dynamic
quenching. For the dynamic quenching, it often involves collisional
quenching, photoinduced electron transfer (PET), radiative energy
transfer and Förster resonance energy transfer (FRET), which do
not require a direct contact of the quencher with fluorophore. In
collisional quenching, higher temperatures result in faster diffusion,
and hence the quenching constants will increase with the
temperature. The fluorescence quenching data of chrysoidine-BSA
binding at different temperatures are analyzed by the Stern–Volmer
equation:
F0=F ¼ 1 þ KSV½Q ð2Þ
where F0 and F are the steady-state fluorescence intensities in
the absence and presence of quencher, respectively; Ksv is the
Stern–Volmer quenching constant; and [Q] is the concentration
of the quencher (chrysoidine). The Stern–Volmer plots are shown
in Fig. 5 and the Ksv values calculated from the Stern–Volmer equation
are presented in Table 3. The results show the Ksv values decrease
with increasing temperature, indicating that the
quenching is not initiated from collisions. Since the interactions
of chrysoidine with BSA induce no discernible change in the protein
(results obtained from UV–vis spectra experiments), and the
fluorescence emission spectrum of BSA overlaps with the UV
absorption spectrum of chrysoidine (see Fig. 6), we inferred that
the quenching of the BSA fluorescence caused by chrysoidine was
attributable to FRET. The reason why the fluorescence of BSA is
quenched to a greater extent at higher temperatures is due to
the chrysoidine-BSA diffusion during the excited-state lifetime.
Lakowicz also observed this phenomenon in the study of FRET
between TMA and TU2D (Lakowicz et al., 1994).
According to the Förster’s non-radiative energy transfer theory,
the parameters related to energy transfer can be calculated based
on the equation as follows:
where E is the efficiency of energy transfer; s0 and s are the relative
lifetimes of the donor, in the absence (s0) and presence (s) of
acceptor; r is the distance between the acceptor and the donor, R0
is the distance at which transfer efficiency equals 50%, which can
be calculated using the following equation:
R6
0 ¼ 8:79  1025K2
n4/J ð4Þ
where K2 is the spatial orientation factor of the dipole, n is the
refractive index of the medium, U is the fluorescence quantum yield
of the donor, and J is the overlap integral of the fluorescence emission
spectrum of the donor and the absorption spectrum of the
acceptor:
J ¼
R 1
0 FðkÞeðkÞk4
dk
R 1
0 FðkÞdk ð5Þ
where F(k) is the fluorescence intensity of the fluorescence donor at
wavelength k, and e(k) is the molar absorption coefficient of the
acceptor at wavelength k. It has been reported for BSA, that
K2 = 2/3, U = 0.15 and n = 1.36 (Deepa and Mishra, 2005; Kandagal
et al., 2006). Thus according to the above equations it is calculated
that J = 3.78  1014 cm3 L mol1
, R0 = 3.21 nm. Values of the r and
E corresponding to the different concentration of chrysoidine are
summarized in Table 4. With the concentration of chrysoidine
increasing, the Förster distance (r) reduces from 5.76 to 4.05, and
accompanied with the increase of transfer efficiency.
According to Förster’s theory, the distance between the acceptor
and donor should be smaller than 10 nm. r, the distance between
chrysoidine and Trp residues of BSA, is in the range of 4–6 nm
and 0.5R0 < r < 2R0, indicating the results are in accordance with
the conditions of FRET and the energy transfer from BSA to chrysoidine
occurs with high probability.
3.5. Conformational changes of BSA monitored by circular dichroism
spectroscopy
To gain a better understanding of the effects of chrysoidine on
the conformational change of BSA, CD spectroscopy were employed
in the present study. Far UV-CD studies revealed that there
were no significant changes in the ellipticity values at 208 or
222 nm, which is characteristic of an a-helical structure of the protein
(Xu et al., 2012; Zhao et al., 2009) (Fig. S1, Supplementary
material). Similarly, the content of secondary structural elements
in BSA was nearly unchanged with increasing the concentration
of chrysoidine (Table 5) indicated that the binding of chrysoidine
to BSA does not lead to an apparent alteration of the secondary
structure of BSA, which is consistent with the results from
UV–vis spectroscopy studies.
3.6. Molecular docking
To predict the binding sites of chrysoidine on BSA, molecular
docking simulations were run with Autodock program (version
4.2), which is available free of charge from The Scripps Research
Institute (http://autodock.scripps.edu/) (Morris et al., 1998). The
structure of chrysoidine was optimization using Gaussian 03 package.
During the docking simulation, 10 conformations were obtained
(Table S1, Supplementary material), and the lowest energy
solution structure of chrysoidine–BSA are shown in Fig. 7. Chrysoidine
binds to BSA at a cavity close to Sudlow site I in domain IIA,
where small organic compounds, including many drugs, were
found to bind to serum albumin (Perry et al., 2006; Saquib et al.,
2011). The distance between tryptophan (the donor) and chrysoidine
(the acceptor) obtained from the docking simulation is less
than 10 nm, which satisfied the condition of FRET (Gadella, 2009;Lakowicz, 2006) and verified the conclusion obtained from fluorescence
quenching analysis. Calculation of the binding energy from
docking simulations revealed that van der Waals and hydrogen
bonding interactions (chrysoidine with Arg 256) play a major role
in the binding of chrysoidine to BSA, in agreement wi

ผลลัพธ์ (ไทย) 3:

3.2 . การเปลี่ยนแปลงโครงสร้างของประเทศ โดย UV UV Vis Vis
––การดูดซึมสเปกโทรสโกปีเป็นง่ายๆ แต่มีประสิทธิภาพซึ่งมักใช้วิธี
เพื่อศึกษาการเปลี่ยนแปลงโครงสร้างของโปรตีน และโปรตีนที่ซับซ้อน ) และการสร้าง บีเอสเอได้สองวงการดูดซึมหลัก
( รูปที่ 3 ) แรงดูดซึมวงรอบ
220 nm เป็นส่วนใหญ่เนื่องจากการพันธะเพปไทด์ P ? ⁄อิเล็กทรอนิกส์
pการเปลี่ยนของเปปไทด์กระดูกสันหลัง ( ขอบเขต , 1974 ) แถบการดูดกลืนอ่อนแอ
ประมาณ 280 nm เป็นส่วนใหญ่เกิดจากการดูดซึม
ของทริปโตเฟนและไทโรซีน ( จังหวะ et al . , 1995 ) การดูดกลืนสเปกตรัมของการแสดงผล (
hypochromic ประมาณ 220 นาโนเมตรกับ
กะ bathochromic เกิดเป็นสมาธิของ chrysoidine
เพิ่มขึ้น เหตุผลสำหรับปรากฏการณ์นี้คือการที่พลังงาน
ที่จําเป็นสําหรับต่อP ⁄เปลี่ยนแปลงลดลงเมื่อขั้วของ BSA
โซลูชั่นเพิ่มขึ้น เพิ่มความเข้มข้น chrysoidine .
ไม่ได้เปลี่ยนแปลงพบว่าสเปกตรัมการดูดกลืน
( ประมาณ 280 nm ในการแสดงตนของ chrysoidine . ผลการศึกษาพบว่า การจับ chrysoidine
เพื่อ BSA ไม่ได้อย่างมาก
เปลี่ยนโครงสร้างของ BSA .
3 . บีเอสเอ โดย chrysoidine
ดับฟลูออเรสเซนซ์สามกลิ่นตกค้าง ( กรดอะมิโนทริปโตเฟนและไทโรซีน , ฟีนิลอะลานีน
) นำไปสู่การแท้จริงของ BSA .
เมื่อพบว่าความยาวคลื่นตั้ง 280 nm , สารมลพิษ
เรืองแสงจาก BSA เป็น dominated โดยทริปโตเฟนและไทโรซีน
ตอนที่ถูกเลือก , ความตื่นเต้นที่ 295 นาโนเมตร จะช่วยลดการปล่อยสารพิษตกค้าง
แต่อย่างใด และเป็นที่รู้จักที่จะเลือกที่ตื่นเต้น
ทริปตกค้าง ( Prasad et al . , 1986 ) ดังแสดงในรูปที่ 4
เมื่อไวโอ้ตื่นเต้นที่ 280 nm , chrysoidine
ดับเรืองแสงด้วยการปล่อยสเปกตรัมของ BSA . บีเอสเอประกอบด้วย
2 ทริปตกค้าง : trp-134 โดเมนและโดเมน trp-213
2 trp-213 ตั้งอยู่ใน ) ผูกกระเป๋า
ส่วน trp-134 มากสัมผัสกับตัวทำละลายและตั้งอยู่บน
ผิวของโมเลกุล ( มัลลิก et al . , 2005 ) เรืองแสง
คุณสมบัติของทริปตกค้างเป็นโคตรไว
สภาพแวดล้อมในท้องถิ่น ทำให้มันเป็นตัวเลือกที่เหมาะสำหรับการตรวจสอบการเปลี่ยนแปลงในโครงสร้างของโปรตีน (

ช์และดอว์สัน , 2008 ) รูปแสดงการเรืองแสง 4B ดับของประเทศ โดย chrysoidine
เมื่อกระตุ้นถูกตั้ง 295 nm .
ความยาวคลื่นสูงสุดของการปล่อยกลุ่มไม่มีความแตกต่างกะ
ด้วยนอกเหนือจาก chrysoidine ซึ่งชี้ให้เห็นว่า chrysoidine
มีผลกระทบเล็กน้อยในโครงสร้างของโปรตีน
( Berger et al . , 1998 ) .
synchronous fluorescence spectroscopy เป็นวิธีที่มีประโยชน์สำหรับการประเมินผลของการเปลี่ยนแปลงโครงสร้างโปรตีน

ซึ่งให้ข้อมูลเกี่ยวกับ การ microenvironments รอบ fluorophore
หมู่ฟังก์ชันโดยพร้อมกันสแกนของความตื่นเต้นและการปล่อย monochromators ด้วย

ช่วงความยาวคลื่นคงที่ เมื่อความยาวคลื่นช่วง ( DK ) ตั้งเป็น 60 nm หรือ
15 nm synchronous เรืองนี้ให้ข้อมูลลักษณะ
ตกค้างทริปตกค้างหรือไทโรซีน )
( หลิวและหลิว , 2012 ) ตามที่เห็นในรูปที่ 4C และ D , ความเข้ม