全 文 ：第38卷第3期
2015年9月
南京师大学报（自然科学版）
JOURNAL OF NANJING NORMAL UNIVERSITY（Natural Science Edition）
Vol. 38 No. 3
Sept，2015
Comparing the Interaction of Platinum-Hypocrellin A
Complex and Hypocrellin A with
Bovine Hemoglobin
Yuan Xiuxue，Chen Enyi，Xiao Mengsi，Zhao Chuanfeng，Zhou Jiahong
（School of Chemistry and Materials Science，Analysis and Testing Centre，Jiangsu Key Laboratory of Biofunctional Materials，
Jiangsu Collaborative Innovation Centre of Biomedical Functional Materials，Key Laboratory of Applied
Photochemistry，Nanjing Normal University，Nanjing 210023，China）
Abstract：The interaction mechanisms of Hypocrellin A（HA）and its platinum complex（Pt-HA）with bovine hemoglobin
（BHb）were compared by ultraviolet-visible（UV-Vis）absorption，time-resolved fluorescence spectra，circular dichroism
（CD）and induced circular dichroism（ICD）spectra，respectively. The UV-Vis results revealed that both HA and Pt-HA
could interact with BHb and the latter interaction was stronger than HA. In addition，quenching mechanism of HA and
Pt-HA were both static process，which were ascribed to the electron transfer. Moreover，CD and ICD spectrum revealed
that the microenvironment and conformation of BHb were changed after conjugation with HA and Pt-HA，as the latter al⁃
tered more acute. Finally，the comparison results demonstrated that the interaction of Pt-HA and BHb was stronger and
more stable than HA.
Key words：hypocrellin A，platinum-hypocrellin A，bovine hemoglobin，interaction
CLC number：O657.3 Document code：A Article ID：1001-4616（2015）03-0014-11
光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素
复合物与牛血红蛋白的相互作用
袁秀雪，陈恩懿，肖梦思，赵传峰，周家宏
（南京师范大学化学与材料科学学院，分析测试中心，江苏省生物功能材料重点实验室，江苏省生物功能材料协同创新中心，
应用光化学重点实验室，江苏南京 210023）
［摘要］ 通过紫外-可见吸收光谱、瞬态荧光光谱以及圆二色谱来比较光疗药物竹红菌甲素及铂竹红菌甲素复
合物与牛血红蛋白的相互作用 .结果表明，虽然竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的反应机理都
是基于电子转移的静态猝灭，但是铂竹红菌甲素复合物与牛血红蛋白的反应不仅要比竹红菌甲素强，而且反应
后牛血红蛋白的微环境和构像的改变也要大于竹红菌甲素 .
［关键词］ 竹红菌甲素，铂竹红菌甲素复合物，牛血红蛋白，相互作用
Photodynamic therapy（PDT）was a noninvasive technique for the treatment of various cancer tumors and
non-cancer diseases. It was a promising modality for the management of various tumors and nonmalignant diseas⁃
es，based on the application of minimal dark toxicity photosensitizer（PS）that was selectively localized in the tar⁃
get tissue，activated by a specific wavelength of light and resulted in photo damage and subsequent cell death via
apoptosis and/or necrosis［1，2］.
As a natural PS，Hypocrellin A（HA），extracted from Hypocrella bambuase，has been receiving intensive in⁃
terest according to its easy preparation and purification，high photo-toxicity but low dark-toxicity，rapid clearance
Received data:2015-03-06.
Foundation item：Project of Department of Science and Technology of Jiangsu Province（BK20131394、BZ201210）.
Corresponding author：Zhao Chuanfeng，senior engineer，majored in applied chemistry. E-mai：zhaochuanfeng@njnu.edu.cn
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袁秀雪，等：光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的相互作用
from normal tissues［3］and the high efficiency in generating reactive oxygen species（ROSs）［4］. Nevertheless，the
poor water solubility has greatly limited its clinic application［5］. In our previous work，we have found that platinum
（VI）and HA can form a complex（Pt-HA）and the complex significantly enhanced the water solubility，light sta⁃
bility and efficiency of 1O2 generation［4］，thereby exhibiting better anti- tumor activity. As a phototherapy medi⁃
cine，Pt-HA was delivered into the body by intravenous injection，which would interact with various proteins and
created effects on the transmission process in the blood. Therefore，it was very important to study the interaction
mechanisms between the Pt-HA and the protein in the blood.
In this study，Bovine hemoglobin（BHb）was selected as a model to study the interaction mechanisms of Pt-
HA under physiological conditions，due to its function of a carrier of oxygen，transporter of carbon dioxide and
regulator of the pH of blood directly or indirectly［6］，taking HA as a control. In addition，it also played an impor⁃
tant role in many biologically relevant processes in life science，clinical medicine and environment［7］. The results
defined the delivery mechanism of the Pt-HA in the blood and have an important practical significance on the
development of the application of the PDT.
1 Materials and Methods
1.1 Materials
The stock solution of HA（19.35 mM）was obtained by adding an appropriate amount of solid HA into
DMSO. Bovine hemoglobin（BHb）was purchased from Sigma，and prepared in phosphate buffers（0.1 M，pH 7.0）
before each experiment without further purification. Moreover，the concentration of BHb was determined by the
absorption at 404 nm（ε=41 000 L·mol-1·cm-1）. The H2PtCl6 was produced by Sigma，and the mother solution
was 19.30 mM after dissolving by methanol. The complex of Pt-HA was prepared as our previous work by mixing
55 μL HA and 110 μL H2PtCl6 in double distilled water for several minutes at the room temperature with minor
stirring in the dark place. Other chemicals were all analytical grade and double distilled water was used through⁃
out. The solution was stored in cool and dark place.
1.2 Spectroscopic measurements
1.2.1 UV-Vis spectroscopy
The UV-Vis absorption data was collected from 200 nm to 600 nm on a Varian Cary 5 000 spectrophotome⁃
ter in a 1.0 cm quartz cuvette. UV-Vis titrations were performed by successive addition of HA and Pt-HA com⁃
plex to 3 mL BHb solution，respectively. When experiments were carried out at different temperatures（298 K，
310 K and 315 K），the solution was equilibrated in a thermostat bath.
1.2.2 Time-resolved fluorescence spectra
The fluorescence lifetime was performed on a Horiba Jobin Yvon Fluoro Max-4 time correlation single pho⁃
ton counting（TCSPC）system，using a 265 nm diode laser excitation source（IBH，Nano LED，pulse Fwhm ~ 3
μs）. The time resolution was estimated at 1.5 ns and repetition rate up to 1 MHz. The time ranges were 0.219 ns /
channel，in 4 096 effective channels. Data were globally fitted to the appropriate exponential model after deconvo⁃
lution of the instrument response function by an iterative deconvolution technique，using the IBH DAS6 fluores⁃
cence decay analysis software.
1.2.3 Steady-state fluorescence spectra
All the Steady-state fluorescence titrations were investigated on Perkin-Elmer LS-50B fluorescence spectro⁃
photometer equipped with a xenon lamp light source，and 1.0 cm quartz cells were used for these measurements
at the room temperature. Every titration was operated manually with gentle stirring.
1.2.4 Circular dichroism（CD）spectra
Circular Dichroism（CD）and Induced circular dichroism（ICD）spectrum were measured with an applied pho⁃
tophysics chriascan circular dichroism spectropolarimeter in 1.0 cm quartz cells at room temperature. Spectra
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南京师大学报（自然科学版） 第38卷第3期（2015年）
were scanned with 1 nm spectral bandwidth and 0.5 nm step resolution. Finally，the data were analyzed by CDNN
programme.
1.2.5 Theory and calculation
1.2.5.1 Binding parameters of BHb and drugs
The binding constant of the BHb and drugs could be calculated according to the following equation［8］：
A0 /A - A0 =(εBHb /εBHb -Drug - εDrug)(1 + 1/Kb[drug])， （1）
where A0 and A were the absorbance of BHb in the absence and presence of drug，respectively. Kb was the binding
constant，εBHb，εDrug，εBHb-Drug were the absorption coefficients of BHb，Drug and the BHb-Drug adduct，respectively.
1.2.5.2 Thermodynamic parameters of the binding forces
The thermodynamic parameters，enthalpy change（DH），entropy change（DS）and free energy change（DG）
could be calculated according to Eq.（2）and Eq.（3）［9，10］，as the reaction enthalpy change was regarded as a con⁃
stant even if the temperature changed little.
lnK1/K2 =(1/T1 - 1/T2)ΔH/R， （2）
ΔG =ΔH - TΔS = -RT lnKb， （3）
where K1 and K2 were the combination constant of composite compound of BHb and the drug at different tempera⁃
tures.
1.2.5.3 Average fluorescence lifetime
The average fluorescence lifetime could be calculated according to the following equations［11，12］：
fi =Biτi /∑
i = 1
3
Biτi， （4）
I(t) =∑
i = 1
3
fi exp(-t/τi)， （5）
τ =∑
i = 1
3
fiτi， （6）
where B was the relative contributions，τ was the lifetimes of the different components to the total decay，fi was
the pre-exponential factor and f1+f2+f3= 1.
2 Results and Discussion
2.1 The UV-Vis absorption analysis
Both UV-Vis and fluorescence spectra were the effective techniques to explore the drug effects on protein.
Moreover，the intrinsic fluorescence of BHb primarily originated from its amino acid residues［13］. However，the
UV-Vis spectroscopy of BHb can present both the amino acid residues，and the heme signal，simultaneously.
Thus，here，UV-Vis absorption measurement was selected to study the interaction of BHb and drugs［14，15］，espe⁃
cially to observe the effects of drugs on heme group of BHb，directly. Hence，absorption spectra of BHb in pres⁃
ence and absence of drugs were recorded as shown in Fig.1. From Fig.1，we could clearly observe that BHb has
three absorption bands，which were located at 274 nm（the phenyl group of tryptophan and tyrosine residues），
300 nm（ε band），and 404 nm（heme or Soret band）［6］in phosphate buffers solution. Upon gradually addition of
HA and Pt-HA into BHb，the absorbance intensity at 274 nm and 300 nm were both increased while the peak at
404 nm decreased in the UV-Vis spectrum，which might conclude that both HA and Pt-HA could interact with
tryptophan，tyrosine residues and heme group，which resulted in disturbing the chemical environment surround⁃
ing BHb. Moreover，the disappeared band of 274 nm of BHb in the presence of Pt-HA implied that Pt-HA inter⁃
acted stronger with the tryptophan and tyrosine residues of BHb and the peptide strands of BHb molecules ex⁃
tended more and the hydrophobicity was decreased［16］. In addition，the absorption maximum of heme group was
decreased significantly after Pt-HA treatment compared to HA，while the maximum absorption wavelengths re⁃
main unchanged. Finally，the effects of HA and Pt-HA on heme group of BHb were compared detail by calculat⁃
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袁秀雪，等：光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的相互作用
ing the absorbance intensity at 404 nm at different temperatures in Fig.2. The results suggested that the interac⁃
tion of BHb with Pt-HA was stronger than HA.
Fig.1 UV-Vis absorption spectra of pure BHb of HA（A-298 K；C-310 K；E-315 K）and UV-Vis absorption spectra of pure BHb of
Pt-HA（B-298 K；D-310 K；F-315 K）；［BHb］=19.9 μmol/L；［HA］=0，3.36，6.71，10.1，13.4，16.8 μmol/L；
［Pt-HA］=0，3.36，6.71，10.1，13.4，16.8 μmol/L；pH=7.0
Fig.2 Plots of I/I0 against HA and Pt-HA with different concentrations at 404 nm；［HA］=0，3.36，6.71，10.1，13.4，16.8 μmol/L；
［Pt-HA］=0，3.36，6.71，10.1，13.4，16.8 μmol/L；pH=7.0，T=298 K（A），T=310 K（B），T=315 K（C）.
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南京师大学报（自然科学版） 第38卷第3期（2015年）
2.2 Binding parameters of interaction
UV-Vis absorption spectroscopy was a simple but powerful way to exhibit the formation of complex when
drug molecules binding with protein［17，18］. The absorption spectrum of BHb upon continuous adding drugs at
298 K, 310 K and 315 K were exhibited in Fig.1. According to Eq.（1），the linear curve of A0/（A-A0）vs/［Drug］
were plotted as shown in Fig.3，while the Kb of HA and Pt-HA with BHb at 298 K，310 K and 315 K were present⁃
ed in Table 1，respectively. The results definitely indicated that the Kb of Pt-HA with BHb were bigger than HA
with BHb，which concluded that the interaction of Pt-HA and BHb was more stable than HA.
Fig.3 Plots of A0/A-A0 vs/［Drug］at different temperature 298 K（A），310 K（B）and 315 K（C）.
Table 1 Estimated values of binding constant of HA with BHb as well as Pt-HA with BHb at different temperature
Samples
HA
Pt-HA
Temperature (K)
298
310
315
298
310
315
Kb/(104 mol/L)-1 )
3.33
1.43
1.36
5.58
3.76
2.58
2.3 Thermodynamics parameters
Binding force of drug molecules to protein could be calculated by the obtained thermodynamic parameters
such as enthalpy change（DH），entropy change（DS）and free energy change（DG）of the reaction［19］. According to
the previous work［20，21］，there were several acting forces between small molecular and biomacromolecule，such as
hydrophobic force，hydrogen bond，van der Waals，electrostatic force and so on. When DH<0 or DH≈0，DS>0，the
mainly acting force was electrostatic force；when DH<0，DS<0，the mainly acting force was van der Waals or hy⁃
drogen bond and when DH>0，DS>0，the mainly force was hydrophobic［22，23］. Considering that DH did not vary sig⁃
nificantly over the working temperature range，the thermodynamic parameters could be calculated by Eq.（2）and
Eq.（3），and the values were listed in Table 2，respectively. On the one hand，the negative DG indicated that the
interaction of drugs and BHb were spontaneous. On the other hand，for HA，the negative of DH and positive of DS
presented that electrostatic and hydrophobic interaction played major role［24］while the negative of DH and DS indi⁃
cated that van der Waals and hydrogen bond were the mainly force for Pt-HA complex.
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袁秀雪，等：光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的相互作用
Table 2 Thermodynamic parameters of the binding reactions of the HA with BHb and Pt-HA with BHb
Drug
HA
Pt-HA
Temperature/K
298
310
315
298
310
315
DG/（KJ/mol）
-25.80
-24.66
-24.93
-27.08
-27.16
-26.60
DH/（KJ/mol）
-8.15
-61.15
DS/（J·mol-1·K-1）
53
-110
2.4 The interaction mechanism analysis between BHb and drugs
To determine the BHb fluorescence quenching mechanism between BHb and drugs，the nanosecond fluores⁃
cence lifetime of BHb was measured by the TCSPC technique，by using 265 nm diode laser excitation where only
tryptophan is excited［25，26］. The lifetimes of systems at 322 nm from BHb were collected and compared. The data
were fitted using a reconvolution method of the instrument response function producing Χ2（Chi. Sq.）fitting values
of 1~1.40. In addition，as shown in Fig.4 and Table 3，the fluorescence evolution of pure BHb can be nicely fitted
by a triple-exponential function，which contributed to three different tryptophan-heme orientations in BHb［27］and
indicated three decay components. According to Eq.（4），Eq.（5）and Eq.（6），the average lifetime of pure BHb in
the experimental conditions was 1.797 ns. After gradually adding HA，the shorter lifetime decreased from 1.811
ns to 1.525 ns，and the proportion of the shorter component didn’t show obvious changing. The lifetime of another
short component（0.356 ns）decreased to 0.294 ns and the proportion of the shorter component increased from
50.2% to 50.5%. The relative longer lifetime decreased from 6.023 ns to 5.561 ns，while the component didn’t
show obvious changing. The average lifetime of systems showed a variance from 1.797 ns to 1.782 ns as the HA
concentration increased from 0 to 3.514 μmol/L. Therefore，for Pt-HA，the shorter lifetime decreased from 1.811
ns to 1.571 ns，and the proportion of the shorter component decreased from 32.8% to 32.5%. The lifetime of anoth⁃
er short component（0.356 ns）decreased to 0.310 ns，and the proportion of the shorter component increased from
50.2% to 50.9%. The relative longer lifetime decreased from 6.023 ns to 5.545 ns，while the proportion of the com⁃
ponent decreased from 17% to 16.6%. The average lifetime of systems showed a variance from 1.797 ns to 1.743
ns as gradually adding Pt-HA from 0 to 3.514 μM. It was noteworthy that in presence of HA and Pt-HA，no signifi⁃
cant change in the tryptophan fluorescence decay parameters were observed（Table 3）. Thus the average lifetimes
（t）computed from the decay parameters remained essentially unchanged. This indicated that the static mecha⁃
nism was principally responsible for this interaction and implied that HA and Pt-HA do indeed bind to BHb and
form the stable complex［28］. Moreover，the relative bigger average lifetime variance of BHb after adding Pt-HA also
certificated the stronger interaction between BHb and Pt-HA compared to HA，which consistent with the UV-Vis
analysis.
Fig.4 Time-resolved fluorescence decays for BHb in the absence and presence of HA（A）and Pt-HA（B）；λex=283 nm，［BHb］=5.0 μM；
［HA］=0.000，1.757，3.514 μmol/L；［Pt-HA］=0.000，1.757，3.514 μmol/L；time calibration=2.194 787×10-10s/ch.
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南京师大学报（自然科学版） 第38卷第3期（2015年）
Table 3 The fitting parameters of fluorescence lifetime of pure BHb，BHb interact with HA and Pt-HA at different concentration
Sample
Pure BHb
BHb-HA
BHb-Pt-HA
C/（μmol/L）
5.000
1.757
3.514
1.757
3.514
f1
0.328
0.326
0.327
0.325
0.325
f2
0.502
0.505
0.505
0.511
0.509
f3
0.170
0.169
0.168
0.165
0.166
t 1/ns
1.811
1.531
1.525
1.584
1.571
t 2/ns
0.356
0.300
0.294
0.314
0.310
t 3/ns
6.023
5.566
5.561
5.662
5.645
/ns
1.797
1.787
1.782
1.746
1.743
To further analyze the interaction mechanism between BHb and drug，their fluorescence properties were
studied. In general，BHb contained three intrinsic fluorophores（tryptophan，tyrosine，and phenylalanine resi⁃
dues）. Besides，it has been reported that the intrinsic fluorescence of BHb primarily originated from the Trp resi⁃
due alone，as the fluorescence quantum yield of phenylalanine was really very low and the tyrosine residue was
mostly quenched near an amino group，a carboxyl group，or a tryptophan［29］，as shown in Fig.5.
Fig.5 Fluorescence spectra of pure BHb with different HA（A）and fluorescence spectra of pure BHb with different Pt-HA（B）；［BHb］=
0.5 μM；［HA］=0.000，1.757，3.514，5.271，7.028，8.758 μmol/L；［Pt-HA］=0.000，1.757，3.514，5.271，7.028，8.758 μmol/L；λex=285 nm，pH=
7.0，T=298 K.
BHb displayed a strong fluorescence emission peaked at 322 nm after being excited with wavelengths of 285
nm in the absence of HA and Pt-HA，while HA and Pt-HA have no
fluorescence signals in this experiment conditions［4］. After gradually
adding HA and Pt-HA into BHb solution，the fluorescence intensity
of BHb reduced gradually and blue- shifted from 322 to 318 nm，
implied that there was an interaction between BHb and drug，and the
changes of the microenvironment around Trp residues may contribute
to the decrease of the fluorescence of BHb. In addition，in order to
obtain an insight into the mechanism of fluorescence quenching，the
Stern-Volmer plot，and the peak intensity as a function of the concen-
trations of drug，were plotted in Fig.6. It was evident that the plot
showed liner behavior. In general，a linear Stern-Volmer reflected
either a dynamic（collisional）or static mechanism. Dynamic quenching
refer to a process that BHb and drug come into contact during the lifetime of the excited state，whereas static
quenching refer to the formation of BHb-drug complex［30］. Thus，combining the results of Time-resolved fluorescence
spectra，we could further conclude that the static mechanism was principally responsible for the fluorescence
quenching. Moreover，the fluorescence quenching possibly could be ascribed to the energy or electron transfer from
the BHb to HA or Pt-HA. Fig.7 presented the fluorescence spectrum of the drug after adding different amount BHb
from 580 nm to 650 nm according to there is no fluorescence of pure BHb in this range. If HA and Pt-HA accepted
the energy from BHb，the fluorescence intensity should be increased. However，it is observed that both HA and
Pt-HA was quenched by BHb，which certificated the electron transfer mechanism between BHb and drug［31］.
Therefore，it could be deduced that quenching mechanism of BHb and HA and Pt-HA were both static process which
ascribed to electron transfer.
Fig.6 Plots of F/F0 vs.［HA］and［Pt-HA］with
different concentrations；［BHb］=0.5 μM；［HA］=
0.000，1.757，3.514，5.271，7.028，8.758 μmol/L；
［Pt-HA］=0.000，1.757，3.514，5.271，7.028，
8.758 μmol/L；pH=7.0，T=298 K.
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袁秀雪，等：光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的相互作用
Fig.7 Fluorescence spectra of HA（A）with different BHb and fluorescence spectra of Pt-HA（B）with different BHb；
［HA］=10 μmol/L；［Pt-HA］=10 μmol/L；［BHb］=0.000，1.757，3.514 μmol/L；λex=285 nm，pH=7.0，T=298 K
2.5 Investigation on the conformation of BHb
CD spectroscopy，a sensitive technique to detect the conformation changes in protein upon interaction with
drug including the changing of secondary and tertiary structures of protein［32］，was employed in the following
study. Fig.8 presented the CD spectra of BHb in the absence and presence HA and Pt-HA at room temperature.
The CD spectrum of pure BHb showed two characteristic peaks of negative peaks at 208 nm and 222 nm，and a
characteristic positive band at 215 nm［33］. The band of 208 nm was contributed to π-π* transfer for the peptide
bound of α-helix，whereas the 222 nm band was contributed to π-π* transfer for both the α-helix and random
coil and the 215 nm band was contributed to β-Sheet［34］. Both HA and Pt-HA have no absorption signals from 200
nm to 240 nm. After adding different amounts of HA and Pt-HA to BHb，the CD spectrum intensity of the two
negative bands was decreased，whereas the positive bands rose. In addition，the CD spectrum of the BHb remains
essentially unchanged upon addition of different concentrations of HA and Pt-HA（BHb concentration remaining
fixed）suggested that the structure of BHb conjugate was still predominately α-helix. According to Fig.8，Pt-HA
was obvious in altering indicated that the interaction between Pt-HA and BHb was stronger. In addition，through
CDNN program，the secondary structural elements of pure BHb，HA-BHb，and Pt-HA-BHb were calculated and
listed in the Table 4 and Table 5. Besides，they were summarized by a bar diagram in Fig.9（A）and Fig.9（B）. On
the basis of Table 4 and Fig.9（A），α-helix of pure BHb was 42.1% and β-sheet was 13.3%，and the amount of α-
helix decreased and β-sheet increased continuously along with the continued addition of HA into BHb. Similarly，
according to Table 5 and Fig.9（B），the values of α-helix decreased and β-sheet increased notably. The results
suggested that both HA and Pt-HA were able to alter the secondary structure of BHb and its percentages composi⁃
tion of conformation，and the latter altered more acute. Moreover，the decrease of α-helix content revealed that
HA and Pt-HA combined with the amino acid residues of the main polypeptide chain and further caused partial
unfolding of BHb［35］，the computation results presented the detail.
Induced circular dichroism（ICD）has been proved to be a sensitive technique for studies of intricate
stereochemical problems related to chiral complexation and which can be applied to small organic molecules as
well as large systems such as biopolymers［36］. In the range of 300 nm to 600 nm，the pure BHb has no bands.
However，both HA and Pt-HA had a positive and a negative band at 355 nm and 455 nm，which were characteristic
of electronic transition of n-π* of the conjugated C=O and π -π* of the large aromatic conjugated system，
respectively. From Fig.10，upon gradual addition BHb into the drug，the original bands both decreased. Both the
interaction indicated that drug must interact within the chiral environment of the protein，and the protein induced
optical activity in the drug［37］. Besides，these changes in the ICD spectra were compared detailly as shown in
Fig.11. The results implied that the BHb interacted stronger with Pt-HA at 455 nm，but slightly more acute of
the interaction at 355 nm than HA due to the polymer-like structure of Pt-HA，it posses bigger aromatic conjugated
system，which further certificated that the aromatic conjugated system facilitate the stronger interaction between
BHb and Pt-HA.
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南京师大学报（自然科学版） 第38卷第3期（2015年）
Fig.8 The CD spectra of pure BHb and HA-BHb system（A）；the CD spectra of pure BHb and Pt-HA-BHb
system（B）in phosphate buffers；［BHb］=0.3 μmol/L，T=298 K.
Table 4 Secondary structural elements of BHb and a function of［HA］from CD data using CDNN program
sample
BHb
BHb-HA
BHb-HA
CHA/(μmol/L)
0.300
0.879
1.757
α-Helix/%
42.1
40.7
39.4
β-Sheet/%
13.3
13.9
14.3
β-Turn/%
15.2
15.5
15.6
Random-coil/%
28.2
29.0
29.9
Table 5 Secondary structural elements of BHb and a function of［Pt-HA］from CD data using CDNN program
sample
BHb
BHb-Pt-HA
BHb-Pt-HA
CPt-HA/(μmol/L)
0.300
0.879
1.757
α-Helix/%
42.1
39.7
38.1
β-Sheet/%
13.3
14.3
14.9
β-Turn/%
15.2
15.6
29.5
Random-coil/%
28.2
29.5
30.4
Fig.9 Bar diagrams of different conformations of BHb and HA-BHb（A）from CD data；Bar diagrams of different conformations of
BHb and Pt-HA-BHb（B）from CD data；［BHb］=0.3μmol/L，［HA］=0.000（dark），0.879（white），1.757（grey）μmol/L
Fig.10 Induced circular dichroism（ICD）spectra of HA（A）on interaction with BHb；Induced circular dichroism（ICD）spectra of
Pt-HA on interaction with BHb（B）.［HA］=33.7μmol/L，［Pt-HA］=33.7μmol/L，［BHb］=0，0.043，0.086，0.129，0.172 μmol/L.
Fig.11 Comparison the interaction of drug with different amount BHb at 455 nm（A）and 355 nm（B）.
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袁秀雪，等：光谱法比较光疗药物竹红菌甲素及铂竹红菌甲素复合物与牛血红蛋白的相互作用
3 Conclusions
The interaction of HA and Pt-HA with pure BHb were studied by employing spectroscopic techniques such
as UV-Vis absorption，Time-resolved fluorescence spectra，CD and ICD spectrum. The UV-Vis spectrum indicat⁃
ed that both HA and Pt-HA could interact with BHb. In addition，the binding constant of Pt-HA with BHb was
significant bigger than HA，and thermodynamic parameters analysis exhibited that the binding force of HA was
mainly electrostatic and hydrophobic force and van der Waals and hydrogen bond force were the mainly force of
Pt-HA complex. Besides，the time-resolved fluorescence and fluorescence spectrum together demonstrated that
the quenching mechanism of HA and Pt-HA were static quenching due to the electron transfer from BHb to HA
and Pt-HA. Finally，CD spectra provided evidences of the reducing of α-helix after interaction，supporting that
the micro-environment and conformation of BHb were changed in the presence of HA or Pt-HA. The all compari⁃
son certificated the interaction of BHb with Pt-HA was more stable and stronger than HA. Finally，the results
also indicated that after chelating with Pt，the prosperities of HA was improved，which would be has an important
practical significance on the development of the application of the PDT.
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