PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences

Recognition of various biomolecules by the environment-sensitive spectralresponses of hypocrellin B

Liming Song,a Jie Xie,a Chunxi Zhang,a Cong Lib and Jingquan Zhao*a

Received 21st December 2006, Accepted 27th March 2007First published as an Advance Article on the web 18th April 2007DOI: 10.1039/b618678e

In this work, the spectral responses of hypocrellin B (HB) to the microenvironments of variousbiomolecules were studied, with human serum albumin (HSA), bovine serum albumin (BSA) andovalbumin (OVA) used as the models for proteins, sodium alginate (SOA) and hyaluronan (HYA) forpolysaccharides and liposomes for lipid membranes. Generally, compared to those in aqueous solution,the absorbance and fluorescence of HB were all strengthened in the model systems except for thefluorescence in HYA. Specially, according to the spectral responses of HB, the microenvironments inbiomolecules and liposomes could be set in a sequence of hydrophobic grades, i.e., liposomes >

proteins > polysaccharides. Further, RF/A, a parameter defined as the ratio of the fluorescence intensityto the absorbance, was proposed to identify the microenvironment quantitatively. It was found that theRF/A could not only distinguish various types of biomolecules but also identify specific binding fromnonspecific binding to proteins or polysaccharides.

1. Introduction

Hypocrellins (HA and HB) (Fig. 1) have been known as a groupof naturally occurring photosensitizers possessing photodynamicactivity to tumors, viruses1–3 and some special vascular diseases.4

Different from the other ICT (intramolecular charge transfer)and TICT (twisted intramolecular charge transfer) indicators,5–7

the fluorescence of hypocrellins, originated from the excitedintramolecular proton transfer so in turn from the intramolecularhydrogen bonds,8–10 is very sensitive to the microenvironment.11, 12

Recently, specific binding of HB to human serum albumin (HSA)and to hyaluronan were reported by taking advantage of theenvironment-sensitive properties of HB fluorescence.13,14 However,whether hypocrellin fluorescence is so sensitive to distinguish onekind of biomolecules from the others is unknown. Generally, itis known that lipid, proteins and polysaccharides are the threetypes of the most abundant biomolecules on cell surfaces, andsome of them would be over-expressed in tumor cells or tissues.For years, people have expected to detect a biological targetspecifically by taking advantages of the fluorescence property ofsome photosensitizers, such as hematoporphyrin derivatives.15–18

It was observed that lipophilic porphyrin derivatives wouldbe concentrated on tumor cells or tissues so the fluorescencewould be much stronger for higher amount of lipids thannormal. However, for the lipid environment of a tumor cellis only quantitatively but not qualitatively different from thenormal one, a tumor target could not be recognized specifically

aBeijing National Laboratory for Molecular Sciences (BNLMS), CAS KeyLaboratory of Photochemistry, Institute of Chemistry, Chinese Academy ofSciences, Beijing, 100080, People’s Republic of ChinabSchool of Engineering and Chemistry Science, Center for Advanced Studiesof Medicinal and Organic Chemistry, Yunnan University, Kunming, 650091,People’s Republic of China. E-mail: zhaojq@iccas.ac.cn; Fax: +86-10-82617315; Tel: +86-10-82617053

Fig. 1 The molecular structure of hypocrellin A (left) and hypocrellin B(right). The dash links denote the intramolecular hydrogen bonds.

by the fluorescence intensity. In this case, some quantitativeinformation may be useful to recognize a target specifically.In the current work, the spectral responses of HB to variousbiomolecules were systemically analyzed with liposomes used asa mimic lipid membrane, HSA, BSA and OVA as model proteins,SOA and HYA as model polysaccharide molecules. Among thebiomolecules, HSA possesses well-determined hydrophobic sites,19

while liposomes, widely used as mimic bio-membrane systems,possess a hydrophobic compartment surrounded by the lipidbilayer.20 On the other hand, the conformation of alginate andHYA (two types of polysaccharides) are not well determinedbut variable in aqueous solution,21 therefore some informationof the conformation may be derived via the fluorescence responsesof HB. Alginate is a linear polysaccharide molecule containing1,4-linked a-L-glucuronate and b-D-mannuronate residues varyingin proportion and sequence. It was proposed that alginatestrands could form a localized hydrophobic compartment (Fig. 2),especially in b-D-mannuronate-rich segments.22 Furthermore, itwas reported that alginate did possess some discrete hydrophobicregions.23

This journal is © The Royal Society of Chemistry and Owner Societies 2007 Photochem. Photobiol. Sci., 2007, 6, 683–688 | 683

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Fig. 2 The molecular structures of the two residues in alginate: 1,4-linkedb-D-mannuronate (left) and a-L-glucuronate residues (right).

2. Materials and methods

2.1. Materials

HB was derived as mentioned previously.24 The egg L-a-phosphatidyl-choline (EPC) and sodium alginate (chemical grade,molecular weight spread from 25000 to 30000) were purchasedfrom Shanghai Chemical Reagent Company, and cholesterol(chemical grade) from China Pharmaceutical Company. HYA(hyaluronan) was purchased from Shandong Freda Biochem.Co., Ltd. (MW >1.5 × 106). HSA (Fraction V), BSA (bovineserum albumin) and OVA (ovalbumin) were purchased from SigmaChemical Co. (molecular weight was 66000, 67000 and 45000respectively), and used as received. Phosphate buffered saline(PBS) at pH 7.0 was prepared by the use of 10 mM KH2PO4

and 10 mM K2HPO4. The working solutions were preparedimmediately before use.

2.2. Preparation of sample

HB was known as a lipid-soluble compound and hardly soluble inwater, however, the definite solubility of HB in aqueous solutionhas to be learnt for study on the binding to the biomoleculesin PBS solution. Based on the linearity of the absorbance toconcentration (figure not shown), 10 lM of HB was well solublein aqueous solution, therefore, 8 lM of HB was used in thiswork. Furthermore, a concentrated solution of HB (2 mg ml−1)in dimethylsulfoxide (DMSO) was added slowly into the PBSsolution of biomolecules in microliter quantity and mixed quickly

by shaking. Liposomes were prepared by EPC and cholesterol byusing the film-ultrasonic technique.25

2.3. Spectral measurements

Absorbance spectra of HB were obtained on a Shimadzu UV-1601 UV-Vis spectrophotometer and fluorescence spectra on anF-4500 spectrofluorimeter (Hitachi, Japan) at room temperature.The fluorescence spectra of HB were measured with selectiveexcitation of HB at 470 nm. The binding constant between HBand various biomolecules was calculated by the Lineweaver–Burkcurve method.26

3. Results and discussionThe fluorescence quantum yield and the e value for HB in aqueoussolution were estimated to be 0.014 and 0.6 × 104 respectively,much lower than 0.07 and 2.08 × 104 in chloroform.27 Theabsorption and fluorescence spectra of HB (8 lM) in PBS solutionand in chloroform are shown in Fig. 3.

Fig. 3 The fluorescence spectra of HB (8 lM) in aqueous solution ( )and in chloroform ( · · · ).

The absorbance and fluorescence spectra of HB (8 lM) inliposomes, HSA and SOA are shown in Fig. 4, and the dependence

Fig. 4 The absorption and fluorescence spectra of HB (8 lM) on concentration of liposomes (A), HSA (B) and sodium alginate (C) in aqueous solution(pH 7.0).

684 | Photochem. Photobiol. Sci., 2007, 6, 683–688 This journal is © The Royal Society of Chemistry and Owner Societies 2007

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of the absorbance and fluorescence intensity of HB (8 lM) on theconcentrations of egg L-a-phosphatidyl-choline (used for buildingliposomes), HSA and SOA is shown in Fig. 5.

Fig. 5 Dependence of the absorbance at 470 nm and fluorescenceintensity at 620 nm of HB (8 lM) on the concentration of liposomes(�), HSA (�) and sodium alginate (�) in aqueous solution (pH 7.0).

It can be seen that the three species possess distinct “saturated”concentration at which all the HB molecules becomes completelybounded to the biomolecules at pH 7.0. Generally, binding tothe biomolecules, both the absorbance and the fluorescence ofHB were strengthened. Specially, the distinct “saturated” spectralintensity reflected individual microenvironment characteristics ofthe biomolecules.

To find if the absorbance or fluorescence is proportional tothe concentration of HB in the biomolecules, Fig. 6 and Fig. 7show the spectra and the plots of the absorbance and fluorescenceintensity to the concentration of HB when the concentrations ofegg L-a-phosphatidyl-choline (used for building liposomes), HSAand SOA were kept at a “saturated” value.

It can be seen that the absorbance and fluorescence intensity arelinearly proportional to concentration of HB so to the absorbedphoton numbers. It should be indicated that the “saturated”

Fig. 7 Dependence of the absorbance at 470 nm and fluorescenceintensity at 620 nm on the concentration of HB (1.00, 2.75, 4.50, 6.25,8.00 lM) at a “saturated” concentration of liposomes (A, 0.08 g l−1), HSA(B, 0.66 g l−1) and sodium alginate (C, 0.75 g l−1) in aqueous solution(pH 7.0).

concentration was used to make sure that all the HB moleculeswould be completely bounded.

Among these biomolecules, the microenvironment in liposomesis the most hydrophobic while the least in SOA with that in HSAbetween. Structurally, liposomes, usually used as drug carriers,could provide a relatively complete hydrophobic surrounding to alipophilic molecule. HSA could bind lipophilic drugs specificallyor nonspecifically in the hydrophobic sites.11,28 Compared toliposomes, the hydrophobic microenvironment in HSA is perhapsincomplete, i.e., partial exposure to aqueous solution. AlthoughSOA possesses some hydrophobic area, which can also be learnt bythe strengthened absorbance and fluorescence, the multi-hydroxylbiomolecules also possess negatively charged carboxyl groups atpH 7.0. In this case, the overall interaction among SOA moleculesshould be a compromise between the hydrophobic attraction andthe electrostatic repulsion. Therefore, the hydrophobic environ-ment in alginate is not so effective as that in liposomes or in HSA,

Fig. 6 The absorption and fluorescence spectra of HB (1.00, 2.75, 4.50, 6.25, 8.00 lM) at the “saturated” concentration of liposomes (A, 0.08 g l−1),HSA (B, 0.66 g l−1) and sodium alginate (C, 0.75 g l−1) in aqueous solution (pH 7.0).

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Fig. 8 The absorption and fluorescence spectra of HB (8 lM) on the time of reaction with liposomes (0.08 g l−1) (A), HSA (0.66 g l−1) (B) and sodiumalginate (0.75 g l−1) (C) in aqueous solution (pH 7.0).

which is why the spectral intensity in SOA is the lowest. In Fig. 8and Fig. 9, the reaction rates of HB to liposomes, HSA and SOA atthe “saturated” concentrations provide information of interactingdynamics. It can be seen that the binding of HB to SOA is farfaster (half an hour) than to liposomes or HSA (6 h), suggesting amore “open” hydrophobic area for the former but a more “closed”hydrophobic compartment for the latter. It was reported that thestructural relaxation was necessary when lipid-soluble drugs weregetting into the hydrophobic compartments in liposomes29 andHSA,30 which may be why the binding of HB has to take muchlonger time.

Fig. 9 Dependence of the absorbance at 470 nm and fluorescenceintensity at 620 nm of HB (8 lM) on the time of reaction with liposomes(0.08 g l−1) (�), HSA (0.66 g l−1) (�) and sodium alginate (0.75 g l−1) (�)in aqueous solution (pH 7.0).

Qualitatively, the environments in liposomes, HSA and SOA canbe set in a sequence of hydrophobicities as liposomes > HSA >

SOA, corresponding to a completely or incompletely “closed”or “half-opened” hydrophobic compartment respectively. In fact,the fluorescence intensity is not a specific parameter to identifythe molecular environments for it depends on concentrationof the fluorescent molecules too. Therefore, to eliminate theconcentration factor, a ratio of the fluorescence intensity of HB

at 620 nm to the absorbance at 470 nm, named as RF/A, wasproposed to evaluate the microenvironments of the biomoleculesquantitatively. In fact, the physical meaning of RF/A is similarbut not exactly the same to the fluorescence quantum yield asexpressed in eqn (1):31,32

φI/φCHCl3= FIACHCl3 N

2I /FCHCl3 AI N 2

CHCl3= (RI/RCHCl3 )

(N2

I /N2CHCl3

)

(1)

where U, F , A stand for the fluorescence quantum yield, intensityand absorbance respectively and N and R the refractive indexand F/A respectively. I and CHCl3 stand for the media I andchloroform respectively.

The fluorescence quantum yield in vivo could hardly be deter-mined for the refractive index in a biomolecule is not derivable,while RF/A could be derived directly by jointly measuring theabsorbance and fluorescence. The plots of the RF/A values versusthe concentrations of various biomolecules are shown in Fig. 10,and the RF/A values at the “saturated” concentrations and thebinding constants are listed in Table 1.

Remarkably, the two kinds of polysaccharides possess the RF/A

values even lower than water, which could clearly distinguish the

Fig. 10 Plot of the RF/A values of HB (8 lM) versus the normalizedconcentrations of biomolecules.

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Table 1 The RF/A values and the binding constants (K/M−1) of HB (8 lM) at “saturated” concentrations of the biomolecules

Polysaccharide Protein Lipid

Solution HYA SOA Aqueous BSA OVA HSA Liposomes CHCl3

RF/A 816 1791 2318 3028 3042 3303 3835 9875K/M−1 5.64 × 106 1.57 × 103 — 6.16 × 103 4.03 × 103 1.21 × 106 — —

binding of HB to polysaccharides from that to other biomolecules.It should be indicated that the fluorescence intensity of HB inSOA is higher than that in water, but the fluorescence increaseis much less than the increase in absorbance, which leads to aneven smaller RF/A value than that in water. Further, comparing thetwo polysaccharides, the far lower RF/A value for HYA than thatfor SOA should account for the specific intermolecular hydrogenbonding for HB with the former9 but nonspecific one with the lat-ter, which was also suggested by the binding constants. Secondly,liposomes possess the largest RF/A value among the three speciesdue to the well-enveloped hydrophobic compartment. However,the value was still far lower than that in pure organic solvent(CHCl3), perhaps due to the fact that the semi-fluid liposomescould not completely protect HB from water molecules. Thirdly,the RF/A values for the three globular proteins located betweenwater and liposome due to the “incompletely closed” hydrophobicenvironment. Among the three proteins, HSA possesses the largestRF/A value and binding constant than the others, most probablydue to a specific binding of HB to the former but nonspecific oneto the latter. In one word, the RF/A value of HB could provide morespecific evaluation of the microenvironments in biomolecules andcould in principle be used to monitor the binding of HB to lipidmembranes, proteins or polysaccharides.

It should be kept in mind that HB possesses not only theenvironment-sensitive spectral properties but also the photo-dynamic activity to some disease targets, therefore, the dualproperties may be valuable for specifically detecting and selectivelydestroying a target tissue at the same time. Of course, a target cell ortissue must contain various biomolecules in variable population,however, for tumor cells or tissues which possess distinct moleculeenvironments from the normal ones, some quantitative evaluationmay be helpful to distinguishable populations of biologicalmolecules. Certainly, more research has to be carried out to achievethis goal.

4. Conclusions

In this work, it was found that the spectral responses ofHB to various biomolecules, including liposomes, proteins andpolysaccharides, were distinctive and could be used specificallyto recognize a microenvironment. Qualitatively, the spectral(absorbance and fluorescence) intensities of HB could be set ina sequence of binding to liposomes > proteins > polysaccharidesdue to the various hydrophobic characteristics. Furthermore, aratio of the fluorescence intensity to the absorbance, named RF/A,was used to evaluate the microenvironments in the biomoleculesquantitatively. It could not only provide a quantitative evaluationof the hydrophobic microenvironments but also differentiate thespecific binding from the nonspecific binding of HB to a same typeof biomolecules. Therefore, it may be a more practical parameterto identify the molecular microenvironments on cells or tissues.

Abbreviations

HA Hypocrellin AHB Hypocrellin BHSA Human serum albuminSOA Sodium alginateEPC Egg L-a-phosphatidyl-cholineHYA HyaluronanBSA Bovine serum albuminOVA OvalbuminPBS Phosphate buffered salineDMSO Dimethylsulfoxide

Acknowledgements

This research was supported by the National Natural Sci-ence Foundation of China (NNSFSC) (No. 20273079 and No.50221201).

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