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Bioconjugate Chem. 2010, 21, 2332–2338
Time-Dependent Enzyme Activity Dominated by Dissociation of J-Aggregates Bound to Protein Surface Kenji Watanabe and Koji Kano* Department of Molecular Chemistry and Biochemistry, Doshisha University, Kyotanabe, Kyoto, 610-0321, Japan. Received August 3, 2010; Revised Manuscript Received October 18, 2010
J-Aggregates of diprotonated 5,10,15,20-tetrakis(4-sulfonatopheny)porphyrin (H4TPPS2-) were stabilized even in a neutral aqueous solution (pH 7.0) containing per-O-methylated β-cyclodextrin by binding to the surface of R-chymotrypsin (ChT). The large J-aggregates covered the active site of ChT and completely inhibited the hydrolysis of the peptides. However, enzyme activity was gradually restored with the dissociation of the J-aggregates attached to the protein surface to monomers. After the completion of dissociation of the aggregates, the enzyme activity was almost completely restored, though the structure of ChT significantly changed. Circular dichroism spectroscopy suggested that the microscopic structure at the active site of ChT was scarcely affected by the J-aggregates, but the binding of J-aggregates to ChT increased the content of the random coils in the enzyme. The present study showed a new type of effector for controlling the function of ChT.
INTRODUCTION Modulation of protein functions by artificial means is an important challenge in the fields of biochemistry, chemical biology, medicinal chemistry, and medical chemistry. Pseudosubstrates that selectively bind to the enzyme active sites have been used as strong inhibitors and can be used to suppress the propagation of viruses (1-3). Artificial receptors that recognize protein surfaces might also be useful for the inhibition of protein-protein and/or protein-substrate interactions. Hamilton et al. designed and prepared several artificial receptors based on calixarene, porphyrin, and G-quartet scaffolds, which recognize the surfaces of proteins such as R-chymotrypsin (ChT) and cytochrome c (cyt c) (4-14). Rotello et al. used functionalized gold nanoparticles to regulate the function of ChT (15-25). These artificial receptors of proteins have larger molecular size than that of pseudosubstrates and, therefore, cover a wider area on the protein surface. Most of these protein receptors are anionic and can nonspecifically recognize the surfaces of basic proteins such as ChT and cyt c. An artificial protein receptor primarily binds to the surface of the protein. If the receptor covers the active site of the protein, it inhibits the activity of the protein. If the receptor causes alteration in protein structure, such receptors act as the denaturant. In the former case, detachment of the receptor from the protein surface restores the protein function. Kano et al. used 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) (FeIIITPPS3-) as a receptor of ChT that resulted in inhibition of the hydrolysis of an amide substrate (26). They found that the ChT function is recovered by detaching FeIIITPPS3- from ChT using heptakis(2,3,6-(triO-methyl)-β-cyclodextrin (TMe-β-CD), which forms a very stable inclusion complex with the porphyrin. In the latter case, refolding of the denatured protein is so difficult that, in most cases, the function of the protein is permanently lost. In the present study, we have introduced a concept of spontaneous restoration of enzyme activity, whose function is initially inhibited by a protein receptor. Recently, we found that J-aggregates of diprotonated H2TPPS4- (H4TPPS2-) are stabilized by ferric myoglobin (metMb) in aqueous solution at pH * E-mail:
[email protected]. Fax: 81-774-65-6845. Phone: 774-65-6624.
7.0 and 25 °C (27). The average half-life of the J-aggregates bound to metMb is approximately 3 h. The J-aggregates gradually dissociate to monomers and the native metMb structure was restored. The results of our previous study prompted us to investigate the time-dependent recovery of ChT activity that is initially inhibited due to covering of the ChT active site by H4TPPS2- J-aggregates. There have been very few studies on time-dependent changes in enzyme activity. Hamilton’s group reported time-dependent inhibition of ChT activity by 4 peptide loops attached to the upper rimes of calix[4]arene and G-quartet scaffolds (4-6). Similar timedependent inhibition of ChT activity by functionalized gold nanoparticles has also been reported (15). These phenomena were attributed to the slow binding of receptors to ChT. Meanwhile, we explained the similar inhibition by FeIIITPPS3as time-dependent irreversible denaturation of ChT (26). At any rate, the enzyme activity lowered by the protein receptors is hardly restored. In the present study, we examined the selfrestoration of ChT activity that is initially inhibited by the J-aggregates of H4TPPS2-.
EXPERIMENTAL PROCEDURES Materials. H2TPPS · nH2O (acidic form, >98%, Tokyo Chem. Ind. Co.) was purchased and used without further purification. The concentration of H2TPPS4- was determined by measuring the absorbance at 414 nm in aqueous NaOH (6 mM) solution using the molar extinction coefficient of ε414 ) 5.33 × 105 M-1 cm-1 (28). ChT from bovine pancreas (Sigma-Aldrich Japan) was used as received. The concentration of ChT was determined by measuring the absorbance at 280 nm using the molar extinction coefficient of ε280 ) 5.06 × 104 M-1cm-1 (29). The building blocks of the substrates, Fmoc-Gly-OH, Fmoc-Glu(tBu)OH, and Fmoc-Ser(tBu)-OH, were purchased from Peptide Institute Inc. (Osaka, Japan). N-Succinyl-L-phenylalanine pnitroanilide (SPNA) was obtained from Sigma-Aldrich Jap. Other reagents and solvents were commercially available and used without further purification. Water was purified using a Millipore Simpack 1 (Nihon Millipore, Tokyo, Japan). Instruments. Absorption and fluorescence spectra were recorded on a Shimadzu UV-2100 spectrophotometer and a Shimadzu RF-5300PC spectrofluorometer, respectively. Circular
10.1021/bc100355v 2010 American Chemical Society Published on Web 11/12/2010
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Scheme 1. Substrates and ChT-Catalyzed Hydrolysis
dichroism (CD) spectra were measured using a Jasco J820 spectropolarimeter. MALDI-TOF MS measurements were performed by using an Shimadzu AXIMA-CFRplus system. A Horiba F-52 pH meter was used to adjust the pH values of the solutions. Synthesis. SPNA3G3E (p-NA-Phe-Suc-Gly-Gly-Gly-GluGlu-Glu-CONH2): The SPNA3G3E peptide (Scheme 1) was prepared by Fmoc solid-phase peptide synthesis on the TGSRAM resin (Shimadzu, Kyoto, Japan). Fmoc deprotection was achieved by treatment with 30% (v/v) piperidine in DMF. Each coupling reaction involved the reaction of the NH2 group on the resin with a carboxy group of Fmoc-amino acid (10 equiv) in DMF containing HBTU (1.2 equiv), HOBt (1.2 equiv), and DIEA (2.4 equiv). The N-terminus of the peptide was conjugated with SPNA (4 equiv) using the coupling agents HBTU (4 equiv), HOBt (4 equiv), and DIEA (4 equiv) in DMF. After the reaction, the peptide was cut off from the resin using a mixture of trifluoroacetic acid/1,2-ethandiol/thioanisole/triisopropylsilane/ water (86:5:5:1.5:2.5, v/v) and precipitated with diethyl ether. ¨ KTApurifier FPLC The crude peptide was purified using an A system (GE Healthcare Jpn. Co., Tokyo, Japan) equipped with a Wakosil-II 5C18 AR reverse phase column (Wako Pure Chem. Ind., Osaka, Japan). The purity of the peptide was confirmed by the FPLC analysis (Supporting Information, Figure S1). Yield 21 mg (76%). MS (MALDI-TOF, R-cyano-4-hydroxycinnamic acid) m/z: calcd for [M - H]- 940.8; found 941.3. SPNA3G3S (p-NA-Phe-Suc-Gly-Gly-Gly-Ser-Ser-SerCONH2): The SPNA3G3S peptide (Scheme 1) was synthesized according to the procedure similar to that followed for SPNA3G3E synthesis. The purity of the peptide was confirmed by FPLC analysis (Supporting Information, Figure S2). Yield 19 mg (79%). MS (MALDI-TOF, R-cyano-4-hydroxycinnamic acid) m/z: Calcd for [M + Na]+ 839.3; found 838.6. Calcd for [M + K]+ 855.3; found 855.6. Sample Preparation. A stock solution of the J-aggregates of H4TPPS2- (2.0 × 10-3 M as the monomer) was prepared by dissolving H2TPPS · nH2O (acidic form) into phosphate buffer (pH 7.0, 5.0 × 10-3 M) and the solution was allowed to stand for a night in dark. Because the buffering effect of the system was not enough, the pH value of the resulting solution decreased to 2.8 due to dissociation of the acidic SO3H groups of the porphyrin. The main species in this solution were the Jaggregates of H4TPPS2- (>80%) as confirmed by the UV-visible absorption spectrum (Supporting Information, Figure S3).
RESULTS AND DISCUSSION Stabilization of J-Aggregates by ChT. Since the pH of the stock solution of H4TPPS2- (pKa ) 4.8) (30) was 2.8, the
J-aggregates were formed through electrostatic interactions between the SO3- groups in the periphery and diprotonated tetrapyrroles of H4TPPS2- (31-35). The aggregation numbers of the J-aggregates have been estimated to be tens to hundreds thousands by resonance light scattering (RLS) (36, 37). When a stock solution of H4TPPS2- (2.0 × 10-3 M of H2TPPS4monomer) was injected into a neutral phosphate buffer solution (pH 7.0, 1.0 × 10-2 M) to achieve the apparent porphyrin concentration of 1 × 10-6 M, the J-aggregates of H4TPPS2immediately dissociated to H2TPPS4- monomers. In the presence of ChT (1.0 × 10-6 M), however, the dissociation of the J-aggregates was strongly slowed down even in a neutral aqueous solution. Figure 1 (in black) shows the UV-visible spectrum of the resulting solution measured immediately after sample preparation. The characteristic narrow and strong absorption bands at 491 and 706 nm were ascribed to the J-aggregates of H4TPPS2-, and the coexisting band at 413 nm was due to the monomer of H2TPPS4- (31-35). These results indicate that ChT stabilizes the J-aggregates of H4TPPS2- in neutral aqueous solution. Similar stabilization of the J-aggregates was observed in the H4TPPS2-/metMb system (27). An image of the H4TPPS2-J-aggregate/ChT complex is shown in Figure
Figure 1. UV-visible spectrum of a sample (sample A) prepared by injecting a stock solution of the J-aggregates of H4TPPS2- (2.0 × 10-3 M as monomer) into 4 mL of phosphate buffer (pH 7.0, 1.0 × 10-2 M) containing ChT (1.0 × 10-6 M) at 25 °C (black solid line) and the spectrum of a sample (spectrum B) prepared by subsequent addition of TMe-β-CD (8.0 × 10-6 M) to sample A (red solid line).
2334 Bioconjugate Chem., Vol. 21, No. 12, 2010
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Figure 2. Image of the complex of J-aggregate of H4TPPS2- with ChT (PDB code 4CHA) drawn by using BioMed CAChe V6. The cationic and anionic amino acid residues of ChT are shown in the red and blue colors, respectively. The light blue color represents the active site of ChT.
2. The J-aggregates stabilized by ChT gradually dissociated to monomers with a t1/2 of 27.2 ( 5.5 min (Supporting Information, Figure S4). We previously found that the FeIIITPPS3- monomer strongly binds with ChT (K ) (4.8 ( 0.3) × 105 M-1) and inhibits its activity (26). The H2TPPS4- monomer also formed a fairly stable complex with ChT (K ) (1.8 ( 0.4) × 105 M-1) (Supporting Information, Figure S5). To prevent the interaction between the H2TPPS4- monomer and ChT, we externally added TMe-β-CD, which forms an extremely stable 2:1 inclusion complex with H2TPPS4- (38), to the system. In the presence of TMe-β-CD (8.0 × 10-6 M), the absorption maximum of the monomer band (λmax ) 413 nm) shifted to 416 nm due to the formation of the 2:1 TMe-β-CD and H2TPPS4- complex (Figure 1, in red), but the absorption band of the J-aggregates was not affected at all. The dissociation of the J-aggregates in the presence of TMeβ-CD was analyzed by UV-visible spectroscopy (Figure 3). The t1/2 in the presence of TMe-β-CD was 26.1 ( 2.9 min, which was similar to that in the absence of TMe-β-CD, indicating that the dissociation of the J-aggregates was not accelerated by TMe-β-CD. Pasternack et al. extensively studied the formation of self-aggregates (not J-aggregates) of a cationic porphyrin (5,10-diphenyl-15,20-bis(N-methylpyridinium)porphyrin) (t-H2Pagg2+) on a polyanionic DNA double strand (39-41). They reported that the cationic porphyrin molecules are detached from the DNA after the addition of β-cyclodextrin (41). The inconsistency in the effects of cyclodextrins between the J-aggregates on ChT and the self-aggregates on DNA might be due to the difference in the regularity of the arrangements of these aggregates. In the case of the J-aggregates, the H4TPPS2molecules are highly ordered so that the insertion of a cyclodextrin molecule into the aggregate is difficult. However, the t-H2Pagg2+ molecules on DNA are arranged in a relatively disordered manner, and therefore, a cyclodextrin molecule can include the phenyl group of the porphyrin (41). Effect of J-Aggregates on Enzyme Activity. ChT is a proteolytic enzyme that catalyzes the hydrolysis of an amide linkage at the C-terminus of a protein. Because ChT recognizes the aromatic moieties of amino acid residues such as Phe, Tyr, and Trp, we introduced a Phe residue into the substrates
Figure 3. Decomposition of the J-aggregates of H4TPPS2- bound to ChT into the TPPS/TMe-β-CD inclusion complex. The spectra were collected with 5 min intervals. Inset: Progressive change in absorbance at 706 nm due to the J-aggregates. The total concentration of the porphyrin in the system was 1.0 × 10-6 M.
(SPNA3G3E and SPNA3G3S, Scheme 1) used in this study. SPNA3G3E and SPNA3G3S are the anionic and nonionic substrates, respectively. SPNA3G3E with a GluGluGlu sequence was designed to allow recognition of the Phe residue by ChT at the C-terminus and to enhance electrostatic repulsion with the J-aggregates. The hydrolysis of the substrate was monitored by following the changes in the absorbance of released pnitroaniline at 355 nm that correspond to the isosbestic point in the UV-visible spectral changes of the H4TPPS2- Jaggregates to the H2TPPS4-/TMe-β-CD inclusion complex (Supporting Information, Figure S6). Figure 4a (in black) shows the progressive changes in the absorbance at 355 nm in the hydrolysis of SPNA3G3E (3.0 × 10-4 M) catalyzed by ChT (1.0 × 10-6 M) in phosphate buffer (pH 7.0, 1.0 × 10-2 M) at 25 °C. The result at the initial stage of hydrolysis, where less than 16% of the substrate molecules were hydrolyzed, is shown in Figure 4. The absorbance at 355
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Figure 4. Hydrolysis profiles of SPNA3G3E (3.0 × 10-4 M) (a) and SPNA3G3S (3.0 × 10-4 M) (b) catalyzed by ChT (1.0 × 10-6 M) in the absence (black) and the presence (red) of both the J-aggregates of H4TPPS2- ([H2TPPS4-] ) 1.5 × 10-5 M) and TMe-β-CD (1.2 × 10-4 M) in phosphate buffer (pH 7.0, 1.0 × 10-2 M) at 25 °C.
Figure 5. Progressive changes in the rate constants for the ChTcatalyzed hydrolyses of SPNA3G3E (a) and SPNA3G3S (b) in the absence (black) and the presence (red) of both the J-aggregates and TMe-β-CD. The progressive change in the absorbance at 706 nm due to the J-aggregates of H4TPPS2- (blue) is also shown.
nm appeared to increase linearly in the case of ChT-catalyzed hydrolysis of SPNA3G3E in the absence of the J aggregates. Meanwhile, the ChT-catalyzed hydrolysis of SPNA3G3E in the presence of both H4TPPS2- J-aggregates (1.5 × 10-5 M as the monomer) and TMe-β-CD (1.2 × 10-4 M) showed a characteristic reaction profile which suggested that the hydrolysis was completely inhibited by the J-aggregates during initial 2 min and the reaction rate gradually increased until 50 min from the start. The inhibition effect depended on the amount of H4TPPS2added to the system (Supporting Information, Figure S7). TMeβ-CD itself as well as the 2:1 complex of TMe-β-CD and H2TPPS4- did not affect the hydrolysis at all. These results clearly indicate that the J-aggregates of H4TPPS2- play a role in the time-dependent inhibition of ChT-catalyzed hydrolysis of SPNA3G3E. The results in Figure 4a were analyzed by the following equations. The change in the absorbance at 355 nm (∆A) is represented as
well with the linear and six-order polynomial equations (eq 3) using the least-squares method. We arbitrarily selected the sixorder polynomial equation. The degrees of fittings were evaluated from the R2 values that were over 0.999.
∆A ) (εSCSl + εPCPl) - εSCS0l
(1)
where εS and εP are the extinction coefficients of the substrate and the product, respectively, at 355 nm, CS0 is the initial concentration of the substrate, CS and CP are the concentrations of the substrate and the product, respectively, at time t, and l is the optical length. Since CP ) (CS0 - CS), the CP can be expressed as CP ) ∆A/(εP - εS)l
(2)
The CP-time profiles obtained for the systems in the absence and the presence of both the J-aggregates and TMe-β-CD fitted
n
CP ) f(t) )
∑a t
m
m
(n ) 1 or 6)
(3)
m)0
Equation 3 was differentiated with time (t) to obtain velocity (V(t)) at time t. V(t) )
df(t) ) dt
n
∑ a mt
m-1
m
(4)
m)1
The second-order rate constant at t was obtained from eq 5 where the enzyme and substrate concentrations were assumed k(t) ) V(t)/[E][S]
(5)
to be the same as their respective initial concentrations. Figure 5a shows the rate constants as a function of time for the ChT-catalyzed hydrolyses of SPNA3G3E in the absence and the presence of both the J-aggregates and TMe-β-CD. In the absence of the J-aggregates, the k(t) value was constant until 50 min from the start (Figure 5a, in black). In the presence of a large excess of the substrate, ChT-catalyzed hydrolysis appeared to proceed linearly at the initial stage. Meanwhile, the k(t) value in the presence of the J-aggregates and TMe-βCD gradually increased and reached the value obtained in the absence of the J-aggregates (Figure 5a, in red). The progressive change in the k(t) was in good agreement with the decrease in
2336 Bioconjugate Chem., Vol. 21, No. 12, 2010
Figure 6. Hydrolysis profiles of SPNA3G3E (3.0 × 10-4 M; black and red) and SPNA3G3S (3.0 × 10-4 M; blue and green) catalyzed by ChT (1.0 × 10-6 M) in the absence and the presence of both the J-aggregates of H4TPPS2- ([H2TPPS4-] ) 1.5 × 10-5 M) and TMeβ-CD (1.2 × 10-4 M) in succinic acid buffer (pH 4.0, 1.0 × 10-2 M) at 25 °C.
the absorbance at 706 nm due to the dissociation of the J-aggregates (Figure 5a, in blue). These results clearly indicate that the enzyme activity of ChT was inhibited by the Jaggregates and that it gradually recovered to the original value with the progressive dissociation of the J-aggregates. The restoration of the enzyme activity was 98% at 50 min from the start of the dissociation of the J-aggregates. Inhibition Mechanism. The following three mechanisms are proposed for the inhibition of ChT activity by the J-aggregates of H4HPPS2-: (1) electrostatic repulsion between the anionic groups of SPNA3G3E and the J-aggregates, (2) covering of the active site of ChT by the J-aggregates, and (3) structural changes in ChT caused by the J-aggregate. To study the contribution of electrostatic interaction to the inhibition of the hydrolysis of anionic SPNA3G3E, we used a neutral SPNA3G3S (Scheme 1) as the substrate. The reaction profile is shown in Figure 4b, which exhibits that the Jaggregates inhibit the hydrolysis of the neutral substrate in the manner similar to that of the anionic substrate, though the inhibitory effect in the case of neutral SPNA3G3S is not as remarkable as that in the case of SPNA3G3E. The degrees of inhibition by the J-aggregates at 500 s from the start were 83% and 35% for SPNA3G3E and SPNA3G3S, respectively. It can be concluded, therefore, that electrostatic repulsion between the polyanionic J-aggregates and anionic SPNA3G3E is one of the important factors that reduces the hydrolysis of this anionic substrate. Since the hydrolysis of the neutral substrate was inhibited by the J-aggregates, the covering effect might also influence the hydrolysis. To this end, we evaluated the inhibition effect of the J-aggregates in aqueous solution at pH 4.0, wherein the J-aggregates do not dissociate to a marked extent. The results are shown in Figure 6. Since the optimum pH range of ChT activity is 8-9, the rates of the hydrolyses of both SPNA3G3E and SPNA3G3S at pH 4.0 were much less than those at pH 7.0. The hydrolyses of both the substrates were completely suppressed in the presence of the J-aggregates. The results clearly show that the J-aggregates of H4TPPS2- cover the active site of the enzyme and block the binding of the substrate leading to complete inhibition of the hydrolysis. At pH 7.0, the partial dissociation of the J-aggregates occurs from the start of the reaction, freeing the active site to interact with the substrate. In this stage, electrostatic interactions influence the approach of the substrate to the open active site of ChT.
Watanabe and Kano
Figure 7. Fluorescence spectra of ChT (1.0 × 10-7 M) before (black) and after (red) the addition of both J-aggregates of H4TPPS2([H2TPPS4-] ) 1.5 × 10-6 M) and TMe-β-CD (1.2 × 10-5 M) (red solid line) in phosphate buffer (pH 7.0, 1.0 × 10-2 M) at 25 °C. The fluorescence intensity gradually recovered to the spectrum indicated in a blue line. The spectra were recorded with 5 min intervals.
Finally, we studied the effect of the J-aggregates on changes in the protein structure of ChT. ChT possesses eight Trp residues, which are located in the core and the surface of the protein (42). The Trp residues exhibit fluorescence and are useful to probe changes in the protein structure, because the fluorescence of these residues greatly depends on the microenvironment (43, 44). Figure 7 shows the fluorescence spectral changes of ChT after the addition of J-aggregates. The fluorescence intensity at 334 nm markedly decreased immediately after the addition of the J-aggregates and gradually recovered with the dissociation of the J-aggregates from the protein surface. The fluorescence maximum of ChT scarcely shifted though the fluorescence intensity markedly diminished, suggesting that the Trp residues in the interior of the protein are not exposed on the protein surface upon binding with the J-aggregates. ChT appears to change its conformation upon binding with J-aggregates without undergoing distinct denaturation. The change in the conformation of ChT might cause enhanced fluorescence quenching of the Trp fluorescence by other amino acid residues (44). The fluorescence was not completely recovered, meaning occurrence of permanent change in the structure of ChT, although the enzyme activity was almost completely restored after the complete dissociation of the J-aggregates (see Figure 5). CD spectra also revealed structural changes in ChT after the addition of J-aggregates (Figure 8). Since the CD measurement required about 10 min, the observed spectrum did not reflect the structure of ChT immediately after binding with the J-aggregates. Upon addition of the J-aggregates, the negative CD bands of ChT at 203 nm shifted to 201 nm along with enhancement of the negative Cotton effect. Meanwhile, the band at 230 nm scarcely changed. The CD spectrum of ChT further changed during the dissociation of the J-aggregates and the CD band finally shifted to 198 nm. The negative CD signal at 198 nm indicates the formation of a random coil structure. The negative band at 230 nm was retained in the final CD spectrum. The negative CD band at 230 nm is characteristic of the Trp 141 that is located close to the His 57 and Ser 195 at the active site of ChT (18, 45). This band has been used as a probe for predicting the enzyme activity of ChT (18, 45). Reduction in the band at 230 nm indicates lowering of the enzyme activity. If the band is not affected by any perturbation, the enzyme activity will be maintained (46). The CD spectral data is
Time-Dependent Enzyme Activity
Figure 8. CD spectra of ChT (1.0 × 10-6 M) before (black) and after the addition (red) of the J-aggregates of H4TPPS2- ([H2TPPS4-] ) 1.5 × 10-5 M) and TMe-β-CD (1.2 × 10-4 M) in phosphate buffer (pH 7.0, 1.0 × 10-2 M) at 25 °C. A spectrum in blue was recorded after complete dissociation of the J-aggregates.
consistent with the fact that the enzyme activity is restored after complete dissociation of the J-aggregates from the surface of ChT.
CONCLUSION The J-aggregates of H4TPPS2- are stabilized by ChT even in neutral aqueous solution. These aggregates cover the active site of the enzyme and completely inhibit the enzyme activity. However, the enzyme activity is restored almost completely with the dissociation of the J-aggregates. The J-aggregates induce a progressive change in enzyme activity from complete inhibition to complete restoration without the aid of a secondary additive. On the basis of the findings of this study, it is possible to develop a “dream drug” with the following features: (1) The drug specifically binds to a target protein. (2) The drug inhibits protein function for a desired time period. (3) The function of the protein is completely restored after the drug has performed its work. (4) The used drug is removed from the body. The present study shows that J-aggregates of H4TPPS2partially fulfill these requirements. The polyanionic J-aggregates selectively bind with a basic protein and do not interact with polyanionic DNA and RNA. The large J-aggregates cover the protein surface and inhibit substrate binding or protein-protein interaction. The J-aggregates spontaneously dissociate to monomers, gradually restoring the protein function. The monomer of H2TPPS4- forms an extremely stable 2:1 inclusion complex with TMe-β-CD that is easily excreted in urine (47). The J-aggregates of H4TPPS2- cannot be actually used as drugs because of difficulty in administration to a living body. However, the present study introduces the concept of preparing a dream drug in the future.
ACKNOWLEDGMENT This study was supported by Grants-in-Aid on Construction of Research Base in Private University from Ministry of Education, Culture, Sports, Science and Technology. Supporting Information Available: Additional experimental data. This material is available free of charge via the Internet at http://pubs.acs.org.
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