Nanoparticulate Glutathione Peroxidase Mimics Based on

Aug 16, 2008 - We synthesized nanoparticulate glutathione peroxidase (GPx) mimics in which ... Glutathione peroxidases (GPxs) are the major antioxidan...
0 downloads 0 Views 383KB Size
Download PDF
Bioconjugate Chem. 2008, 19, 1831–1839

1831

Nanoparticulate Glutathione Peroxidase Mimics Based on Selenocystine-Pullulan Conjugates Mamoru Haratake,* Shinya Matsumoto, Masahiro Ono, and Morio Nakayama* Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan. Received January 4, 2008; Revised Manuscript Received June 23, 2008

We synthesized nanoparticulate glutathione peroxidase (GPx) mimics in which selenocystine (SeCyst) was conjugated to a hydrophilic linear polysaccharide, pullulan (Pul). The SeCyst ester-conjugated Pul derivatives (SeCyst-Pul) in phosphate buffer (pH 7) were treated with a sonicator to spontaneously form particulate materials. Dynamic light scattering measurements revealed that the SeCyst-Pul conjugates could form particulate materials with diameters between 100 and 300 nm. Distinctive endothermic peaks were observed for the SeCyst-Pul aggregate solutions based on a differential scanning calorimetric analysis. The tryptophan (Trp) fluorescence intensity of SeCyst benzyl ester-tryptophanyl-Pul (SeCyst-Bz-Trp-Pul) mostly decreased in comparison to those of the TrpPul (its precursor) and free Trp, which indicates that the Trp residues come close to each other during the aggregation of the conjugates. Formation of SeCyst-Pul aggregates could be induced by the hydrophobic interactions between the SeCyst esters and the amino acid residues on Pul. The GPx-like activity of SeCyst-Bz-Trp-Pul aggregates for the reduction of H2O2 was enhanced nearly 20-fold higher than that of free SeCyst. The double-reciprocal plots of the SeCyst-Bz-Trp-Pul aggregate-catalyzed reduction yielded parallel lines by varying the substrate concentrations, indicating a “ping-pong” mechanism that is similar to those of the natural GPxs. The enhanced GPx activity of the SeCyst-Bz-Trp-Pul aggregate was also supported by higher kinetic parameters, kcat/KmGSH and kcat/KmH2O2. Overall, the enhanced activity of the SeCyst-Bz-Trp-Pul aggregate would be attributed to a hydrophobic environment that was formed at the vicinity of the SeCyst.

INTRODUCTION Glutathione peroxidases (GPxs) are the major antioxidant defense in living systems, which catalyze the reduction of hydrogen peroxide and phospholipid hydroperoxides using reduced glutathione (GSH) (2GSH + R-OOH f GSSG + R-OH + H2O) (1). Selenium-dependent GPxs (GPx-1, GPx-2, GPx-3, GPx-4, and GPx-6), except for GPx-5, contain the selenocysteine (Sec) residue in their catalytic centers (2–4). The Sec residue, which is called the twenty-first amino acid, is encoded by the UGA stop codon that normally functions as a stop codon. The biosynthesis of Sec incorporated in response to the UGA codon is distinctive from the twenty standard amino acids. Translation of the Sec residue is a complicated mechanism which is controlled by the specific stem-loop Sec insertion sequence (SECIS) located in the downstream of the UGA codon and four execution elements [Sec synthase (selA), an elongation factor (selB), seryl-tRNAsec (selC), and selenophosphate synthase (selD)] (5–9). As the Sec insertion into the selenoproteins occurs by such a specific translational control process, at present, it is still quite difficult to express the Sec-containing proteins by the state-of-the-art genetic engineering techniques (10).1 In the X-ray crystal structures of the GPxs, the residues that could be functionally important in the vicinity of the catalytic Sec are some aromatic amino acids [tryptophan (Trp), pheny* To whom correspondence should be addressed. E-mail: haratake@ nagasaki-u.ac.jp for M. Haratake; [email protected] for M. Nakayama. 1 Abbreviations: GPx, glutathione peroxidase; GSH, reduced glutathione; Sec, selenocysteine; SeCyst, D,L-selenocystine; Pul, pullulan; DAN, 2,3-diaminonaphtharene; EEDQ, N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline; DMF, N,N-dimethylformamide; NADPH, nicotinamide adenine dinucleotide phosphate in reduced form; GR, glutathione reductase.

lalanine and tyrosine] and glutamine (Gln) and arginine presumably forming salt bridges and/or hydrogen bonding to the glutathione molecule (11, 12). A common structure around the catalytic center, i.e., the “catalytic triad”, was also found in the X-ray crystal structures of the GPxs, which involves Sec and the adjacent Gln and Trp residues. The reactive selenol group of the Sec residue is thought to be stabilized by the formation of the hydrogen-bonding with the imino group of Trp and/or the carbamoyl group of Gln, and the catalytic triad of these three residues represent a catalytic center, whose integrity is essential for the full catalytic function of the GPxs (13). To explore the catalytic mechanism of natural GPxs and/or apply it to medicine, low mass GPx mimics, such as Ebselen, have been synthesized (14–21). A report by Wilson et al. described that an improved GPx-like activity of diselenide compounds (R-Se-Se-R) resulted from intramolecular interactions of the selenium atom with the nitrogen atom that stabilizes the reaction intermediates (18). Some researchers also suggested that noncovalent interactions between selenium and nitrogen or oxygen atoms increase the GPx-like activity (19, 20). Natural GPxs, except for GPx-4, comprise four identical subunits of 21 kDa that contains one Sec residue as the active center, while most of the GPx mimics previously known was low mass compounds and not yet comparable with the natural GPxs regarding the GPx-like activity. The binding affinity of the substrates in the vicinity of the selenium atom must also be an important element for improving the GPx-like activity. Ren et al. reported that the selenocystine (SeCyst)-conjugated β-cyclodextrin provided the GPx-like activity for the organic cumene hydroperoxide that can be included in the hydrophobic cavity of β-cyclodextrin (16). Sun et al. developed the diselenide-linked 15-mer selenopeptide with a high GPx-like activity by incorporating a specific amino acid sequence for the binding of glutathione (21). However, there are only a few reports on the

10.1021/bc800086z CCC: $40.75  2008 American Chemical Society Published on Web 08/16/2008

1832 Bioconjugate Chem., Vol. 19, No. 9, 2008

GPx mimics that are designed from the viewpoint of the substrate binding. In this study, we synthesized nanosized particulate GPx mimics based on the conjugates of which the selenocystine (SeCyst) as a functional element was attached to the hydrophilic linear polysaccharide, pullulan (Pul). The structural features of the particulate GPx mimics formed by the self-aggregation of the SeCyst-Pul conjugates were characterized by the dynamic light scattering, differential scanning calorimetric, and fluorescence spectroscopic techniques. Their GPx-like activity and steady state kinetics were further examined.

EXPERIMENTAL PROCEDURES Materials. Pullulan (average Mw 73000, Mw/Mn ) 1.54, Pul) used as a conjugate polymer was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Seleno-D,L-cystine (SeCyst) was obtained from Sigma Co., Ltd. (St. Louis, MO) The amino acid derivatives were from either Merck Biosciences AG Novabiochem (La¨ufelfingen, Switzerland) or Kanto Chemical Co., Inc. (Tokyo, Japan). 2,3-Diaminonaphtharene (DAN) and reduced glutathione (GSH) were obtained from Tokyo Chemical Ind. Co., Ltd. (Tokyo, Japan). Nicotinamide adenine dinucleotide phosphate in the reduced form (NADPH) and glutathione reductase (GR) were from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). H2O2, used for the substrate to determine GPxlike activity, was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). An Ultrafree-MC (Millipore Corp., nominal molecular weight cutoff: 5000) was used for ultracentrifugation of the polymer solutions. Water, used for the preparations of the SeCyst-Pul aggregates and their GPx-like activity measurements, was generated by a Milli-Q Biocel system (Millipore Corp., Billerica, MA). All other chemicals were of commercial reagent grade and used as received. Synthesis of Carboxymethylated Pullulan (CM-Pul) (22). Pul (1.5 g) dissolved in a 6 M sodium hydroxide aqueous solution (25 mL) and chloroacetic acid (0.375, 0.75, 1.5, 3, or 4.5 g) were combined at 70 °C, and allowed to react for 2 h with stirring. The reaction mixture was neutralized with a 1 M acetic acid aqueous solution, and then dialyzed against deionized water for 24 h. The dialysate was poured into strong cationexchange resins (DIAION SK1B, Mitsubishi Chemical Co., Tokyo, Japan) in the free form to transform it from sodium form to free form, and then were subjected to lyophilization (CMPul, yield: 57.5%). CM-Pul (DS ) 0.44) - IR (ATR, cm-1): 3354 (O-H stretching), 1734 (CdO stretching), 1026 (C-O-C bending). 1H NMR (DMSO-d6): δ 4.42 (carboxymethyl H), 2.56-4.58 (glucose 2H, 3H, 4H, 5H, and 6H), 4.62 (glucose 1H (1f4)), 5.12 (glucose 1H (1f6)). The degree of substitution of the carboxymethyl group in CMPul (DS) was estimated by pH titration; an appropriate amount of CM-Pul in free form (50-100 mg, Pul-CH2COOH) dissolved in 10 mL of a 0.1 M sodium hydroxide aqueous solution was titrated with a 0.1 M HCl aqueous solution. The temperature of the titration vessels was maintained at 30 °C throughout each titration by a circulator RTE-111 (NESLAB Instruments, Inc., Newington, NH). The pH-metric titrations were performed on a titrator DL25 (Mettler-Toledo, Inc., Columbus, OH). The DS values were calculated from the difference between VS and VB using the following equation (eq 1); where F is the titer of the 0.1 M HCl, VB is the volume of the titrant consumed for the sample-free blank (mL), VS is the volume of the titrant consumed for the samples (mL), W is the sample weight used for the titration (mg), 162 is the molar mass of a glucose unit in Pul (C6H10O5), and 59 is the molar mass of carboxymethyl group (C2H3O2). One milliliter of 0.1 M sodium hydroxide corresponds to 0.1 mmol carboxymethyl group in this titration.

Haratake et al.

DS ) 162 × 0.1 × F × (VB - VS) ⁄ [W - 58 × 0.1 × F × (VB - VS)] (1) Synthesis of SeCyst Esters. The SeCyst esters were synthesized according to the procedures reported by Bondanszky (23) as follows: For the SeCyst alkyl esters, a mixture of SeCyst (30 mg) and p-toluenesulfonic acid (300 mg) dissolved in 10 mL of absolute methanol (ethanol or isopropanol) was refluxed for 40 h. After the removal of the alcohol, the resultant was left for 24 h in diethyl ether. The obtained solid materials were washed several times with diethyl ether, and dried in Vacuo. For the synthesis of the SeCyst benzyl ester (SeCyst-Bz), SeCyst (30 mg), p-toluenesulfonic acid (300 mg) and benzyl alcohol (7.5 mL) were mixed in benzene (15 mL), and refluxed for 3 h. After removal of the benzene, the residual material was left in a 1:1 mixture of petroleum ether and diethyl ether (100 mL) for 24 h. The obtained solid material was left in diethyl ether for another 24 h (yield: 75.3%). IR (ATR, cm-1): SeCyst-Me 1702 (ester CdO stretching), SeCyst-Et 1712 (ester CdO stretching), SeCyst-iPro 1710 (ester CdO stretching), SeCystBz 1716 (ester CdO stretching). 1H NMR (CD3OD): SeCystMe; δ 2.37 (s, 3H), 3.29 (s, 3H), 3.30-3.31 (m, 2H), 3.85 (s, 3H), 4.40 (m, 1H), 7.22-7.25 (d, 2H), 7.69-7.72 (d, 2H), SeCyst-Et; δ 1.30 (m, 3H), 2.37 (s, 3H), 3.40 (m, 2H), 4.29 (q, 2H), 4.32 (m, 1H), 7.25 (d, 2H), 7.70 (d, 2H), SeCyst-iPro; δ 1.32 (d, 6H), 2.37 (s, 3H), 3.20-3.31 (m, 2H), 4.35 (m, 1H), 5.08-5.19 (q, 1H), 7.23 (d, 2H), 7.70 (d, 2H). SeCyst-Bz; δ 2.36 (s, 3H), 3.29 (m, 1H), 4.46 (m, 1H), 5.27 (s, 2H), 7.22 (d, 2H), 7.36 (m, 5H), 7.70 (d, 2H). Conjugation of SeCyst Esters and Amino Acids to CM-Puls. CM-Pul (50 mg) and amino acid methyl ester (50 mg) were dissolved in 10 mL of deionized water. Triethylamine and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 100 mg) dissolved in N,N-dimethylformamide (DMF, 10 mL) were sequentially added to the mixture, and stirred for 2 h at room temperature. After the dialysis against deionized water for 24 h, an equal volume mixture of the dialysate and a 2 M sodium hydroxide aqueous solution was stirred for another 24 h at room temperature to remove any of the unreacted amino acid esters. The resulting solution was dialyzed against deionized water, followed by lyophilization (white powder, yield: 55.1%). The degree of introduction of the Trp and Tyr residues in Pul was estimated by measuring the intrinsic absorbance at 280 nm. His residue (24) was also spectrophotometrically estimated by measuring the absorbance at 240 nm using diethyl bicarbonate. CM-Pul or Trp-Pul was combined with the SeCyst esters in 10 mL deionized water in a flask, and then an appropriate amount of triethylamine and EEDQ dissolved in 10 mL DMF was added to the flask. The mixture was allowed to react for 2 h with stirring at room temperature. The resultant material was dialyzed against deionized water for 24 h, and then subjected to lyophilization (pale yellow powder, yield: 69.3%). All the SeCyst-Pul conjugates were stored in a desiccator at ambient temperature until use. SeCyst-Me-Pul (DS ) 0.44) IR (ATR, cm-1): 3341 (O-H stretching), 1715 (ester CdO stretching), 1650 (amide CdO stretching), 1522 (amide N-H bending), 1038 (C-O-C bending). 1H NMR (DMSO-d6): δ 2.37-2.58, 7.30-7.72 (SeCyst-Me H), 2.60-4.60 (glucose 2H, 3H, 4H, 5H, and 6H), 4.62 (glucose 1H (1f4)), 5.10 (glucose 1H (1f6)). SeCyst-Me-Trp-Pul (DS ) 0.44) - IR (ATR, cm-1): 3353 (O-H stretching), 1706 (ester CdO stretching), 1642 (amide CdO stretching), 1541 (amide N-H bending), 1050 (C-O-C bending). 1H NMR (DMSO-d6): δ 2.37-2.58, 7.30-7.72 (SeCyst-Me H), 2.60-4.60 (glucose 2H, 3H, 4H, 5H, and 6H), 4.62 (glucose 1H (1f4)), 5.10 (glucose 1H (1f6)), 6.80-7.23 (tryptophan imidazole H). SeCyst-Bz-TrpPul (DS ) 0.36) - IR (ATR, cm-1): 3350 (O-H stretching), 1716 (ester CdO stretching), 1650 (amide CdO stretching),

Nanoparticulate Glutathione Peroxidase Mimics

1540 (amide N-H bending), 1030 (C-O-C bending), 1H NMR (DMSO-d6): δ 2.37-2.58, 7.30-7.72 (SeCyst-Bz H), 2.58-4.60 (glucose 2H, 3H, 4H, 5H, and 6H), 4.52 (glucose 1H (1f4)), 5.12 (glucose 1H (1f6)), 6.71-7.30 (tryptophan imidazole H). Determination of Selenium Concentration. The selenium concentrations in the sample solutions were fluorometrically determined using DAN after digestion using a 1:4 mixture of HClO4 and HNO3 (25). Dynamic Light Scattering (DLS) Measurements. The particle diameter and distribution of the SeCyst-Pul aggregates were measured by a DLS technique. The DLS measurements were carried out at 25 °C using a Zetasizer Nano ZS (Malvern Instruments, Worcs, UK) at 90° to the incident beam (a wavelength 633 nm from a 4 mW He-Ne laser tube) in a 1 cm length quartz cuvette. The SeCyst-Pul conjugates dispersed in 0.05 M phosphate buffer (pH 7) (0.2 mg/mL) were treated by a Sonifier 250D probe-type sonicator (Branson Danburg, CT) at 120 W for 5 min in an ice bath 24 h before the measurements. Data fitting was performed using a multimodal algorithm supplied by Malvern Instruments. The collected correlelograms were fitted to diffusion coefficients and converted to a hydrodynamic diameter using the Einstein-Stokes equation. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) was performed using an MC-2 ultrasensitive scanning calorimeter (Microcal, Inc., Amherst, MA). The MC-2 contains a fixed pair of matched tantalum cells (cell volume 1.2 mL), which are separately filled with the sample and the reference solution. The SeCyst-Pul aggregate solution samples (0.01 wt %) were injected into the sample cell and the reference cell was filled with 0.05 M phosphate buffer (pH 7). A nitrogen cylinder normally set at 4 kg/cm maintained pressure in the cells to suppress the formation of air bubbles during scanning. Temperature scans were performed at the rate of 1 °C/min and in the range 30-100 °C. The baseline was drawn with the help of ORIGIN software supplied by Microcal, Inc. The DSC thermograms of the pullulan in 0.05 M phosphate buffer (pH 7) were subtracted from those of the SeCyst-Pul aggregates. Determination of GPx-Like Activity (18, 26). The SeCystPul aggregate solution (final selenium concentration: 6.25 µM) was sequentially combined with ethylenediaminetetraacetic acid (EDTA, 0.63 mM), sodium azide (0.63 mM), glutathione reductase solution (0.63 unit), reduced glutathione solution (0.63 mM), and NADPH solution (2.34 mM) in 0.05 M phosphate buffer (pH 7). The reaction was initiated by the addition of H2O2 solutions (3.14 mM). Absorbance at 340 nm due to the NADPH was recorded every 10 s just after mixing by inversion. The GPx-like activity was calculated using the following equation (eq 2) as µmoles NADPH oxidized per minute, where ∆ASMP is the decrease in absorbance at 340 nm of sample solutions between 10 and 70 s after addition of the substrates, ∆ABLK is the decrease in absorbance at 340 nm per minute of the solutions without the SeCyst-Pul conjugates, εmM is the extinction coefficient for 1 mM NADPH solution [6.22/(mM · cm)], and c is final selenium concentration (µM). GPx-like activity ) (∆ASMP - ∆ABLK) × 1000 ⁄ εmM ⁄ c (2) Kinetic Study of SeCyst-Pul Aggregate-Catalyzed Reduction. The initial reaction rate of the SeCyst-Pul aggregates was determined by varying the H2O2 and GSH concentrations: at several concentrations of one substrate while the concentration of the other substrate remained the same (H2O2 concentrations, 0.25, 0.5, and 1.0 mM; GSH concentrations, 0.2, 0.4, 0.6, and 1.0 mM). The other reaction conditions and operating procedures were the same as those described above. By the doublereciprocal plotting (1/[GSH] versus 1/v), the kinetic parameters were calculated from the following equation (eq 3) (27).

Bioconjugate Chem., Vol. 19, No. 9, 2008 1833

(

)

GSH H2O2 1 1 Km 1 [H2O2] + Km ) + (3) ν [GSH] Vmax Vmax [H2O2] where [H2O2] and [GSH] are the concentrations of hydrogen peroxide and glutathione, respectively, KmS is the Michaelis constant for substrate S, Vmax ) kcat[E0], kcat, and [E0] are a general rate constant and a total concentration of selenium, respectively. Statistical Analysis. All data were presented as mean ( standard deviation (n ) 5 or more). Statistical analyses were performed using PRISM 4 (GraphPad Software Inc., San Diego, CA). Multiple mean values were compared by a two-way ANOVA with a Bonferroni posthoc test with the treatment and selenium concentration in the diets as factors. Comparisons were considered statistically significant at P < 0.05.

RESULTS AND DISCUSSION Synthesis of SeCyst-Pul Conjugates (Scheme 1). Natural GPxs have a Sec residue in their active center. The direct Sec introduction to polymers is quite difficult, because of its poor chemical stability. In this study, SeCyst (Sec-Se-Se-Sec) was used as a catalytic site to be attached to the polymers. Pullulan (Pul, average Mw 73 000) was employed as a polymer material to improve the chemical stability and the solubility of SeCyst in an aqueous medium. The Pul molecule is a hydrophilic linear polysaccharide consisting of a maltotriose repeating unit in which three glucose molecules are connected by an R-1,4 glycosidic bond. Pul molecule was first carboxymethylated in order to sequentially attach the SeCyst ester moieties through an amide linkage (CM-Pul). As the highly substituted SeCystPul conjugates were thought to become water-insoluble due to the poor solubility of SeCyst, the CM-Pul derivatives with less than a 0.5 substitution degree of the carboxymethyl group per glucose unit of Pul were employed for the further chemical modifications. We tried to examine the substituent distribution of the carboxymethyl group in the CM-Pul by NMR spectroscopy, but it could not be clearly detected probably due to the low degree of substitution. It seems common knowledge that the primary alcohol at C-6 (OH-6) is the most reactive hydroxyl group of glucose in most substitution reactions. However, Glinel et al. recently reported that the reactivity order of hydroxyl groups in the carboxymethylation of Pul was found to be OH-2 > OH-4 > OH-6 > OH-3 using the hydrolyzed pullulan fragment (28). As the CM-Pul used in this study was also thought to be substituted by carboxymethyl groups at several positions of the hydroxyl groups, the same batches of the CMPul were used for the subsequent conjugation reactions. The free SeCyst was decomposed to the red elemental selenium in both the solid and aqueous solution states. However, the conjugation of the SeCyst esters to Pul fairly improved their stability; when they were exposed to sonication and/or heating, no elemental selenium was observed throughout the experiments and during storage. The selenium contents of the SeCyst-Pul conjugates increased with an increase in the degree of substitution of the carboxymethyl group (Table 1). When the SeCystPul conjugate ethanolic aqueous solutions were ultrafiltered through a 5000 molecular weight cutoff filter, the selenium amount in the filtrates was less than 1% of the total selenium amount listed in Table 1. Thus, the SeCyst esters noncovalently attached to Pul were thought to be a negligible amount in the conjugates. In addition, the residual carboxymethyl groups in the SeCyst-Pul conjugates could hardly be detected. The selenium contents of SeCyst-Me-His-Pul, SeCyst-Me-Tyr-Pul, SeCyst-Me-Trp-Pul, and SeCyst-Bz-Trp-Pul conjugates were almost consistent with those that were calculated from the contents of the amino acids in the respective precursors (97-105%), indicating that SeCyst and these amino acids were introduced into these conjugates in equal molar ratios.

1834 Bioconjugate Chem., Vol. 19, No. 9, 2008 Scheme 1. Synthetic Route of SeCyst-Pul Conjugates

Haratake et al.

Nanoparticulate Glutathione Peroxidase Mimics

Bioconjugate Chem., Vol. 19, No. 9, 2008 1835

Table 1. Degree of Substitution of Carboxymethyl Group in CM-Pul and Selenium Content of SeCyst-Pul Conjugates

Conjugate SeCyst-Me-Pul SeCyst-Et-Pul SeCyst-iPr-Pul SeCyst-Bz-Pul SeCyst-Me-His-Pul SeCyst-Me-Leu-Pul SeCyst-Me-Tyr-Pul SeCyst-Me-Trp-Pul SeCyst-Bz-Trp-Pul SeCyst-Me-His-Trp-Pul SeCyst-Me-Leu-Trp-Pul SeCyst-Me-Tyr-Trp-Pul

Degree of substitution of CM group (/glucose unit of Pul)

Selenium content (mg/g conjugate)

0.06 0.21 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.36 0.36 0.36 0.36

12.4 ( 0.3 45.4 ( 0.7 79.7 ( 0.5 82.2 ( 1.8 70.9 ( 0.9 79.1 ( 1.1 44.1 ( 0.3 57.9 ( 1.1 56.4 ( 1.9 49.7 ( 0.7 56.6 ( 0.7 49.9 ( 1.5 56.2 ( 0.7 45.2 ( 2.7

The SeCyst esters have two chemically equivalent primary amino groups at both ends. Both amino groups could be possibly reacted intra- and/or intermolecularly with the carboxymethyl groups of the CM-Puls. To obtain structural information on the SeCyst-Pul conjugates, the particle diameters of the SeCystPul conjugates were measured by the DLS technique. The mean particle diameters of SeCyst-Me-Pul (DS ) 0.44), SeCyst-MeTrp-Pul, and SeCyst-Bz-Trp-Pul conjugates were almost comparable to each other, and were close to that of Pul (Table 2 and Figure 1). The particle diameter distributions of these conjugates were also similar to that of Pul. The diselenide linkage (R-Se-Se-R) can react with free thiol compounds (R′SH) to form selenosulfide (R-Se-S-R′). When the SeCyst-Pul conjugates are treated with an excess molar amount of glutathione (GSH), the Sec fragment (Sec-Se-SG) could be released from the SeCyst esters bound to Pul through its one end amino group. Practically, such low molecular mass selenium-containing fragments were not released from the SeCyst-Pul conjugates. Overall, the reaction of the CM-Pul with the bifunctional SeCyst esters appears to mainly result in intramolecular substitution rather than intermolecular linkage. Aggregation of SeCyst-Pul Conjugates. The SeCyst esters have hydrophobic characteristics, and hydrophobic amino acids were also introduced to a hydrophilic Pul molecule. As the SeCyst-Pul conjugates have amphiphilic structure elements within the molecules, these conjugates could possibly form self-

Figure 1. Particle diameter distribution of Pul (A), SeCyst-Me-Pul conjugate (B), SeCyst-Me-Trp-Pul conjugate (C), SeCyst-Bz-TrpPul conjugate (D), SeCyst-Me-Pul aggregate (E), SeCyst-Me-TrpPul aggregate (F), and SeCyst-Bz-Trp-Pul aggregate (G). Degree of substitution of carboxymethyl group for SeCyst-Me-Pul conjugate: 0.44.

Table 2. Particle Diameter of SeCyst-Pul Aggregates and Conjugates

Aggregate/conjugate SeCyst-Me-Pul aggregate

SeCyst-Et-Pul aggregate SeCyst-iPr-Pul aggregate SeCyst-Bz-Pul aggregate SeCyst-Me-His-Pul aggregate SeCyst-Me-Leu-Pul aggregate SeCyst-Me-Tyr-Pul aggregate SeCyst-Me-Trp-Pul aggregate SeCyst-Bz-Trp-Pul aggregate SeCyst-Me-His-Trp-Pul aggregate SeCyst-Me-Leu-Trp-Pul aggregate SeCyst-Me-Tyr-Trp-Pul aggregate SeCyst-Me-Pul conjugate SeCyst-Me-Trp-Pul conjugate SeCyst-Bz-Trp-Pul conjugate Pul

Degree of substitution of CM group Particle (/glucose unit of Pul) diameter (nm) 0.06 0.21 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.36 0.36 0.36 0.36 0.44 0.44 0.36 0.00

446.0 ( 25.6 602.4 ( 16.8 106.5 ( 0.9 211.1 ( 5.7 118.9 ( 1.2 175.8 ( 0.9 151.1 ( 2.2 240.9 ( 3.0 189.9 ( 3.5 162.8 ( 1.3 253.1 ( 1.6 194.2 ( 18.4 185.7 ( 0.7 128.5 ( 3.6 18.9 ( 0.8 15.8 ( 2.8 15.6 ( 1.2 13.3 ( 1.0

aggregates. The aggregation behavior of the SeCyst-Pul conjugates in an aqueous medium was preliminarily checked by the static light scattering method (Supporting Information Figure S1). Scattered lights were observed from all the tested SeCystPul conjugate solutions, and their intensities were apparently greater than that from the unmodified Pul solution. In comparison to the SeCyst-Me-Pul conjugates with different selenium contents, the scattered light intensity increased with the increasing selenium content (≈ degree of substitution of SeCyst ester moiety). The SeCyst-Me-Trp-Pul and SeCyst-Bz-Trp-Pul conjugates provided higher scattered light intensities than the SeCyst-Me-Pul conjugate. These facts suggest the presence of particulate materials that were formed by the self-aggregation of the SeCyst-Pul conjugates. The SeCyst-Pul aggregates were characterized by the DLS technique (Table 2). Pul used as the starting material had a mean diameter of 13.3 nm, which indicates that Pul without any chemical modifications does not form self-aggregates. The mean particle diameters of the SeCyst-Pul aggregates were in the range from 100 to 300 nm, which were apparently greater than that of Pul. The particle diameters of the SeCyst-Me-Pul conjugates tended to decrease with an increase in the degree of substitution of the SeCyst ester moiety. The mean particle diameters of the SeCyst-Me-Pul, SeCyst-Me-Trp-Pul, and SeCyst-Bz-Trp-Pul aggregates also became greater than those of the corresponding conjugates that are dissolved in a molecularly dispersed state. The particle diameter distributions of the SeCyst-Me-Pul, SeCyst-Me-Trp-Pul, and SeCyst-Bz-Trp-Pul aggregates were evidently shifted to larger diameter ranges than those of Pul and these conjugates (Figure 1). In general, the morphology of the stable aggregate of the amphiphiles depends on their molecular orientation and freedom; the key factors are to control the hydrophile-lipophile balance, the hydrophobic interactions and dispersion of the hydrophilic moiety in the aqueous medium for formation of the structured self-aggregates (29, 30). The SeCyst-Me-Pul conjugates with a low DS produced larger aggregates with diameters of 400-600 nm, while the SeCystBz-Pul conjugate with a high DS and benzyl group and amino acid residue produced smaller aggregate diameters (100-300 nm). Self-aggregation of the SeCyst-Pul conjugates is dependent on the degree of introduction of the SeCyst and its chemical structures, although no unambiguous relation between the hydrophobicity of the substituents and the size of the formed aggregates was found. Since an intramolecular linkage of Pul molecules through the SeCyst esters could be cleaved by GSH treatment, the treatment of the SeCyst-Pul aggregates with GSH may give rise to

1836 Bioconjugate Chem., Vol. 19, No. 9, 2008

Haratake et al.

Table 3. Changes in Particle Diameter of SeCyst-Puls Aggregates after the Glutathione Treatmenta

Aggregate

Degree of substitution of CM group (/glucose unit of Pul)

SeCyst-Me-Pul SeCyst-Bz-Pul SeCyst-Me-Trp-Pul SeCyst-Bz-Trp-Pul

0.44 0.44 0.44 0.36

a

Particle diameter (nm) Before

After

106.5 ( 0.9 175.8 ( 0.9 162.8 ( 1.3 253.1 ( 1.6

108.6 ( 0.4 184.8 ( 8.8 154.2 ( 1.9 272.1 ( 3.8

Aggregate solutions were treated with 2 mM GSH at 37 °C for 30

min.

Figure 2. Tryptophan fluorescence spectra of free Trp (A), Trp-Pul (B), SeCyst-Me-Trp-Pul aggregate (C), and SeCyst-Bz-Trp-Pul aggregate (D). Trp concentration: 60 µM (*Trp concentrations were adjusted by UV absorbance at 280 nm). Excitation wavelength: 280 nm.

structural changes in the SeCyst-Pul aggregates. The particle diameters of the SeCyst-Pul aggregates were compared before and after the treatment with an excess amount of GSH. No remarkable changes in the particle diameters were found for the SeCyst-Me-Pul, SeCyst-Bz-Pul, SeCyst-Me-Trp-Pul, and SeCyst-Bz-Trp-Pul aggregates after the GSH treatment (Table 3). Therefore, the intramolecular linkage of Pul through amino groups at both ends of the SeCyst esters does not appear to be a dominant structural element for formation of the nanosized aggregates. Consequently, the structures of the SeCyst-Pul aggregates are likely to involve noncovalent interactions such as the hydrophobic interactions between the SeCyst esters and amino acid residues rather than the diselenide linkage. To obtain information on the structures of the SeCyst-Pul aggregates from another viewpoint, the Trp intrinsic fluorescence spectra of the SeCyst-Trp-Pul conjugates were compared to the free Trp and their precursor (Trp-Pul). Self-aggregation in aqueous medium would transfer Trp and SeCyst moieties to a hydrophobic environment. The fluorescence intensity of the SeCyst-Me-Trp-Pul aggregate mostly decreased as compared to those of the free Trp and its precursor, Trp-Pul (Figure 2). Furthermore, aggregation of SeCyst-Bz-Trp-Pul almost quenched its Trp fluorescence, demonstrating that the SeCyst-Bz-Trp moieties in Pul are associated with each other in the formed aggregates. Independently, the SeCyst-Me-Pul, SeCyst-Me-TrpPul, and SeCyst-Bz-Trp-Pul aggregates were prepared in the presence of a fluorescent 8-anilino-1-naphthalene sulfonic acid (ANS), and the fluorescence spectra of ANS were measured at the excitation wavelength of 380 nm. The Trp-containing SeCyst-Me-Trp-Pul and SeCyst-Bz-Trp-Pul aggregates provided a 60 nm blue shift in their fluorescence spectra (Supporting Information Figure S2). Consequently, self-aggregation of the SeCyst-Pul conjugates appears to be driven by both intramolecular chemical linking of Pul with SeCyst and the hydrophobic

Figure 3. DSC thermograms of SeCyst-Me-Pul aggregate (A) and SeCyst-Bz-Trp-Pul aggregate (B). *Degree of substitution of carboxymethyl group for SeCyst-Me-Pul conjugate: 0.44. Heating rate: 1 °C/ min. The broken lines in panel (B) were drawn by ORIGIN software.

interactions that occur between the SeCyst side groups and amino acid residues. The SeCyst-Pul aggregates were further characterized by a DSC analysis. When supplying heat energy to the aggregates or assemblies (e.g., lipid membranes) that are formed by noncovalent interactions, an enthalpy change due to the decomposition (disassembly) of the aggregates would be observed. Cholesterol-conjugated pullulan (CHP) can spontaneously form hydrogel aggregates in an aqueous medium with diameters ranging between 20 and 30 nm (31–34). The CHP self-aggregates contain hydrophobic domains that are formed by the noncovalent hydrophobic interactions among the cholesterol moieties. When the CHP aggregates were subjected to the DSC analysis, a characteristic endothermic peak was observed in the temperature range between 60 and 80 °C (Supporting Information Figure S3). This is due to the dissociation of the cholesterol hydrophobic domains in the CHP hydrogel aggregates. SeCyst-Me-Pul and SeCyst-Bz-Trp-Pul (DS of carboxymethyl group: 43.78%) also gave similar endothermic peaks at 60-80 °C (Figure 3). The observed thermal behavior resulted from the hydrophobic domains structured in the SeCyst-Pul aggregates. The Trp-containing SeCyst-Pul aggregates such as SeCyst-Me-Trp-Pul and SeCystBz-Trp-Pul also produced other endothermic peaks at different temperature ranging from 50 to 70 °C. The appearance of these endothermic peaks suggests that Trp residue-associated hydrophobic interactions occur in the Trp-containing SeCyst-Pul aggregates.

Nanoparticulate Glutathione Peroxidase Mimics

Bioconjugate Chem., Vol. 19, No. 9, 2008 1837

Figure 4. GPx-like activity of SeCyst-Pul aggregates for hydrogen peroxide. #: Degree of substitution of carboxymethyl group. * and **: significantly different from SeCyst at P < 0.05 and P < 0.01, respectively.

GPx-like Activity and Steady State Kinetics of SeCystPul Aggregates. The GPx-like activity of the SeCyst-Pul aggregates was determined according to the conventional procedure, in which oxidation of NADPH is spectrophotometrically monitored by coupling to the reduction of the oxidized glutathione (GSSG) catalyzed by glutathione reductase. The GPx-like activity was compared among three SeCyst-Me-Pul aggregates with different degrees of SeCyst methyl ester loading. There was no significant difference in the activity among the three aggregates, and their activities were almost comparable to that of the free SeCyst (Figure 4). The SeCyst-Me-Pul, SeCyst-Et-Pul, and SeCyst-iPr-Pul aggregates showed 2-fold higher activities than the free SeCyst, while the SeCyst-Bz-Pul aggregate provided 3-fold higher activity. The activities of the amino acid-inserted SeCyst-Me-His-Pul, SeCyst-Me-Tyr-Pul, and SeCyst-Me-Leu-Pul aggregates were almost twice that of the free SeCyst, whereas the Trp-containing SeCyst-Me-TrpPul aggregate was more effective than the other three. When SeCyst was attached to Pul through a dipeptide linkage consisting of Trp and His, Tyr, or Leu, no remarkable enhancement in the activity was also observed for SeCyst-Me-His-TrpPul, SeCyst-Me-Tyr-Trp-Pul, and SeCyst-Me-Leu-Trp-Pul. On the basis of these results, the benzyl side groups of the SeCyst and Trp residue appear to be good substituents for enhancing the GPx-like activity in our system. The SeCyst benzyl ester and Trp residue were, therefore, introduced to Pul. The SeCystBz-Trp-Pul aggregate synergistically exhibited nearly a 20-fold higher GPx-like activity than the free SeCyst. The enhanced activity of the SeCyst-Bz-Trp-Pul aggregate was not inhibited by the radical trap 2,6-di-tert-butyl-4-methylphenol, which suggests that this aggregate catalyzes the reduction of H2O2 by the SeCyst moiety via a nonradical mechanism (35). The steady state kinetics of the SeCyst-Bz-Trp-Pul aggregatecatalyzed reduction was analyzed using the double-reciprocal equation with varying concentrations of both H2O2 and GSH (Figure 5). The double-reciprocal plots (1/[GSH] versus 1/v) of the SeCyst-Bz-Trp-Pul aggregate-catalyzed reduction yielded parallel straight lines for three different H2O2 concentrations, suggesting that its reaction pathway is a “ping-pong” (double-

Figure 5. Double-reciprocal plots for SeCyst-Me-Pul (A) and SeCystBz-Trp-Pul (B) aggregate-catalyzed H2O2 reduction. Selenium concentrations in SeCyst-Me-Pul and SeCyst-Bz-Trp-Pul aggregate samples: 43.6 and 2.1 µM. [H2O2] ) 0.25 (2), 0.5 (9), and 1 mM ([). Table 4. Kinetic Parameters of SeCyst-Me-Pul and SeCyst-Bz-Trp-Pul Aggregate-Catalyzed H2O2 Reduction Km (mM)

kcat/Km [1/(mM · min)]

Aggregate

kcat(/min)

GSH

H2O2

GSH

H2O2

SeCyst-Me-Pula SeCyst-Bz-Trp-Pul

4.12 49.26

2.04 0.76

4.64 3.76

2.02 64.82

0.82 13.10

a Degree of substitution of carboxymethyl group for SeCyst-Me-Pul conjugate: 0.44.

displacement) reaction pathway analogous to those of the natural GPxs. A similar result was also obtained for the SeCyst-MePul aggregate. The kinetic parameters of the two aggregates were calculated from the slope and Y-axis intercept of the double-reciprocal plots. The apparent reduction rate constant, kcat, for the SeCyst-Bz-Trp-Pul was greater than that for SeCystMe-Pul (Table 4). This kinetic advantage can be supportive evidence for the enhanced GPx-like activity of the SeCyst-BzTrp-Pul aggregate in comparison to that of the SeCyst-Me-Pul aggregate. In addition, the SeCyst-Bz-Trp-Pul aggregate provided a higher binding affinity for GSH (KmGSH) and H2O2 (KmH2O2), as compared to the SeCyst-Me-Pul aggregate. Taken together with the characterization data of the SeCyst-Bz-TrpPul aggregate, its activity enhancement would be attributed to the hydrophobic environments that were formed together with the benzyl group and Trp residue in the vicinity of the SeCyst moiety. The GPx-like activity of SeCyst-Bz-Trp-Pul aggregate for organic tert-butylhydroperoxide (t-BuOOH) as a substrate was also examined (Supporting Information Figure S4). SeCyst-BzTrp-Pul aggregate did not provide enhancements in the activity

1838 Bioconjugate Chem., Vol. 19, No. 9, 2008

for this substrate as much as that for hydrogen peroxide (nearly a 4-fold increase). A previous paper by Ren et al. (16) reported that SeCyst bound to the primary side of β-cyclodextrin (βCD) via the two amino groups resulted in enhanced GPx-like activity for t-BuOOH and cumene hydroperoxide (CuOOH), which is ascribed to the inclusion interactions of these substrates into the hydrophobic cavity of β-CD. Although the SeCyst moieties in SeCyst-Bz-Trp-Pul aggregate exist in hydrophobic environments, t-BuOOH may be less accessible to the SeCyst moieties than hydrogen peroxide due to hydrophilic surrounds by Pul molecules. Consequently, the hydrophobic regions that are formed in the SeCyst-Bz-Trp-Pul aggregate do not seem to provide binding sites for such an organic substrate. Actually, selenium-dependent native GPxs possess the substrate specificity; GPx-1 is active for inorganic hydrogen peroxide, but not for organic phospholipid hydroperoxides that are substrates of GPx-4 (36). In conclusion, we synthesized nanoparticulate glutathione peroxidase mimics based on the SeCyst esters-attached Pul conjugates. The chemical stability of SeCyst in an aqueous medium was somewhat improved by attachment to the Pul polymer. SeCyst-Pul conjugates were spontaneously formed into self-aggregates with diameters of several hundred nanometers. The results of the physicochemical characterization suggest the formation of hydrophobic microenvironments in the vicinity of SeCyst in the SeCyst-Pul aggregates. In comparison to the free SeCyst, improvements in the activity of the H2O2 reduction were observed for all the SeCyst-Pul aggregates. Even better, the most effective SeCyst-Bz-Trp-Pul aggregate produced a nearly 20fold activity enhancement. The double-reciprocal plot analysis of the SeCyst-Bz-Trp-Pul aggregate-catalyzed reduction revealed that the catalytic mechanism of SeCyst-Bz-Trp-Pul aggregate was analogous to those of the natural GPxs (a “ping-pong” reaction pathway), and the kinetic parameters of the SeCystBz-Trp-Pul aggregate were improved. Consequently, several features of our nanosized aggregate system were found to be different from the low mass GPx-mimics: (i) to improve the water solubility and stability of the liable selenium compounds in order to function as the active site; (ii) to be capable of forming a hydrophobic environment in the vicinity of the selenium compound; and (iii) to concentrate the local selenium compound in the self-aggregates. We are now designing more efficacious SeCyst-Pul aggregates with GPx-like activity. In addition, we will try to apply these aggregates to medicine as a particulate antioxidant material. Supporting Information Available: Scattered light intensity of SeCyst-Pul conjugates (Figure S1), fluorescence spectra of SeCyst-Bz-Trp-Pul and cholesterol-conjugated Pul aggregates prepared in the presence of ANS and ANS alone (Figure S2), DSC thermogram of cholesterol-conjugated Pul self-aggregate (Figure S3), GPx-like activity of SeCyst-Pul aggregates for tert-butylhydroperoxide (t-BuOOH) (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Rayman, M. R. (2000) The importance of selenium to human health. Lancet 356, 233–241. (2) Chu, F. F. (1994) The human glutathione peroxidase genes GPX2, GPX3, and GPX4 map to chromosomes 14, 5 and 19, respectively. Cytogenet. Cell Genet. 66, 96–98. (3) Drevet, J. R. (2006) The antioxidant peroxidase family and spermatozoa: A complex story. Mol. Cell. Endoclinol. 250, 70– 79. (4) Singh, A. K., and Shichi, H. (1998) A novel glutathione peroxidase in bovine eye. J. Biol. Chem. 273, 26171–26178.

Haratake et al. (5) Berry, M. J., Banu, L., Chen, Y., Mandel, S. K., Kieffer, J. D., Harney, J. W., and Larsen, P. R. (1991) Recognition of UGA as a Sec codon in Type I deiodinase requires sequences in the 3′ untranslated region. Nature 353, 273–276. (6) Bock, A. (2000) Biocynthesis of selenoproteins. BioFactors 11, 77–78. (7) Atokins, F. J., and Gesteland, F. R. (2000) The twenty-first amino acid. Nature 407, 463–464. (8) Hatfield, L. D., and Gradyshev, N. V. (2002) How selenium has altered our understanding of the genetic code. Mol. Cell. Biol. 22, 3565–3576. (9) Mizutani, T., and Fujiwara, T. (2000) SBP, SECIS binding protein, binds to the RNA fragment upstream of the Sec UGA codon in glutathione peroxidase mRNA. Mol. Cell. Reports 27, 99–105. (10) Hendrickson, T. L. (2007) Easing selenocysteine into proteins. Nat. Struct. Mol. Biol. 14, 100–101. (11) Epp, O., Ladenstein, R., and Wendel, A. (1983) The refined structure of the seleno-enzyme glutathione peroxidase at 0.2nm resolution. Eur. J. Biochem. 133, 51–69. (12) Ren, B., Huang, W., Akesson, B., and Ladenstein, R. (1997) The crystal structure of seleno- glutathione peroxidase from human plasma at 2.9-angstrom resolution. J. Mol. Biol. 268, 869– 885. (13) Maiorino, M., Aumann, D. K., Flohe, B. R., Doria, D., McCarthy, J., Roveri, A., Ursini, F., and Flohe, L. (1995) Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide gulutathione peroxidase (PHGPx). Biol. Chem. Hoppe-Seyler 376, 651–660. (14) Klayman, D. L., and Griffin, S. T. (1973) Reaction of selenium with sodium borohydride in protic solvents. A facile method for the introduction of selenium into organic molecules. J. Am. Chem. Soc. 95, 197–199. (15) Luo, G., Ren, J., Liu, J., Mu, Y., and Shen, J. (2003) Towards more efficient glutathione peroxidase mimics: Substrate recognition and catalytic group assembly. Curr. Med. Chem. 10, 1151– 1183. (16) Ren, X., Liu, J. Q., Luo, G. M., Zhang, Y., Luo, Y., Yan, G., and Shen, J. (2000) A novel selenocystine-β-cyclodextrin conjugate that acts as a glutathione peroxidase mimic. Bioconjugate Chem. 11, 682–687. (17) Liu, J., Gao, S., Luo, G., Yan, G., and Shen, J. C. (1998) Artificial imitation of glutathione peroxidase with 6-seleniumbridged β-cyclodextrin. Biochem. Biophys. Res. Commun. 247, 397–400. (18) Wilson, S. R., Zucker, A. P., Huang, R. C., and Spector, A. (1989) Development of synthetic compounds with glutathione peroxidase activity. J. Am. Chem. Soc. 111, 5936–5939. (19) Iwaoka, M., and Tomoda, S. (1994) A model study on the effect of an amino group on the antioxidant activity of glutathione peroxidase. J. Am. Chem. Soc. 116, 2557–2561. (20) Zade, S.S., Sighn, B. H., and Butcher, J. B. (2004) The isolation and crystal structure of a cyclic selenenate este derived from bis(2,6-diformyl-4-tert-butylphenyl) diselenide and its glutathione peroxidase-like activity. Angew. Chem., Int. Ed. 43, 4513–4515. (21) Sun, Y., Li, T., Chen, H., Zhang, K., Zheng, K., Mu, Y., Yan, G., Li, W., Shen, J., and Luo, G. (2004) Selenium-containing 15-mer peptides with high glutathione peroxidase-like activity. J. Biol. Chem. 279, 37235–37240. (22) Nogusa, H., Yano, T., Okuno, S., Hamana, H., and Inoue, K. (1995) Synthesis of carboxymethylpullulan-peptide-doxorubicin conjugates and their properties. Chem. Pharm. Bull. 43, 1931– 1936. (23) Bondanszky, M. (1984) Alkyl esters of amino acids. Int. J. Pept. Protein Res. 23, 111. (24) Burstein, Y., Walsh, K. A., and Neurath, H. (1974) Evidence of an essential histidine residue in thermolysin. Biochemistry 13, 205–210.

Nanoparticulate Glutathione Peroxidase Mimics (25) Watkinson, J. H. (1966) Fluorometric determination of selenium in biological material with 2,3-diaminonaphtalene. Anal. Chem. 38, 92–97. (26) Mannervik, B. (1985) Glutathione peroxidase. Methods Enzymol. 133, 490–495. (27) Cleland, W. W. (2002)Enzyme kinetics: steady state, In Nature Encyclopedia of Life Sciences, Vol. 6, pp 421-425. (28) Glinel, K., Sauvage, J. P., Oulyadi, H., and Huguet, J. (2000) Determination of substituents distribution in carboxymethylpullulans by NMR spectroscopy. Carbohydr. Res. 382, 343–354. (29) Kunitake, T., Okahata, Y., Shmomura, M., Yasunami, S., and Takarabe, K. (1981) Fomation of stable bilayer assemblies in water from single-chain amphiphiles. Relationship between the amphiphile structure and the aggregate morphology. J. Am. Chem. Soc. 103, 5401–5413. (30) Park, S. J., Han, H. T., Lee, Y. K., Han, S. S., Hwang, J. J., Moon, H. D., Kim, Y. S., and Cho, W. Y. (2006) N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis, exocytosis and drug release. J. Controlled Release 115, 37–45.

Bioconjugate Chem., Vol. 19, No. 9, 2008 1839 (31) Akiyoshi, K., Yamaguchi, S., and Sunamoto, J. (1991) Selfaggregates of hydrophobic polysaccharide derivatives. Chem. Lett. 20, 1263–1266. (32) Akiyoshi, K., Deguchi, S., Moriguchi, N., Yamaguchi, S., and Sunamoto, J. (1993) Self-aggregates of hydrophobized polysaccharides in water. Fomation and characteristics of nanoparticles. Macromolecules 26, 3062–3068. (33) Akiyoshi, K., Deguchi, S., Tajima, H., Nishikawa, T., and Sunamoto, J. (1995) Self-assembly of hydrophobized polysaccharide. Proc. Jpn. Acad. 71, 15–19. (34) Akiyoshi, K., Deguchi, S., Tajima, H., Nishikawa, T., and Sunamoto, J. (1997) Microscopic structure and thermoresponsiveness of a hydrogel nanoparticle by self-assembly of a hydrophobized polysaccharide. Macromolecules 30, 857–861. (35) Bell, I. M., and Hilvert, D. (1993) Peroxide dependence of the semisynthetic enzyme selenosubtilisin. Biochemistry 32, 13969–13973. (36) Brigelius-Flohe´, R. (1999) Tissue-specific functions of individual glutathione peroxidases. Free Radic. Biol. Med. 27, 951–965. BC800086Z