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Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease
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Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 180–186 www.elsevier.com/locate/jinorgbio
Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease Tatsuo Maruyama *, Saori Sonokawa, Hironari Matsushita, Masahiro Goto
*
Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan Received 31 July 2006; received in revised form 18 September 2006; accepted 18 September 2006 Available online 1 October 2006
Abstract Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Gold ion; DNase; Precious metal ions; Reversible inhibition; RNase
1. Introduction The remarkable progress of genetic engineering and DNA- and RNA-based nanotechnology has rendered DNA/RNA-relevant enzymes highly valuable. Genetic engineering is mainly based on amplification, cleavage, digestion and ligation of nucleotides. The handling and modification of nucleotides relies heavily on enzyme functions. In addition, in the field of gene diagnostics, nucleotide-relevant enzymes play important roles in the precise detection of a nucleobase mutation and the amplification of detection signals [1–5]. The nucleotide-relevant enzymes should work under targeted conditions on desired occasions. This means that artificial control of these enzyme * Corresponding authors. Tel.: +81 0 92 802 2806; fax: +81 0 92 802 2810. E-mail addresses: [email protected] (T. Maruyama), [email protected] (M. Goto).
0162-0134/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.09.021
activities is required, so that the enzymes can be used for a broad range of applications. Ribonuclease A (RNase A), which cleaves a phosphodiester bond at the 5 0 -ribose of a nucleotide, was the first protein to have its amino acid sequence determined (124 amino acids) [6,7] and one of the first proteins to yield an X-ray structure [8]. Deoxyribonuclease I (DNase I) is an endonuclease that degrades double-stranded DNA through hydrolysis of a P–O3 0 bond [9]. Since DNase and RNase are present in various environments, there is always a high risk that sample nucleotides in genetic engineering and in genetic analyses can be digested unexpectedly by contaminant DNase or RNase. To prevent unexpected enzymatic digestion of DNA and RNA, DNase and RNase inhibitors are of great importance. DNase inhibition can be simply achieved by using EDTA [10]. RNase inhibition is much more difficult. In particular, RNase A is outstandingly stable in an aqueous solution [11]. Even if it is denatured by heat or a denaturant, removal of the denaturing factors
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causes rapid refolding of RNase A [12], meaning that artificial control of RNase A activity is a difficult task. RNase inhibitor proteins have been found in the human placenta and porcine liver, and these reversibly form 1:1 complexes with RNase A to mask RNase activity [13,14]. The RNase inhibitors have been characterized well and are commercially available. However, the high cost and low oxidative stability of the RNase inhibitor proteins limits their range of applications. There have been many studies on the interactions between RNase A and transition metal ions such as copper, zinc, and magnesium. Two histidine residues in the active site play an important role in the catalytic performance [15–17]. The above-mentioned metal ions exhibit an inhibitory effect on RNase A. The inhibition of RNase activity, however, requires high concentrations of the transition metal ions (approx. 1000 molar equivalents to RNase A). A vanadium complex is an effective inhibitor for RNase A [18]. Heavy metal ions are good candidates as a RNase inhibitor. Several research groups reported the anti-tumor activity of gold compounds [19,20], which indicates the remarkable interaction of gold ions with biomolecules. Indeed, there are reports describing strong interactions between Au(III) and histidine-containing peptides [21,22]. Their results suggested to us that these precious metal ions would be strongly coordinated to a histidine-containing sequence in a protein. If a protein is an enzyme with an active site that contains a histidine residue, its activity is expected to be affected by precious metal ions. Furthermore, removal of coordinated metal ions from the enzyme’s active site would recover the enzyme activity. RNase A and DNase I have two histidine residues in their active site [23]. These nucleases are expected to be inhibited by the precious metal ions. Indeed, there has been a report on the reaction between Au(III) ions and RNase A [24]. The reaction caused aggregation of RNase A, which affected the RNase activity at low molar equivalents of Au(III) ions. However, there is still considerable uncertainty on the reaction between RNase A and Au(III) ions. In the present study, we investigated the inhibitory effects of Au(III) ions on RNase A and DNase I. Au(III) ions were coordinated to the nucleases, resulting in effective inhibition of the nuclease activities. We then attempted removal of the bound Au(III) ions from RNase A molecules to recover the inhibited enzyme activity. The present study reports that Au(III) ions facilitate effective and partially reversible inhibition of these nucleases at low concentrations of Au(III) ions.
(from bovine pancreas), DNase I (from bovine pancreas), cytidine 2 0 ,3 0 -cyclic monophosphate and DNA from calf thymus were purchased from Sigma (St. Luis, MO). Tripeptide (Gly-Gly-His) was purchased from Peptide Institute Inc., Osaka. All other chemicals were of analytical grade. All experiments were carried out in triplicate except for atomic absorbance measurements. Error bars represent the standard deviations.
2. Materials and methods
The substrate solution of 2.4 ml (HEPES buffer, 10 mM, pH 7.5) containing cytidine 2 0 ,3 0 -cyclic monophosphate (2 g/l) was put in a cuvette. Addition of an RNase A solution (33 lM, 0.2 ml) to the substrate solution started the hydrolysis of cytidine 2 0 ,3 0 -cyclic monophosphate. After 10 min, a Au(III) solution (5 mM, 6 ll) was added to the cuvette to give 11.5 lM Au(III) ions (the RNase A concentration was 2.5 lM). Twenty min after the addition of
2.1. Materials HAuCl4/HCl standard solution (Au(III) ions 1000 ppm; HCl 1 M) was purchased from Merck (Darmstadt, Germany). Thiourea and amino acids were purchased from Kishida Chemicals Co. Ltd. (Osaka, Japan). RNase A
2.2. Inhibition of RNase A by Au(III) ions and RNase activity assay Au(III) standard solution (1000 ppm) was diluted with water and 0.2 M NaOH solution to prepare 20 ppm Au(III) solutions (100 lM) with varied pH values. RNase A was typically dissolved in the Au(III) solution to give a 33 lM RNase A solution, followed by stirring for 1 h at 25 °C. To investigate the effect of the molar ratio of Au(III) ions/RNase A, the RNase A concentration was varied from 10 to 130 lM while the Au(III) concentration was fixed at 100 lM. The RNase activity assay was carried out using cytidine 2 0 ,3 0 -cyclic monophosphate (0.2 mg/ml) as a substrate in Tris–HCl buffer (pH 7.5) at 25 °C [25]. The RNase A solution (0.2 ml) prepared above was added to the substrate solution (0.8 ml) to start the hydrolysis reaction, followed by the measurements of the absorbance at 286 nm using a spectrophotometer. The RNase activity in this article is described as the value relative to that of native RNase A at each pH. The kcat value of native RNase activity at pH 7 was determined to be 1.2 s1 based on the Lineweaver-Burk plot (supplementary material, S1) [26,27]. 2.3. Elimination of Au(III) ions from RNase A After RNase A (33 lM) had been inhibited by Au(III) ions (100 lM) at pH 7 as described above, elimination of Au(III) ions from RNase A by an Au-complexing agent (typically thiourea) was conducted as follows. Thiourea (4 mM) was added to the Au(III) ion/RNase A solution and the solution was gently stirred for 1 h 25 °C. Cysteine, histidine, glutamic acid, phenylalanine, methionine, lysine, asparagine, glycine, a tripeptide (Gly-Gly-His), mercaptoethanol and EDTA(2Na) (ethylenediamine tetraacetic acid (2Na)) were also investigated as Au-complexing agents. 2.4. On/off switching of RNase A activity
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Au(III) ions, a thiourea solution (0.1 M, 10 ll) was added to the reaction solution to give a thiourea concentration of 0.38 mM. The reaction was monitored at 25 °C by measuring the absorbance at 286 nm.
3. Results
2.5. Determination of Au(III) ions coordinated to RNase A
The effect of Au(III) ions on RNase A activity was investigated at pH 7. The RNase activity decreased with an increasing Au(III) ion/RNase A molar ratio (Fig. 1a). Just one molar equivalent of Au(III) ions caused 85% inhibition of RNase A. Au(III) ion/RNase A molar ratios above three resulted in complete inhibition. These results are in agreement with those reported by Isab and Sadler [24]. As the Au(III) ion/RNase A molar ratio increased, the number of Au(III) ions bound to an RNase A molecule also increased (Fig. 1a). The maximal number of Au(III) ions bound to an RNase A molecule was 8, and this occurred when the Au(III) ion/RNase A molar ratio exceeded 50 (data not shown). The RNase A modified with iodoacetic acid bound approx. 5 Au ions at a Au(III)/RNase A molar ratio of 10, while native RNase A bound 7 Au ions. Since the alkylation by iodoacetic acid below pH 5.5 occurs mainly on a
2.6. CD spectra measurements CD spectra of RNase A were measured using a JASCO J-725 spectropolarimeter at 25 °C. The CD spectra bandwidth was set at 2 nm and the scanning speed was set to 10 nm/min. Cuvettes with a 10 mm path length were used. The concentration of RNase A was set at 0.66 lM. All spectra shown are the averages of eight scans. 2.7. DNase I activity assay [29] DNA from calf thymus (2 mg) was dissolved in 22.5 ml water containing 6.25 mM MgSO4 followed by incubation for 12 h at 25 °C. The DNA solution was added by 2.5 ml acetate buffer (1 M, pH 5.0) to prepare a substrate solution. DNA hydrolysis was started by the addition of 0.5 ml DNase I solution to 2.5 ml substrate solution in a quartz cuvette. The digestion of DNA catalyzed by DNase I was monitored by measurement of the absorbance at 260 nm. The Au(III) concentration was 100 lM and the DNase concentration was varied to give the different Au(III)/ RNase A molar ratios. The thiourea concentration was 4 mM for recovering of the inhibited activity. It was confirmed that this activity assay was not affected by thiourea.
50
8
40
6
30 4 20 2
10 0
Bound Au ions per RNase A molecule
Alkylation of histidine residues in RNase A was carried out using iodoacetic acid [28]. Briefly, iodoacetic acid solution (4 g/l, pH 5.5) of 0.5 ml was mixed with 0.1 ml RNase solution (200 g/l), followed by incubation over nigh at 25 °C. The His-alkylated RNase A was purified by a GPC column (PD-10, GE-Healthcare). It was confirmed that His-alkylated RNase A lost the intrinsic activity thoroughly. The Au(III)-binding was also investigated for the His-alkylated RNase A.
0 0
2
4
6
8
10
Au(III) / RNase A molar ratio 100
10
80
8
60
6
40
4
20
2
0
Au ions remaining per RNase A molecule
=½Initial AuðIIIÞ ion concentration
RNase A activity [%]
R ¼ 100 ð½Initial AuðIIIÞ ion concentration ½AuðIIIÞ ion concentration in the filtrateÞ
RNase A activity [%]
After RNase A had been inhibited by Au(III) ions for 1 h at pH 7.0, the RNase A solution was filtered through an ultrafiltration membrane with a molecular cut-off of 10,000 Da (Amicon Ultra-4, Millipore Co.). It was confirmed that Au(III) ions were not bound to the filtration membrane. The filtrate was subjected to atomic absorbance spectrophotometry (AA6700, Shimadzu, Kyoto) using flame to determine the concentration of free Au(III) ions. The binding percentage (R) of Au(III) ions was calculated by the following equation.
3.1. Au(III)-induced inhibition of RNase A and recovery of the activity by thiourea
0 0
2
4
6
8
10
Au(III) / RNase A molar ratio Fig. 1. Effect of Au(III) ions on RNase A activity. (a) Au(III) binding on RNase A. (b) Au(III) elimination from RNase A by thiourea. Closed squares represent RNase activity and open circles represent the number of bound Au(III) ions per RNase A molecule. The final Au(III) concentration was fixed at 20 lM and the RNase A concentration was varied. The thiourea concentration was 4 mM and the pH was 7.0. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions.
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3.2. Effect of pH on the inhibition of RNase A by Au(III) ions The Au(III)-induced inhibition of RNase A and the thiourea-induced renaturation of the inhibited RNase A were investigated at various pH values (Fig. 2a). The Au(III)-induced inhibition was effective over a wide range of neutral pH values (pH 6–10). Recovery of the RNase activity by thiourea was not affected much by the pH (Fig. 2a). Fig. 2b shows binding ratios of Au(III) ions in the inhibition of RNase A, where 3 molar equivalents of Au(III) ions were used. The binding of Au(III) ions was relatively high at pH 6–10, which coincided with the inhibition efficiency. 3.3. Selection of Au-complexing or Au(III)-reducing agent for elimination of Au(III) ions from RNase A A tripeptide, mercaptoethanol and EDTA were employed at 4 mM (40-fold greater than Au(III)) for eliminating Au(III) ions from the Au(III)-inhibited RNase A. Thiourea exhibited the highest Au(III)-eliminating ability (Fig. 3a) and the highest recovery of RNase activity (Fig. 3b). Of the various amino acids, Cys most effectively facilitated the elimination of Au(III) ions from RNase A, resulting in 56% elimination of Au(III) and 86% recovery of its intrinsic activity. His and Met also exhibited Au(III)-eliminating ability and recovered approx. 30% of the intrinsic RNase activity. Other amino acids did not eliminate Au(III) ions from RNase A and did not recover RNase activity either. Addition of a tripeptide (Gly-GlyHis), which forms a Au(III)-tripeptide complex [21], eliminated 40% of the Au(III) ions from RNase A and recovered 30% of the intrinsic RNase activity. EDTA is a good chelator for transition metal ions, but EDTA did not facilitate the elimination of Au(III) ions from RNase
RNase A activity [%]
100 80 60 40 20 0 4
6
8
10
12
10
12
pH 120
Au(III)-binding ratio [%]
histidine residue [28], a histidine residue could be one of the Au-binding sites in RNase. We next investigated the elimination of Au(III) ions from a Au(III)-RNase A complex using thiourea. Thiourea has a strong interaction with precious metal ions [30]. It reduces Au(III) ions to Au(I) ions and forms a 2:1 complex [31]. Addition of thiourea to the Au(III)-inhibited RNase A resulted in the recovery of RNase activity (Fig. 1b). At a Au(III) ion/RNase A molar ratio of 3, 85% of the intrinsic activity was recovered from the completely inhibited RNase A. Under these conditions, 90% of the Au(III) ions bound to RNase A were eliminated from RNase. The recovered activity decreased with an increasing Au(III) ion/RNase A molar ratio. This was because the elimination of Au(III) ions from RNase A was not sufficient at high Au(III) ion/RNase A molar ratios. In fact, more than two Au(III) ions remained per RNase A molecule after the addition of thiourea at a molar ratio of 10, while less than one Au(III) ion remained at a Au(III) ion/RNase A molar ratio of 3.
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100 80 60 40 20 0
4
6
8
pH Fig. 2. (a) Effect of pH on the Au(III)-induced inhibition and renaturation of RNase A. Closed circles represent the Au(III)-inhibited RNase activity and open circles represent the recovered RNase A activity. (b) Effect of pH on the Au(III) binding ratio. The RNase A concentration was 33 lM, the Au(III) concentration was 100 lM and the thiourea concentration was 4 mM. Hundred percent RNase activity was defined as the native RNase A activity at each pH in the absence of Au(III) ions.
A. Mercaptoethanol showed relatively high recovery of the RNase activity. The results for Cys and mercaptoethanol mean that a thiol group is effective at eliminating Au(III) ions, resulting in recovering of the activity of the Au(III)-inhibited RNase A. The recovery of RNase activity coinciding with Au(III)-elimination suggests that the interaction of RNase with Au(III) ions did not reduce Au(III) ions to produce gold nanoparticles. Since Cys and mercaptoethanol can also act as reducing compounds that break disulfide bonds in RNase A molecules, the following experiments were carried out using thiourea as the Au-eliminating agent. 3.4. Effect of the thiourea/Au(III) ion molar ratio on the recovery of the Au(III)-inhibited RNase A activity The effect of the thiourea/Au(III) ion molar ratio was investigated (Fig. 4). As the thiourea/Au ratio increased, the relative activity of the renatured RNase A increased. When the molar ratio exceeded 10, the recovered activity reached a maximum of around 90%. We confirmed that thiourea itself does not affect the activity of native RNase
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0.5 80 60 40 20
Th io
Ly s As n G ly G G H M E ED TA
ea C ys M et H is G lu Ph e
0
Absorbance at 286 nm
100
ur
Au(III) elimination ratio [%]
184
0.4 0.3 0.2 0.1 0 0
100
10
20
30
40
Time [min] Fig. 5. On/off switching of hydrolysis of cytidine 2 0 ,3 0 -cyclic monophosphate (2 g/l) catalyzed by RNase A (2.5 lM). White arrow indicates the addition of a Au(III) solution (6 ll, 5 mM) to the reaction solution (2.6 ml). Black arrow indicates the addition of a thiourea solution (10 ll, 0.1 M) to the reaction solution.
80 60 40 20
Th io u
Ly s As n G ly G G H M E ED TA
0 re a C ys M et H is G lu Ph e
Recovery of RNase A Activity [%]
Au(III)-complexing or reducing reagent
Au(III)-complexing or reducing reagent
Fig. 3. (a) Au(III) elimination from Au(III)-inhibited RNase A by addition of various Au-complexing agents. (b) Activity recovery of Au(III)-inhibited RNase A by addition of various complexing agents. The pH was 7.0 and other experimental conditions were the same as those in Fig. 2. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions. Abbreviations: GGH, Gly-Gly-His; ME, mercaptoethanol and EDTA, ethylenediamine tetraacetic acid (2Na).
monophosphate was started by the addition of RNase A. The addition of Au(III) ions markedly reduced the hydrolysis rate, to only 8% of its initial reaction rate. As soon as the thiourea was added, the reaction was stimulated and progressed sharply. The reaction rate was 8-fold faster than that observed prior to the addition of thiourea. It should be noted that the addition of Au(III) ions and thiourea had little effect on the pH of the reaction medium and also that the Au(III) and thiourea solutions did not have detectable absorbance at 286 nm.
100
The conformational change of RNase A was investigated by measuring CD spectra of native, Au(III)-coordinated and renatured RNase A (Fig. 6). The addition of Au(III) ions reduced the negative bands at 208 nm,
80 60 40 20 0
10
20
30
40
50
Thiourea/Au molar ratio Fig. 4. Effect of thiourea concentration on the recovery of RNase activity. The experimental conditions were the same as those in Fig. 3. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions.
A (data not shown). Atomic absorbance analyses demonstrated that excess thiourea (40 molar equivalents) resulted in a 90% Au(III) elimination ratio, meaning that 0.3 Au(III) ions per RNase A molecule were still coordinated. 3.5. On/off switching of RNase activity We next studied the on/off switching of an RNase A-catalyzed reaction based on the binding and elimination of Au(III) ions (Fig. 5). The hydrolysis of cytidine 2 0 ,3 0 -cyclic
Molecular rotation:
10
0
10-3 × θ [deg•cm2/dmol]
Recovery of RNase A activity [%]
3.6. CD spectra of RNase A in the absence and presence of Au(III) ions
5 0 -5 -10 -15 190 200 210 220
230 240 250
Wavelength [nm] Fig. 6. Circular dichroism spectra of RNase A. The red line with circles represents the spectrum of native RNase A, the blue line with squares represents that of Au(III)-inhibited RNase A and the green line with triangles represents that of RNase A renatured by thiourea. The RNase A concentration was 0.66 lM, the Au(III) concentration was 2.0 lM and the thiourea concentration was 80 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
T. Maruyama et al. / Journal of Inorganic Biochemistry 101 (2007) 180–186
DNase I activity [%]
100 Au Thiourea
80 60 40 20 0
0
20
40
60
Au(III)/DNase I molar ratio Fig. 7. Effect of Au(III) binding (closed circles) and Au(III) elimination (open circles) on DNase I. Hundred percent DNase activity was defined as the native DNase I activity in the absence of Au(III) ions. The Au(III) concentration was fixed at 100 lM and the pH was 5.0. Other experimental conditions were the same as those in Fig. 1.
217 nm and 222 nm. Since these negative bands represent a-helix and b-sheet contents, the addition of Au(III) ions induced drastic conformational changes in the RNase A to reduce a-helix and b-sheet contents. Additionally, the largest negative band shifted from 208 nm to 204 nm. This implies an increase in the random coils present in the Au(III)-coordinated RNase A. After addition of thiourea, the negative bands at 208 nm, 217 nm and 222 nm increased and the largest negative band returned to 208 nm. However, the spectrum of the renatured RNase A was not the same as that of native RNase A. It should be noted that the addition of thiourea did not change the CD spectrum of native RNase A solution. 3.7. Reversible inhibition of DNase I by Au(III) ions The Au(III)-induced inhibition and thiourea-induced renaturation were also investigated for DNase I. Similar to the results for RNase A, the DNase activity decreased with an increasing Au(III) ion/DNase I ratio (Fig. 7). The DNase activity was depressed to less than 5% of its intrinsic activity at Au(III) ion/DNase I ratios over 10. Addition of thiourea also recovered the DNase activity. When the Au(III) ion/DNase I molar ratio was 10, approx. 80% of the intrinsic DNase activity was recovered. DNase I from bovine pancreas has 6 His residues and 4 Met residues but no free thiol group [32,33]. More Au(III) ions were, therefore, required for the inhibition of DNase I than for that of RNase A. 4. Discussion While the inhibition of RNase A by transition metal ions requires thousands of molar equivalents of those metal ions to RNase A [34,35], only 3 or 4 molar equivalents of Au(III) ions effectively inhibited RNase activity. Isab et al. and Craig reported that Au(III) ions caused protein aggregation at a high protein concentration (2 mM)
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[24,36]. We also confirmed the aggregation of RNase A at such a high concentration. Under our typical conditions, the RNase concentrations were around 33 lM, where no aggregation was observed. The addition of thiourea recovered the Au(III)-inhibited RNase activity, These results indicate that the inhibitory effect of Au(III) ions on RNase A is induced by complexation of Au(III) ions with RNase A molecules, neither to the aggregation of the RNase nor to the oxidation of RNase A molecules, although Au(III) ions are strong oxidants. The Lineweaver-Burk plot shows two sets of parallel lines (a supplementary material), suggesting two sets of the uncompetitive inhibition models. Alkylation of His residues by iodoacetic acid decreased Au-binding but did not lead to no Au-binding. These results indicate the binding mechanisms more than 2, but there is considerable uncertainty to discuss the inhibition manner. The study of Au(III) elimination using amino acids revealed that Cys, His and Met facilitated the elimination of Au(III) ions from an RNase A molecule. RNase A has 8 Cys, 4 His and 4 Met residues. All the Cys residues in an RNase A molecule form disulfide bonds. So the amino acid residues that could possibly interact with Au(III) ions in an RNase A molecule are His and Met residues. Several groups reported a strong interaction between a Au(III) ion and a histidine-containing peptide to form a Au(III)-peptide complex [21,22,37]. Zou et al first reported the crystal structure of gold/protein complex and demonstrated the specific Au(I)-binding to histidine residue [38]. The active site of RNase A has two histidine residues and the sub˚ wide strate pocket of RNase A is very narrow, approx. 5 A [39]. The bond lengths between Au(III) and the nitrogen ˚ , and the atomic radii of the Au atoms were 1.9–2.1 A ˚ and 0.5 A ˚ , respectively, and nitrogen atoms were 1.44 A meaning that the size of the substrate pocket matched the Au(III) coordination. These allow us to speculate that Au(III) ions are likely to be coordinated to His residues in RNase A. Initially, Au(III) ions were dissolved as [AuCl4] in an aqueous solution before formation of the Au(III)-RNase A complex. The stability of [AuCl4] tends to be influenced by the pH and the Cl concentration [40]. At higher pH, the Cl ligand can be exchanged for OH to produce [AuCl3(OH)], [AuCl2(OH)2] and so on. It is necessary to exchange Cl (or OH) ligands for the formation of the Au(III)-RNase A complex. This implies that the unstable [AuCln(OH)4n] complex tends to form the Au(III)RNase A complex. On the other hand, a high pH above 10 (high OH concentration) would also stabilize [Au(OH)4] by not allowing the exchange of OH ligands. Furthermore, an RNase A molecule is negatively charged at such a pH (pI of RNase A is 9.45). The negatively charged RNase A molecule might prevent [Au(OH)4] from accessing it, due to electronic repulsion. CD spectra suggest that the secondary structure of an RNase A molecule was denatured by Au(III) ions and was renatured by the addition of thiourea. However, the
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conformational refolding of the Au(III)-inhibited RNase A was only partial. Since Au(III) ions can oxidize amino acid residues [41], the irreversible oxidation of amino acid residues in a RNase molecule could be another reason for the incomplete recovery of RNase activity. In particular, Met and disulfide bonds are likely to be oxidized [24,42,43]. However, 90% of RNase activity was recovered by the addition of thiourea, suggesting that the majority of the Au-binding sites in RNase A would not be irreversibly oxidized by Au(III) ions. The present study shows that Au(III) ions were coordinated to RNase A and DNase I. The binding of Au(III) ions induced inhibition of the nuclease activity, accompanied by conformational change of the nuclease molecule. Au-complexing agents such as thiourea effectively eliminated Au(III) ions from the nucleases, resulting in recovery of the inhibited nuclease activity. This study suggests that Au(III) ions effectively facilitate partially reversible inhibition of RNase A and DNase I. Acknowledgements This research was supported by a Grant-in-Aid for the 21st Century COE Program, ‘‘Functional Innovation of Molecular Informatics’’ from the Ministry of Education, Culture, Science, Sports and Technology of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio. 2006.09.021. References [1] G.R. Taylor, J. Deeble, Biomol. Eng. 14 (1999) 181–186. [2] J.G. Hall, P.S. Eis, S.M. Law, L.P. Reynaldo, J.R. Prudent, D.J. Marshall, H.T. Allawi, A.L. Mast, J.E. Dahlberg, R.W. Kwiatkowski, M. de Arruda, B.P. Neri, V.I. Lyamichev, Proc. Natl. Acad. Sci. USA 97 (2000) 8272–8277. [3] M.V. Myakishev, Y. Khripin, S. Hu, D.H. Hamer, Genome Res. 11 (2001) 163–169. [4] M.M. Goldrick, Hum. Mutat. 18 (2001) 190–204. [5] H. Ichinose, M. Kitaoka, N. Okamura, T. Maruyama, N. Kamiya, M. Goto, Anal. Chem. 7 (2005) 7047–7053. [6] J.T. Potts, A. Berger, J. Cooke, C.B. Anfinsen, J. Biol. Chem. 237 (1962) 1851–1855. [7] D.G. Smyth, W.H. Stein, S. Moore, J. Biol. Chem. 238 (1963) 277– 284. [8] G. Kartha, J. Bello, D. Harker, Nature 213 (1967) 862–865. [9] S. Moore, Third ed., in: P.D. Boyer, H. Lardy, K. Myrback (Eds.), The Enzymes, vol. 14, Academic Press, New York, 1981, pp. 281–294.
[10] M. Laskowski, Second ed., in: P.D. Boyer, H. Lardy, K. Myrback (Eds.), The Enzymes, vol. 5, Academic Press, New York, 1961, pp. 123–147. [11] F. Delben, V. Crescenz, F. Quadrifo, Int. J. Protein Res. 1 (1969) 145–149. [12] E.A. Barnard, J. Mol. Biol. 10 (1964) 235–262. [13] R. Shapiro, B.L. Vallee, Proc. Natl. Acad. Sci. 84 (1987) 2238–2241. [14] F.S. Lee, R. Shapiro, B.L. Vallee, Biochemistry 28 (1989) 225–230. [15] T. Takahashi, M. Irie, T. Ukita, J. Biochem. 61 (1967) 669–678. [16] G.L. Eichhorn, P. Clark, E. Tarien, J. Biol. Chem. 44 (1969) 937–942. [17] N. Nishimura, Y. Kitaoka, M. Niwano, J. Ferment. Bioeng. 78 (1994) 130–133. [18] D.C. Crans, R.A. Felty, M.M. Miller, J. Am. Chem. Soc. 113 (1991) 265–269. [19] R. Eisler, Inflamm. Res. 52 (2003) 487–501. [20] C.M. Che, R.W.Y. Sun, W.Y. Yu, C.B. Ko, N.Y. Zhu, H.Z. Sun, Chem. Commun. (2003) 1718–1719. [21] S.L. Best, T.K. Chattopadhyay, M.I. Djuran, R.A. Palmer, P.J. Sadler, I. Sovago, K. Varnagy, J. Chem. Soc., Dalton Trans. (1997) 2587–2596. [22] M. Wienken, B. Lippert, E. Zangrando, L. Randaccio, Inorg. Chem. 31 (1992) 1983–1985. [23] H.K. Paudel, T.H. Liao, J. Biol. Chem. 261 (1986) 6012–6017. [24] A.A. Isab, P.J. Sadler, Biochim. Biophys. Acta 492 (1977) 322–330. [25] E.M. Crook, A.P. Mathias, B.R. Rabin, Biochem. J. 74 (1960) 234– 238. [26] M. Litt, J. Biol. Chem. 236 (1961) 1786–1790. [27] M. Moussaoui, M.V. Nogues, A. Guasch, T. Barman, F. Travers, C.M. Cuchillo, J. Biol. Chem. 273 (1998) 25565–25572. [28] A.M. Crestfield, W.H. Stein, S. Moore, J. Biol. Chem. 238 (1963) 2413–2420. [29] M. Kunitz, J. Gen. Physiol. 33 (1950) 349–362. [30] J. Li, J.D. Miller, Mine. Proc. Extractive Metall. 27 (2006) 177–214. [31] O.E. Piro, E.E. Castellano, R.C.V. Piatti, A.E. Bolzan, A.J. Arvia, Acta Cryst. C58 (2002) m252–m255. [32] J. Salnikow, T.H. Liao, S. Moore, W.H. Stein, J. Biol. Chem. 248 (1973) 1480–1488. [33] T.H. Liao, J. Salnikow, S. Moore, W.H. Stein, J. Biol. Chem. 248 (1973) 1489–1495. [34] F.F. Davis, F.W. Allen, J. Biol. Chem. 217 (1955) 13–22. [35] T.D. Alger, Biochemistry 9 (1970) 3248–3255. [36] J.P. Craig, A.G. Garrett Jr., H.B. Williams, J. Am. Chem. Soc. 76 (1954) 1570–1575. [37] J.A. Cuadrado, W.X. Zhang, W. Hang, V. Majidi, J. Environ. Monit. 2 (2000) 355–359. [38] J. Zou, P. Taylor, J. Dornan, S.P. Robinson, M.D. Walkinshaw, P.J. Sadler, Angew. Chem., Int. Ed. 39 (2000) 2931–2934. [39] C.H. Carlisle, R.A. Palmer, S.K. Mazumdar, B.A. Gorinsky, D.G.R. Yeates, J. Mol. Biol. 85 (1974) 1–18. [40] F.H. Fry, G.A. Hamilton, J. Turkevich, Inorg. Chem. 5 (1966) 1943– 1946. [41] J. Zou, Z. Guo, J.A. Parkinson, Y. Chen, P.J. Sadler, Chem. Commun. (1999) 1359–1361. [42] E. Bordigno, L. Cattalin, G. Natile, A. Scatturi, J. Chem. Soc., Chem. Commun. (1973) 878–879. [43] J. Darkwa, R. Olojo, E. Chikwana, R.H. Simoyi, J. Phys. Chem. A 108 (2004) 5576–5587.
Inorganic Biochemistry Journal of Inorganic Biochemistry 101 (2007) 180–186 www.elsevier.com/locate/jinorgbio
Inhibitiory effects of gold(III) ions on ribonuclease and deoxyribonuclease Tatsuo Maruyama *, Saori Sonokawa, Hironari Matsushita, Masahiro Goto
*
Department of Applied Chemistry, Graduate School of Engineering and Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka 819-0395, Japan Received 31 July 2006; received in revised form 18 September 2006; accepted 18 September 2006 Available online 1 October 2006
Abstract Inhibitory effects of gold(III) ions (Au(III)) on ribonuclease A (RNase A) and deoxyribonuclease I (DNase I) were investigated at neutral pH. RNase A was completely inhibited by 3 molar equivalents of Au(III) ions. DNase I was inhibited by 10 molar equivalents of Au(III) ions. Stoichiometric analyses suggest that Au(III) ions were coordinated to RNase A molecules. The Au(III)-inhibited RNase A and DNase I were renatured to exhibit 80% and 60% of their intrinsic activity, when the bound Au(III) ions were eliminated from the nucleases by addition of thiourea, which forms a strong complex with gold ions. This suggests that RNase A and DNase I were not oxidized to lose their activity, but reversibly complexed with Au(III) ions to lose their activity. Au(III) ions were probably considered to be bound to histidine and methionine residues in the nucleases, resulting in the inhibition of their activity. CD spectra revealed that the Au(III)-induced inhibition caused a conformational change in RNase A molecules and that the addition of thiourea induced refolding of the Au(III)-inhibited RNase A. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Gold ion; DNase; Precious metal ions; Reversible inhibition; RNase
1. Introduction The remarkable progress of genetic engineering and DNA- and RNA-based nanotechnology has rendered DNA/RNA-relevant enzymes highly valuable. Genetic engineering is mainly based on amplification, cleavage, digestion and ligation of nucleotides. The handling and modification of nucleotides relies heavily on enzyme functions. In addition, in the field of gene diagnostics, nucleotide-relevant enzymes play important roles in the precise detection of a nucleobase mutation and the amplification of detection signals [1–5]. The nucleotide-relevant enzymes should work under targeted conditions on desired occasions. This means that artificial control of these enzyme * Corresponding authors. Tel.: +81 0 92 802 2806; fax: +81 0 92 802 2810. E-mail addresses: [email protected] (T. Maruyama), [email protected] (M. Goto).
0162-0134/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.09.021
activities is required, so that the enzymes can be used for a broad range of applications. Ribonuclease A (RNase A), which cleaves a phosphodiester bond at the 5 0 -ribose of a nucleotide, was the first protein to have its amino acid sequence determined (124 amino acids) [6,7] and one of the first proteins to yield an X-ray structure [8]. Deoxyribonuclease I (DNase I) is an endonuclease that degrades double-stranded DNA through hydrolysis of a P–O3 0 bond [9]. Since DNase and RNase are present in various environments, there is always a high risk that sample nucleotides in genetic engineering and in genetic analyses can be digested unexpectedly by contaminant DNase or RNase. To prevent unexpected enzymatic digestion of DNA and RNA, DNase and RNase inhibitors are of great importance. DNase inhibition can be simply achieved by using EDTA [10]. RNase inhibition is much more difficult. In particular, RNase A is outstandingly stable in an aqueous solution [11]. Even if it is denatured by heat or a denaturant, removal of the denaturing factors
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181
causes rapid refolding of RNase A [12], meaning that artificial control of RNase A activity is a difficult task. RNase inhibitor proteins have been found in the human placenta and porcine liver, and these reversibly form 1:1 complexes with RNase A to mask RNase activity [13,14]. The RNase inhibitors have been characterized well and are commercially available. However, the high cost and low oxidative stability of the RNase inhibitor proteins limits their range of applications. There have been many studies on the interactions between RNase A and transition metal ions such as copper, zinc, and magnesium. Two histidine residues in the active site play an important role in the catalytic performance [15–17]. The above-mentioned metal ions exhibit an inhibitory effect on RNase A. The inhibition of RNase activity, however, requires high concentrations of the transition metal ions (approx. 1000 molar equivalents to RNase A). A vanadium complex is an effective inhibitor for RNase A [18]. Heavy metal ions are good candidates as a RNase inhibitor. Several research groups reported the anti-tumor activity of gold compounds [19,20], which indicates the remarkable interaction of gold ions with biomolecules. Indeed, there are reports describing strong interactions between Au(III) and histidine-containing peptides [21,22]. Their results suggested to us that these precious metal ions would be strongly coordinated to a histidine-containing sequence in a protein. If a protein is an enzyme with an active site that contains a histidine residue, its activity is expected to be affected by precious metal ions. Furthermore, removal of coordinated metal ions from the enzyme’s active site would recover the enzyme activity. RNase A and DNase I have two histidine residues in their active site [23]. These nucleases are expected to be inhibited by the precious metal ions. Indeed, there has been a report on the reaction between Au(III) ions and RNase A [24]. The reaction caused aggregation of RNase A, which affected the RNase activity at low molar equivalents of Au(III) ions. However, there is still considerable uncertainty on the reaction between RNase A and Au(III) ions. In the present study, we investigated the inhibitory effects of Au(III) ions on RNase A and DNase I. Au(III) ions were coordinated to the nucleases, resulting in effective inhibition of the nuclease activities. We then attempted removal of the bound Au(III) ions from RNase A molecules to recover the inhibited enzyme activity. The present study reports that Au(III) ions facilitate effective and partially reversible inhibition of these nucleases at low concentrations of Au(III) ions.
(from bovine pancreas), DNase I (from bovine pancreas), cytidine 2 0 ,3 0 -cyclic monophosphate and DNA from calf thymus were purchased from Sigma (St. Luis, MO). Tripeptide (Gly-Gly-His) was purchased from Peptide Institute Inc., Osaka. All other chemicals were of analytical grade. All experiments were carried out in triplicate except for atomic absorbance measurements. Error bars represent the standard deviations.
2. Materials and methods
The substrate solution of 2.4 ml (HEPES buffer, 10 mM, pH 7.5) containing cytidine 2 0 ,3 0 -cyclic monophosphate (2 g/l) was put in a cuvette. Addition of an RNase A solution (33 lM, 0.2 ml) to the substrate solution started the hydrolysis of cytidine 2 0 ,3 0 -cyclic monophosphate. After 10 min, a Au(III) solution (5 mM, 6 ll) was added to the cuvette to give 11.5 lM Au(III) ions (the RNase A concentration was 2.5 lM). Twenty min after the addition of
2.1. Materials HAuCl4/HCl standard solution (Au(III) ions 1000 ppm; HCl 1 M) was purchased from Merck (Darmstadt, Germany). Thiourea and amino acids were purchased from Kishida Chemicals Co. Ltd. (Osaka, Japan). RNase A
2.2. Inhibition of RNase A by Au(III) ions and RNase activity assay Au(III) standard solution (1000 ppm) was diluted with water and 0.2 M NaOH solution to prepare 20 ppm Au(III) solutions (100 lM) with varied pH values. RNase A was typically dissolved in the Au(III) solution to give a 33 lM RNase A solution, followed by stirring for 1 h at 25 °C. To investigate the effect of the molar ratio of Au(III) ions/RNase A, the RNase A concentration was varied from 10 to 130 lM while the Au(III) concentration was fixed at 100 lM. The RNase activity assay was carried out using cytidine 2 0 ,3 0 -cyclic monophosphate (0.2 mg/ml) as a substrate in Tris–HCl buffer (pH 7.5) at 25 °C [25]. The RNase A solution (0.2 ml) prepared above was added to the substrate solution (0.8 ml) to start the hydrolysis reaction, followed by the measurements of the absorbance at 286 nm using a spectrophotometer. The RNase activity in this article is described as the value relative to that of native RNase A at each pH. The kcat value of native RNase activity at pH 7 was determined to be 1.2 s1 based on the Lineweaver-Burk plot (supplementary material, S1) [26,27]. 2.3. Elimination of Au(III) ions from RNase A After RNase A (33 lM) had been inhibited by Au(III) ions (100 lM) at pH 7 as described above, elimination of Au(III) ions from RNase A by an Au-complexing agent (typically thiourea) was conducted as follows. Thiourea (4 mM) was added to the Au(III) ion/RNase A solution and the solution was gently stirred for 1 h 25 °C. Cysteine, histidine, glutamic acid, phenylalanine, methionine, lysine, asparagine, glycine, a tripeptide (Gly-Gly-His), mercaptoethanol and EDTA(2Na) (ethylenediamine tetraacetic acid (2Na)) were also investigated as Au-complexing agents. 2.4. On/off switching of RNase A activity
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Au(III) ions, a thiourea solution (0.1 M, 10 ll) was added to the reaction solution to give a thiourea concentration of 0.38 mM. The reaction was monitored at 25 °C by measuring the absorbance at 286 nm.
3. Results
2.5. Determination of Au(III) ions coordinated to RNase A
The effect of Au(III) ions on RNase A activity was investigated at pH 7. The RNase activity decreased with an increasing Au(III) ion/RNase A molar ratio (Fig. 1a). Just one molar equivalent of Au(III) ions caused 85% inhibition of RNase A. Au(III) ion/RNase A molar ratios above three resulted in complete inhibition. These results are in agreement with those reported by Isab and Sadler [24]. As the Au(III) ion/RNase A molar ratio increased, the number of Au(III) ions bound to an RNase A molecule also increased (Fig. 1a). The maximal number of Au(III) ions bound to an RNase A molecule was 8, and this occurred when the Au(III) ion/RNase A molar ratio exceeded 50 (data not shown). The RNase A modified with iodoacetic acid bound approx. 5 Au ions at a Au(III)/RNase A molar ratio of 10, while native RNase A bound 7 Au ions. Since the alkylation by iodoacetic acid below pH 5.5 occurs mainly on a
2.6. CD spectra measurements CD spectra of RNase A were measured using a JASCO J-725 spectropolarimeter at 25 °C. The CD spectra bandwidth was set at 2 nm and the scanning speed was set to 10 nm/min. Cuvettes with a 10 mm path length were used. The concentration of RNase A was set at 0.66 lM. All spectra shown are the averages of eight scans. 2.7. DNase I activity assay [29] DNA from calf thymus (2 mg) was dissolved in 22.5 ml water containing 6.25 mM MgSO4 followed by incubation for 12 h at 25 °C. The DNA solution was added by 2.5 ml acetate buffer (1 M, pH 5.0) to prepare a substrate solution. DNA hydrolysis was started by the addition of 0.5 ml DNase I solution to 2.5 ml substrate solution in a quartz cuvette. The digestion of DNA catalyzed by DNase I was monitored by measurement of the absorbance at 260 nm. The Au(III) concentration was 100 lM and the DNase concentration was varied to give the different Au(III)/ RNase A molar ratios. The thiourea concentration was 4 mM for recovering of the inhibited activity. It was confirmed that this activity assay was not affected by thiourea.
50
8
40
6
30 4 20 2
10 0
Bound Au ions per RNase A molecule
Alkylation of histidine residues in RNase A was carried out using iodoacetic acid [28]. Briefly, iodoacetic acid solution (4 g/l, pH 5.5) of 0.5 ml was mixed with 0.1 ml RNase solution (200 g/l), followed by incubation over nigh at 25 °C. The His-alkylated RNase A was purified by a GPC column (PD-10, GE-Healthcare). It was confirmed that His-alkylated RNase A lost the intrinsic activity thoroughly. The Au(III)-binding was also investigated for the His-alkylated RNase A.
0 0
2
4
6
8
10
Au(III) / RNase A molar ratio 100
10
80
8
60
6
40
4
20
2
0
Au ions remaining per RNase A molecule
=½Initial AuðIIIÞ ion concentration
RNase A activity [%]
R ¼ 100 ð½Initial AuðIIIÞ ion concentration ½AuðIIIÞ ion concentration in the filtrateÞ
RNase A activity [%]
After RNase A had been inhibited by Au(III) ions for 1 h at pH 7.0, the RNase A solution was filtered through an ultrafiltration membrane with a molecular cut-off of 10,000 Da (Amicon Ultra-4, Millipore Co.). It was confirmed that Au(III) ions were not bound to the filtration membrane. The filtrate was subjected to atomic absorbance spectrophotometry (AA6700, Shimadzu, Kyoto) using flame to determine the concentration of free Au(III) ions. The binding percentage (R) of Au(III) ions was calculated by the following equation.
3.1. Au(III)-induced inhibition of RNase A and recovery of the activity by thiourea
0 0
2
4
6
8
10
Au(III) / RNase A molar ratio Fig. 1. Effect of Au(III) ions on RNase A activity. (a) Au(III) binding on RNase A. (b) Au(III) elimination from RNase A by thiourea. Closed squares represent RNase activity and open circles represent the number of bound Au(III) ions per RNase A molecule. The final Au(III) concentration was fixed at 20 lM and the RNase A concentration was varied. The thiourea concentration was 4 mM and the pH was 7.0. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions.
T. Maruyama et al. / Journal of Inorganic Biochemistry 101 (2007) 180–186
3.2. Effect of pH on the inhibition of RNase A by Au(III) ions The Au(III)-induced inhibition of RNase A and the thiourea-induced renaturation of the inhibited RNase A were investigated at various pH values (Fig. 2a). The Au(III)-induced inhibition was effective over a wide range of neutral pH values (pH 6–10). Recovery of the RNase activity by thiourea was not affected much by the pH (Fig. 2a). Fig. 2b shows binding ratios of Au(III) ions in the inhibition of RNase A, where 3 molar equivalents of Au(III) ions were used. The binding of Au(III) ions was relatively high at pH 6–10, which coincided with the inhibition efficiency. 3.3. Selection of Au-complexing or Au(III)-reducing agent for elimination of Au(III) ions from RNase A A tripeptide, mercaptoethanol and EDTA were employed at 4 mM (40-fold greater than Au(III)) for eliminating Au(III) ions from the Au(III)-inhibited RNase A. Thiourea exhibited the highest Au(III)-eliminating ability (Fig. 3a) and the highest recovery of RNase activity (Fig. 3b). Of the various amino acids, Cys most effectively facilitated the elimination of Au(III) ions from RNase A, resulting in 56% elimination of Au(III) and 86% recovery of its intrinsic activity. His and Met also exhibited Au(III)-eliminating ability and recovered approx. 30% of the intrinsic RNase activity. Other amino acids did not eliminate Au(III) ions from RNase A and did not recover RNase activity either. Addition of a tripeptide (Gly-GlyHis), which forms a Au(III)-tripeptide complex [21], eliminated 40% of the Au(III) ions from RNase A and recovered 30% of the intrinsic RNase activity. EDTA is a good chelator for transition metal ions, but EDTA did not facilitate the elimination of Au(III) ions from RNase
RNase A activity [%]
100 80 60 40 20 0 4
6
8
10
12
10
12
pH 120
Au(III)-binding ratio [%]
histidine residue [28], a histidine residue could be one of the Au-binding sites in RNase. We next investigated the elimination of Au(III) ions from a Au(III)-RNase A complex using thiourea. Thiourea has a strong interaction with precious metal ions [30]. It reduces Au(III) ions to Au(I) ions and forms a 2:1 complex [31]. Addition of thiourea to the Au(III)-inhibited RNase A resulted in the recovery of RNase activity (Fig. 1b). At a Au(III) ion/RNase A molar ratio of 3, 85% of the intrinsic activity was recovered from the completely inhibited RNase A. Under these conditions, 90% of the Au(III) ions bound to RNase A were eliminated from RNase. The recovered activity decreased with an increasing Au(III) ion/RNase A molar ratio. This was because the elimination of Au(III) ions from RNase A was not sufficient at high Au(III) ion/RNase A molar ratios. In fact, more than two Au(III) ions remained per RNase A molecule after the addition of thiourea at a molar ratio of 10, while less than one Au(III) ion remained at a Au(III) ion/RNase A molar ratio of 3.
183
100 80 60 40 20 0
4
6
8
pH Fig. 2. (a) Effect of pH on the Au(III)-induced inhibition and renaturation of RNase A. Closed circles represent the Au(III)-inhibited RNase activity and open circles represent the recovered RNase A activity. (b) Effect of pH on the Au(III) binding ratio. The RNase A concentration was 33 lM, the Au(III) concentration was 100 lM and the thiourea concentration was 4 mM. Hundred percent RNase activity was defined as the native RNase A activity at each pH in the absence of Au(III) ions.
A. Mercaptoethanol showed relatively high recovery of the RNase activity. The results for Cys and mercaptoethanol mean that a thiol group is effective at eliminating Au(III) ions, resulting in recovering of the activity of the Au(III)-inhibited RNase A. The recovery of RNase activity coinciding with Au(III)-elimination suggests that the interaction of RNase with Au(III) ions did not reduce Au(III) ions to produce gold nanoparticles. Since Cys and mercaptoethanol can also act as reducing compounds that break disulfide bonds in RNase A molecules, the following experiments were carried out using thiourea as the Au-eliminating agent. 3.4. Effect of the thiourea/Au(III) ion molar ratio on the recovery of the Au(III)-inhibited RNase A activity The effect of the thiourea/Au(III) ion molar ratio was investigated (Fig. 4). As the thiourea/Au ratio increased, the relative activity of the renatured RNase A increased. When the molar ratio exceeded 10, the recovered activity reached a maximum of around 90%. We confirmed that thiourea itself does not affect the activity of native RNase
T. Maruyama et al. / Journal of Inorganic Biochemistry 101 (2007) 180–186
0.5 80 60 40 20
Th io
Ly s As n G ly G G H M E ED TA
ea C ys M et H is G lu Ph e
0
Absorbance at 286 nm
100
ur
Au(III) elimination ratio [%]
184
0.4 0.3 0.2 0.1 0 0
100
10
20
30
40
Time [min] Fig. 5. On/off switching of hydrolysis of cytidine 2 0 ,3 0 -cyclic monophosphate (2 g/l) catalyzed by RNase A (2.5 lM). White arrow indicates the addition of a Au(III) solution (6 ll, 5 mM) to the reaction solution (2.6 ml). Black arrow indicates the addition of a thiourea solution (10 ll, 0.1 M) to the reaction solution.
80 60 40 20
Th io u
Ly s As n G ly G G H M E ED TA
0 re a C ys M et H is G lu Ph e
Recovery of RNase A Activity [%]
Au(III)-complexing or reducing reagent
Au(III)-complexing or reducing reagent
Fig. 3. (a) Au(III) elimination from Au(III)-inhibited RNase A by addition of various Au-complexing agents. (b) Activity recovery of Au(III)-inhibited RNase A by addition of various complexing agents. The pH was 7.0 and other experimental conditions were the same as those in Fig. 2. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions. Abbreviations: GGH, Gly-Gly-His; ME, mercaptoethanol and EDTA, ethylenediamine tetraacetic acid (2Na).
monophosphate was started by the addition of RNase A. The addition of Au(III) ions markedly reduced the hydrolysis rate, to only 8% of its initial reaction rate. As soon as the thiourea was added, the reaction was stimulated and progressed sharply. The reaction rate was 8-fold faster than that observed prior to the addition of thiourea. It should be noted that the addition of Au(III) ions and thiourea had little effect on the pH of the reaction medium and also that the Au(III) and thiourea solutions did not have detectable absorbance at 286 nm.
100
The conformational change of RNase A was investigated by measuring CD spectra of native, Au(III)-coordinated and renatured RNase A (Fig. 6). The addition of Au(III) ions reduced the negative bands at 208 nm,
80 60 40 20 0
10
20
30
40
50
Thiourea/Au molar ratio Fig. 4. Effect of thiourea concentration on the recovery of RNase activity. The experimental conditions were the same as those in Fig. 3. Hundred percent RNase activity was defined as the native RNase A activity in the absence of Au(III) ions.
A (data not shown). Atomic absorbance analyses demonstrated that excess thiourea (40 molar equivalents) resulted in a 90% Au(III) elimination ratio, meaning that 0.3 Au(III) ions per RNase A molecule were still coordinated. 3.5. On/off switching of RNase activity We next studied the on/off switching of an RNase A-catalyzed reaction based on the binding and elimination of Au(III) ions (Fig. 5). The hydrolysis of cytidine 2 0 ,3 0 -cyclic
Molecular rotation:
10
0
10-3 × θ [deg•cm2/dmol]
Recovery of RNase A activity [%]
3.6. CD spectra of RNase A in the absence and presence of Au(III) ions
5 0 -5 -10 -15 190 200 210 220
230 240 250
Wavelength [nm] Fig. 6. Circular dichroism spectra of RNase A. The red line with circles represents the spectrum of native RNase A, the blue line with squares represents that of Au(III)-inhibited RNase A and the green line with triangles represents that of RNase A renatured by thiourea. The RNase A concentration was 0.66 lM, the Au(III) concentration was 2.0 lM and the thiourea concentration was 80 lM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
T. Maruyama et al. / Journal of Inorganic Biochemistry 101 (2007) 180–186
DNase I activity [%]
100 Au Thiourea
80 60 40 20 0
0
20
40
60
Au(III)/DNase I molar ratio Fig. 7. Effect of Au(III) binding (closed circles) and Au(III) elimination (open circles) on DNase I. Hundred percent DNase activity was defined as the native DNase I activity in the absence of Au(III) ions. The Au(III) concentration was fixed at 100 lM and the pH was 5.0. Other experimental conditions were the same as those in Fig. 1.
217 nm and 222 nm. Since these negative bands represent a-helix and b-sheet contents, the addition of Au(III) ions induced drastic conformational changes in the RNase A to reduce a-helix and b-sheet contents. Additionally, the largest negative band shifted from 208 nm to 204 nm. This implies an increase in the random coils present in the Au(III)-coordinated RNase A. After addition of thiourea, the negative bands at 208 nm, 217 nm and 222 nm increased and the largest negative band returned to 208 nm. However, the spectrum of the renatured RNase A was not the same as that of native RNase A. It should be noted that the addition of thiourea did not change the CD spectrum of native RNase A solution. 3.7. Reversible inhibition of DNase I by Au(III) ions The Au(III)-induced inhibition and thiourea-induced renaturation were also investigated for DNase I. Similar to the results for RNase A, the DNase activity decreased with an increasing Au(III) ion/DNase I ratio (Fig. 7). The DNase activity was depressed to less than 5% of its intrinsic activity at Au(III) ion/DNase I ratios over 10. Addition of thiourea also recovered the DNase activity. When the Au(III) ion/DNase I molar ratio was 10, approx. 80% of the intrinsic DNase activity was recovered. DNase I from bovine pancreas has 6 His residues and 4 Met residues but no free thiol group [32,33]. More Au(III) ions were, therefore, required for the inhibition of DNase I than for that of RNase A. 4. Discussion While the inhibition of RNase A by transition metal ions requires thousands of molar equivalents of those metal ions to RNase A [34,35], only 3 or 4 molar equivalents of Au(III) ions effectively inhibited RNase activity. Isab et al. and Craig reported that Au(III) ions caused protein aggregation at a high protein concentration (2 mM)
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[24,36]. We also confirmed the aggregation of RNase A at such a high concentration. Under our typical conditions, the RNase concentrations were around 33 lM, where no aggregation was observed. The addition of thiourea recovered the Au(III)-inhibited RNase activity, These results indicate that the inhibitory effect of Au(III) ions on RNase A is induced by complexation of Au(III) ions with RNase A molecules, neither to the aggregation of the RNase nor to the oxidation of RNase A molecules, although Au(III) ions are strong oxidants. The Lineweaver-Burk plot shows two sets of parallel lines (a supplementary material), suggesting two sets of the uncompetitive inhibition models. Alkylation of His residues by iodoacetic acid decreased Au-binding but did not lead to no Au-binding. These results indicate the binding mechanisms more than 2, but there is considerable uncertainty to discuss the inhibition manner. The study of Au(III) elimination using amino acids revealed that Cys, His and Met facilitated the elimination of Au(III) ions from an RNase A molecule. RNase A has 8 Cys, 4 His and 4 Met residues. All the Cys residues in an RNase A molecule form disulfide bonds. So the amino acid residues that could possibly interact with Au(III) ions in an RNase A molecule are His and Met residues. Several groups reported a strong interaction between a Au(III) ion and a histidine-containing peptide to form a Au(III)-peptide complex [21,22,37]. Zou et al first reported the crystal structure of gold/protein complex and demonstrated the specific Au(I)-binding to histidine residue [38]. The active site of RNase A has two histidine residues and the sub˚ wide strate pocket of RNase A is very narrow, approx. 5 A [39]. The bond lengths between Au(III) and the nitrogen ˚ , and the atomic radii of the Au atoms were 1.9–2.1 A ˚ and 0.5 A ˚ , respectively, and nitrogen atoms were 1.44 A meaning that the size of the substrate pocket matched the Au(III) coordination. These allow us to speculate that Au(III) ions are likely to be coordinated to His residues in RNase A. Initially, Au(III) ions were dissolved as [AuCl4] in an aqueous solution before formation of the Au(III)-RNase A complex. The stability of [AuCl4] tends to be influenced by the pH and the Cl concentration [40]. At higher pH, the Cl ligand can be exchanged for OH to produce [AuCl3(OH)], [AuCl2(OH)2] and so on. It is necessary to exchange Cl (or OH) ligands for the formation of the Au(III)-RNase A complex. This implies that the unstable [AuCln(OH)4n] complex tends to form the Au(III)RNase A complex. On the other hand, a high pH above 10 (high OH concentration) would also stabilize [Au(OH)4] by not allowing the exchange of OH ligands. Furthermore, an RNase A molecule is negatively charged at such a pH (pI of RNase A is 9.45). The negatively charged RNase A molecule might prevent [Au(OH)4] from accessing it, due to electronic repulsion. CD spectra suggest that the secondary structure of an RNase A molecule was denatured by Au(III) ions and was renatured by the addition of thiourea. However, the
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conformational refolding of the Au(III)-inhibited RNase A was only partial. Since Au(III) ions can oxidize amino acid residues [41], the irreversible oxidation of amino acid residues in a RNase molecule could be another reason for the incomplete recovery of RNase activity. In particular, Met and disulfide bonds are likely to be oxidized [24,42,43]. However, 90% of RNase activity was recovered by the addition of thiourea, suggesting that the majority of the Au-binding sites in RNase A would not be irreversibly oxidized by Au(III) ions. The present study shows that Au(III) ions were coordinated to RNase A and DNase I. The binding of Au(III) ions induced inhibition of the nuclease activity, accompanied by conformational change of the nuclease molecule. Au-complexing agents such as thiourea effectively eliminated Au(III) ions from the nucleases, resulting in recovery of the inhibited nuclease activity. This study suggests that Au(III) ions effectively facilitate partially reversible inhibition of RNase A and DNase I. Acknowledgements This research was supported by a Grant-in-Aid for the 21st Century COE Program, ‘‘Functional Innovation of Molecular Informatics’’ from the Ministry of Education, Culture, Science, Sports and Technology of Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio. 2006.09.021. References [1] G.R. Taylor, J. Deeble, Biomol. Eng. 14 (1999) 181–186. [2] J.G. Hall, P.S. Eis, S.M. Law, L.P. Reynaldo, J.R. Prudent, D.J. Marshall, H.T. Allawi, A.L. Mast, J.E. Dahlberg, R.W. Kwiatkowski, M. de Arruda, B.P. Neri, V.I. Lyamichev, Proc. Natl. Acad. Sci. USA 97 (2000) 8272–8277. [3] M.V. Myakishev, Y. Khripin, S. Hu, D.H. Hamer, Genome Res. 11 (2001) 163–169. [4] M.M. Goldrick, Hum. Mutat. 18 (2001) 190–204. [5] H. Ichinose, M. Kitaoka, N. Okamura, T. Maruyama, N. Kamiya, M. Goto, Anal. Chem. 7 (2005) 7047–7053. [6] J.T. Potts, A. Berger, J. Cooke, C.B. Anfinsen, J. Biol. Chem. 237 (1962) 1851–1855. [7] D.G. Smyth, W.H. Stein, S. Moore, J. Biol. Chem. 238 (1963) 277– 284. [8] G. Kartha, J. Bello, D. Harker, Nature 213 (1967) 862–865. [9] S. Moore, Third ed., in: P.D. Boyer, H. Lardy, K. Myrback (Eds.), The Enzymes, vol. 14, Academic Press, New York, 1981, pp. 281–294.
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