From the
Departments of Biochemistry and ¶Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
Received for publication, February 21, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Similar to most known ribonucleases, RNase A can make multiple contacts with an RNA substrate (Fig. 1). The enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions (5). The most potent RNase A inhibitors take advantage of this extended interface. For example, a pyrophosphate-linked oligonucleotide (pdUppA-3'-p), which occupies three subsites, is the tightest known small-molecule inhibitor of RNase A (4). Nature also uses this strategy to inhibit RNase A and its homologs. The 50-kDa ribonuclease inhibitor protein forms a tight 1:1 complex with RNase A (Kd 10-14 M) (6), chelating all of its phosphoryl group binding subsites (7). The utility of pyrophosphate-linked oligonucleotides and ribonuclease inhibitor is limited, both by the difficulty and expense of their production and by their intrinsic instability. For example, pyrophosphate-linked oligonucleotides are susceptible to hydrolysis (8), and ribonuclease inhibitor is readily inactivated by oxidation (9).
|
While studying RNase A catalysis as a function of salt concentration, we found that a contaminant in common biological buffers was a potent inhibitor in solutions of low salt concentrations (10). We estimated that Ki for this inhibitor was 0.1 nM, which is 102-fold lower than that for any other small-molecule RNase A inhibitor. Herein, we identified this inhibitor as a byproduct of the synthesis of commercial buffers containing sulfonylethyl groups. Next, we found that this inhibitor acts in a competitive manner and is the most potent known small-molecule inhibitor of a ribonuclease. In addition, we identified the number of Coulombic interactions that the inhibitor makes upon binding to RNase A. Finally, we examined RNase A inhibition by analogs of the inhibitor.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synthesis of Diethanesulfonic Acid Ether (3)2-Mercaptoethylether (5.0 g, 36.2 mmol, Caution: Stench!, Aldrich) was dissolved in glacial acetic acid (5 ml). The resulting solution was then cooled to 0 °C. While stirring at 0 °C, a mixture (50:45 ml) of glacial acetic acid and aqueous hydrogen peroxide (30% v/v) was added to the solution dropwise over 1 h. The reaction mixture was then heated at 60 °C for 90 min. The solvent was removed under reduced pressure. The addition of toluene enabled residual acetic acid to form azeotropes of low boiling point. The resulting yellow oil was used without further purification (spectral data: 1H NMR (300 MHz, D2O) 2.88 (t, J = 7.1 Hz, 4H), 2.17 (t, J = 7.0 Hz, 4H) ppm; mass spectrometry (electrospray ionization) m/z 232.9791 (M-H [C4H9O7S2] = 232.9795)).
InstrumentsUV absorbance measurements were made with a Cary Model 3 spectrophotometer (Varian, Palo Alto, CA). Fluorescence measurements were made with a QuantaMaster 1 photon counting fluorometer equipped with sample stirring (Photon Technology International, South Brunswick, NJ).
Production of RNase APlasmid pBXR (11) directs the production of RNase A in Escherichia coli. RNase A was produced and purified as described previously (12) with the following modifications. E. coli strain BL21(DE3) transformed with pBXR was grown to an optical density of 1.8 at 600 nm in terrific broth medium containing ampicillin (0.40 mg/ml). The expression of the RNase A cDNA was induced by the addition of isopropyl-1-thio--D-galactopyranoside to 0.5 mM. Cells were collected 4 h after induction and lysed with a French pressure cell. Inclusion bodies were recovered by centrifugation and resuspended for 2 h in 20 mM Tris-HCl buffer, pH 8.0, containing guanidine-HCl (7 M), dithiothreitol (0.10 M), and EDTA (10 mM). The protein solution was diluted 10-fold with aqueous acetic acid (20 mM), subjected to centrifugation to remove any precipitate, and dialyzed overnight against aqueous acetic acid (20 mM). Any precipitate was removed again by centrifugation. The supernatant was diluted to a protein concentration near 0.5 mg/ml in a refolding solution of 0.10 M Tris-HCl buffer, pH 8.0, containing NaCl (0.10 M), reduced glutathione (1.0 mM), and oxidized glutathione (0.2 mM). RNase A was refolded for 16 h and concentrated by ultrafiltration with a YM10 membrane (Mr 10,000 cut-off, Millipore, Bedford, MA). Concentrated RNase A was applied to a Superdex G-75 gel filtration fast protein liquid chromatography column (Amersham Biosciences) in 50 mM sodium acetate buffer, pH 5.0, containing NaCl (0.10 M) and NaN3 (0.02% w/v). Protein from the major A280 peak was collected and applied to a Mono S cation-exchange fast protein liquid chromatography column. RNase A was eluted from the column with a linear gradient of NaCl (0.20.4 M) in 50 mM sodium acetate buffer, pH 5.0. Protein concentration was determined by UV spectroscopy using
= 0.72 ml mg-1 cm-1 at 278 nm (13).
Inhibition of RNase A CatalysisInhibition of ribonucleolytic activity was measured by using either poly(C) or a fluorogenic substrate. The total cytidyl concentration of poly(C) was quantitated using = 6,200 M-1 cm-1 at 268 nm (14). The cleavage of poly(C) was monitored by the decrease in ultraviolet hypochromicity. The
value for this reaction calculated from the difference in molar absorptivity of the polymeric substrate and the mononucleotide cyclic phosphate product was 2,380 M M-1 cm-1 at 250 nm (15). Assays were performed at 25 °C in 50 mM imidazole-HCl buffer, pH 6.0, containing NaCl (0.10 M), poly(C) (10 µM-1.5 mM), OVS (01.43 µM), and enzyme (1.0 nM). Molar values of OVS were calculated by using its average molecular mass of 2,000 g/mol. It is possible that a polymer of this size could bind two enzymes. Thus, the actual Ki values could be 2-fold higher. Kinetic parameters were determined from initial velocity data with the program DELTAGRAPH 4.0 (DeltaPoint, Monterey, CA).
For the fluorescence assay, the inhibition of ribonucleolytic activity was assessed at 25 °C in 2.0 ml of 50 mM imidazole-HCl buffer, pH 6.0, containing NaCl (00.25 M), 6-FAMdArUdAdA
6-TAMRA (60 nM), and RNase A (15pM) as described previously (16, 17). Fluorescence (F) was measured using 493 and 515 nm as the excitation and emission wavelengths, respectively. The value of
F/
t was measured for 3 min after the addition of RNase A. An aliquot of inhibitor (I) dissolved in the assay buffer was added next, and
F/
t was measured in the presence of the inhibitor for 3 min. The concentration of inhibitor in the assay was doubled repeatedly in 3-min intervals. Excess RNase A was then added to the mixture to ensure that <10% substrate had been cleaved prior to completion of the inhibition assay. Apparent changes in ribonucleolytic activity due to dilution were corrected by comparing values to an assay in which aliquots of buffer were added to the assay. Values of Ki were determined by non-linear least squares regression analysis of data fitted to Equation 1 (16, 17).
![]() | (Eq. 1) |
At 0 M NaCl, the enzyme concentration ([E]total) caused a significant depletion in inhibitor concentration, thus the data were fitted to Equation 2, which describes tight-binding inhibition (18).
![]() | (Eq. 2) |
In Equations 1 and 2, (F/
t)0 was the ribonucleolytic activity prior to inhibitor addition.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of InhibitorIn ethanesulfonic acid buffer synthesis, a nucleophile attacks 2-bromoethanesulfonic acid in H2O to yield the buffer product (Fig. 2). Hydrolysis or -elimination of 2-bromoethanesulfonic acid could yield 2-hydroxyethanesulfonic acid (1) or vinylsulfonic acid (2). Nucleophilic attack of 2-hydroxyethanesulfonic acid on 2-bromoethanesulfonic acid or Michael addition to vinylsulfonic acid could generate diethanesulfonic acid ether (3). Indeed, all three of these byproducts were identified by NMR spectroscopy and mass spectrometry in the material purified from MES buffer (data not shown).
|
Commercial 2-hydroxyethanesulfonic acid (1) and vinylsulfonic acid (2) were tested as inhibitors of RNase A. Neither was a potent inhibitor in solutions of low salt concentration (Fig. 2). 3 Diethanesulfonic acid ether (3) was synthesized (vide supra) but likewise failed to inhibit RNase A. Thus, the sought-after inhibitor was not byproduct 1, 2, or 3.
We next used a Vivaspin concentrator (5,000 molecular weight cut-off, Vivascience AG, Hannover, Germany) to purify the inhibitor based on its affinity for RNase A. RNase A (10 mg) was mixed in ddH2O (10 ml) with the inhibitor (10 mg) that had been purified by anion-exchange chromatography. The sample was subjected to centrifugation at 6,000 rpm for 15 min, washed with ddH2O (3 x 15 ml), and subjected again to centrifugation. Molecules that bind tightly to RNase A remained in the retentate, whereas impurities were washed into the eluate. Matrix-assisted laser desorption ionization mass spectrometry of the retentate containing RNase A and the inhibitor revealed a heterogeneous mixture of small molecules of a molecular mass of 9002,000 g/mol. The inhibitor was then separated from RNase A by adding a solution of ammonium acetate (0.10 M) to the mixture. After repeatedly concentrating and adding ammonium acetate solution to the mixture, unbound inhibitor moved to the eluate, whereas RNase A remained in the retentate. Matrix-assisted laser desorption ionization mass spectrometry of the free inhibitor revealed the same heterogeneous distribution of molecular mass with individual peaks separated by 108 g/mol (Fig. 3). Because the molecular mass of vinylsulfonic acid (2) is 108 g/mol, we reasoned that the inhibitor was probably an oligomer of vinylsulfonic acid (OVS, 4). Similar to byproducts 1-3, OVS is probably a byproduct of ethanesulfonic acid buffer synthesis with ultraviolet light possibly initiating the radical-mediated polymerization of vinylsulfonic acid (3) (Fig. 2) (23).
|
Characterization of Inhibition by OVSInhibition of RNase A activity was measured in 0.05 M imidazole-HCl buffer, pH 6.0, containing NaCl (0.10 M) and commercial OVS (Mr 2,000, 01.43 µM). OVS inhibition of RNase A is not time-dependent (data not shown). The addition of NaCl diminishes the OVS inhibition of RNase A, indicating that OVS is a reversible inhibitor of the enzyme. OVS inhibits RNase A at concentrations well below that of substrate; thus, inhibition by OVS is not attributed to its sequestering of RNA. Double-reciprocal plots of RNase A catalytic activity versus the concentration of poly(C) at different OVS concentrations reveal that OVS inhibits RNase A in a competitive manner (Fig. 4). With poly(C) as a substrate, the apparent Ki = (0.40 ± 0.03) µM at 0.10 M NaCl. A replot of (Km/Vmax)app versus [OVS] reveals a straight line, which is indicative of simple competitive inhibition (24).
|
Salt Dependence of Inhibition by OVSThe Ki of OVS was measured at four different salt concentrations in 50 mM imidazole-HCl buffer, pH 6.0. Because OVS inhibits RNase A in a competitive manner, we were able to use a sensitive fluorescent assay to assess inhibition by OVS. OVS inhibition of RNase A is highly salt-dependent (Fig. 5A). At 0 M NaCl, OVS inhibits catalysis by RNase A with an astonishingly low inhibition constant of Ki = (11 ± 2) pM. At 0 M NaCl, the inhibition curve was fitted to a tight-binding inhibitor equation, yet the curve still exhibits some cooperativity. At 0.10 M NaCl, OVS inhibits RNase A with an inhibition constant of Ki = (120 ± 10) nM.4
|
According to polyelectrolyte theory, the slope of a plot of log(Ki) versus log([cation]) reveals the number of Coulombic interactions between a ligand and a polyanion (28). OVS makes on average 7.8 ionic interactions with RNase A (Fig. 5B). Poly-(vinylsulfuric acid), an OVS analog, exhibits a similar salt dependence (data not shown).
RNase A Inhibition by OVS AnalogsTo assess the importance of the sulfonic acid group for RNase A inhibition, we tested poly(vinylphosphonic acid) (PVP) and poly(vinylsulfuric acid) (PVOS) for inhibition of ribonucleolytic activity in our fluorescent assay. These analogs are also good inhibitors of RNase A but are slightly less effective than is OVS (Table I). The average molecular masses of PVP and PVOS were 20,000 and 170,000 g/mol, respectively. Nevertheless, by mass spectrometry, the minimum number of OVS units that bound tightly to RNase A was nine. Thus, each chain of commercial OVS (2,000 g/mol) could tightly bind to two RNase A molecules per chain, whereas each chain of PVP or PVOS could tightly bind to more. Hence, to enable a direct comparison of inhibition by OVS, PVP, and PVOS, the data listed in Table I are in units of mass rather than moles.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After our identification of OVS in MES buffer, we found a previous report (29) that large polymers (50,000 g/mol) of poly(vinylsulfonic acid) comprised
1% of a single lot of MES buffer (29). This lot of MES buffer inhibited the catalytic activity of 6-phosphogluconate dehydrogenase. Other lots of MES buffer failed to inhibit the enzyme because only long polymers were inhibitory (30). We suspect that oligo(vinylsulfonic acid) and occasionally poly(vinylsulfonic acid) contaminate commercial MES buffer and other ethanesulfonic acid buffers and that the amount of these contaminants varies from lot to lot.
Kinetic AnalysesOVS inhibition of RNase A follows a simple competitive model (Fig. 4). Because OVS (2000 g/mol) has on average only 18 monomer units per molecule, it is on the cusp of consideration as a polyelectrolyte (28). Nonetheless, a double-log plot of Ki versus [cation] indicates that OVS forms 7.8 Coulombic interactions with RNase A (Fig. 5B). The inhibition of RNase A by poly(vinylsulfuric acid) (
170,000 g/mol) shows a similar salt dependence (data not shown). The number of Coulombic interactions between OVS and RNase A is in gratifying agreement with a previous report (28) that single-stranded DNA forms 7 Coulombic interactions with RNase A. Thus, OVS probably saturates the same phosphoryl group binding subsites as does a single-stranded nucleic acid (Fig. 1).
Multivalent InhibitionPolyanions are known to be effective inhibitors of RNase A (27). Heparin, tyrosine-glutamate copolymers, and many different polysulfates and polyphosphates have been shown previously to inhibit catalysis by the enzyme (26, 31, 32). We were surprised to learn that 40 years ago, even poly(vinylsulfonic acid) had been tested as an inhibitor of RNase A. Those data suggested that poly(vinylsulfonic acid) was a worse inhibitor of RNase A than other polyanions (33, 34), or alternatively, that only long polymers (>9,000 g/mol) were good inhibitors of RNase A (35). We do not know the basis for the disparity with our data.
OVS, like PVP and PVOS, is similar to a nucleic acid backbone in having anionic non-bridging oxygen atoms. In addition, the phosphorous atoms in a nucleic acid and alternating sulfur atoms in OVS are separated by five other atoms. However, there is a major difference between OVS and a nucleic acid. With its three non-bridging oxygens per monomer unit, OVS provides many more opportunities to form strong hydrogen bonds than does a nucleic acid. Pyrophosphate-linked ribonuclease inhibitors also display extra non-bridging oxygens, which probably enhance their affinity for RNase A (8).
OVS compares favorably with the most potent known small-molecule inhibitor of RNase A, a pyrophosphate-linked oligonucleotide, pdUppA-3'-p (4). Under similar buffer conditions with 0.10 M NaCl, each has a Ki near 120 nM. Yet, unlike pdUppA-3'-p, OVS is simple to prepare and is extremely stable. Accordingly, OVS could be useful in preventing incidental ribonuclease contamination and RNA degradation in experiments involving RNA. Indeed, poly(vinylsulfuric acid), an OVS analog, has been added to experiments involving the isolation of mRNA (36) or cell-free translation (37).
Other enzymes are known to be inhibited by poly(vinylsulfonic acid). For example, poly(vinylsulfonic acid) inhibits catalysis by RNA polymerase and reverse transcriptase (38, 39). We believe that OVS could be an inhibitor of any enzyme that binds strands of RNA or DNA.
Buffer ContaminationThe presence of OVS in all lots of MES buffer tested herein and in many other ethanesulfonic acid buffers is troubling. The amount of OVS varies from lot to lot, and thus, some lots of buffers could contain high concentrations of OVS. We recommend that all ethansulfonic acid buffers be purified by anion-exchange chromatography prior to their use in assays of enzymatic activity. Alternatively, an OVS-free buffer should be used instead. Imidazole, bis-tris, and Tris buffer are suitable alternatives, depending on the pH of the assay.
MES buffer has been the buffer of choice in assays of the catalytic activity of RNase A, as the pKa of MES buffer (pKa = 6.15) (19) is near the pH of maximal activity (pH = 6.0) (40). Many RNase A assays are performed in the presence of 0.10 M NaCl at which the Ki of OVS is 120 nM (Fig. 5A). We find that the OVS concentration in many lots of MES buffer is near 2 ppm. In 0.10 M MES buffer, the concentration of OVS is near 0.2 µM, which is greater than its Ki value. Historically, RNase A has been reported to have a bell-shaped salt-rate profile with an optimum salt concentration near 0.1 M NaCl (41, 42, 43). We believe that this observed bell shape is an artifact because of contaminating OVS in MES buffer. Indeed, the salt-rate profile of RNase A has been measured recently in bis-tris buffer, revealing that ribonucleolytic activity increases to the diffusion limit as salt concentration decreases (10, 44, 45).
![]() |
CONCLUSIONS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
Supported by a Wisconsin Alumni Research Foundation predoctoral fellowship and Biotechnology Training Grant 08349 from the National Institutes of Health.
|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-262-8588; Fax: 608-262-3453; E-mail: Raines{at}biochem.wisc.edu.
1 The abbreviations used are: RNase A, ribonuclease A; 6-FAM, 6-carboxyfluorescein; 6-TAMRA, 6-carboxytetramethylrhodamine; MES, 2-(N-morpholino)ethanesulfonic acid; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; OVS, oligo(vinylsulfonic acid); poly(C), poly(cytidylic acid); PVP, poly(vinylphosphonic acid); PVOS, poly(vinylsulfuric acid); F, fluorescence; ddH2O, double distilled H2O; bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
2 Of note, the flow-through of this column can be recrystallized from water to yield MES buffer that is devoid of inhibitor.
3 In contrast, divinylsulfone (CH2CHS(O)2CHCH2) is an irreversible inhibitor of RNase A, forming covalent bonds to active-site residues by Michael addition (21). Mechanism-based inactivation of RNase A by Michael addition has also been described previously (22).
4 In theory, the value of Ki for a competitive inhibitor should be independent of the substrate used in the assay. Yet, the observed value of Ki for OVS is 3-fold higher when poly(C) rather than 6-FAMdArUdAdA
6-TAMRA is the substrate for RNase A. This effect of polymeric substrates has much precedence and several proposed explanations (25, 26, 27).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|