Measuring Dissociation Constants of RNA and Aminoglycoside Antibiotics by Electrospray Ionization Mass Spectrometry

Analytical Biochemistry 280, 264 –271 (2000) doi:10.1006/abio.2000.4550, available online at http://www.idealibrary.com on

Measuring Dissociation Constants of RNA and Aminoglycoside Antibiotics by Electrospray Ionization Mass Spectrometry Kristin A. Sannes-Lowery, Richard H. Griffey, and Steven A. Hofstadler 1 Ibis Therapeutics, Division of Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, California 92008

Received November 11, 1999

Electrospray ionization mass spectrometry (ESIMS) has been used to determine the dissociation constants (K Ds) and binding stoichiometry for tobramycin and paromomycin with a 27-nucleotide RNA construct representing the A-site of the 16S ribosomal RNA. K D values determined by holding the ligand concentration fixed are compared with K D values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt present in the solution on the K D measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS is holding the RNA target concentration fixed below the expected K D and titrating the ligand. K D measurements should also be carried out at as high as possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding sites were found with K D1 ⴝ 352 nM and K D2 ⴝ 9 ␮M. For paromomycin, there is only one binding site with K D ⴝ 52 nM. © 2000 Academic Press

Many important biological functions are mediated through noncovalent interactions between proteins, RNA, DNA, enzymes, and cofactors. Traditionally, proteins have been targeted for drug discovery efforts because folded proteins provide structures for specific recognition by ligands. RNA also has well-defined tertiary structures that make RNA an attractive candidate for small molecule binding (1). For example, the 16S ribosomal RNA is part of the 30S ribosomal subunit involved in prokaryotic translation. The A-site of 1 To whom correspondence should be addressed. Fax: (760) 4312768. E-mail: [email protected].

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the 16S rRNA binds the charged aminoacyl tRNA corresponding to the next mRNA codon in the transcript. Aminoglycoside antibiotics are known to bind to the A-site of the 16S rRNA and interfere with protein translation in the ribosome, which leads to cell death (2, 3). The specificity of the interaction between the aminoglycoside antibiotic and the 16S A-site is governed by the three-dimensional structure of the rRNA as well as electrostatic and base-specific interactions between the antibiotic and the 16S A-site. By determining solution dissociation constants (K D) and stoichiometry of the complexes formed, information about the specificity and strength of the interactions is obtained. Electrospray ionization mass spectrometry (ESIMS) 2 has shown promise for studying noncovalent interactions including ligand–RNA interactions (4 –7). ESI is a very gentle ionization technique that propagates noncovalent complexes formed in solution into the gas phase where they can be characterized by mass spectrometric analysis. ESI-MS has previously been used to determine the stoichiometry and dissociation constants for protein–protein interactions, protein–ligand interactions, and protein– oligonucleotide interactions (8 –15). Using mass spectrometry it is possible to simultaneously detect free ligand, free target, and ligand–target complex(es). In contrast, solution-phase methods such as radioimmunoassays, filter assays, and surface plasmon resonance assays typically measure only the equilibrium concentration of either the free ligand or the complex(es) and provide little or no information pertaining to the binding stoichiometry of the complexes (16). In a few cases gel mobility shift assays have been used to show that multiple aminoglycosides bind to TAR RNA, but in those studies ESI 2 Abbreviations used: ESI-MS, electrospray ionization mass spectrometry; DMSO, dimethyl sulfoxide.

0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

MEASURING DISSOCIATION CONSTANTS OF RNA AND AMINOGLYCOSIDE

mass spectrometry was still needed to determine that the correct binding stoichiometry was three aminoglycosides per RNA (and not two as indicated by the assay) (17–19). Furthermore, gel mobility shift assay cannot be generally used for monitoring small ligand binding to RNA; the highly charged aminoglycosides are a special case. In this work we investigate three ESI-MS-based experimental protocols employing different solution conditions to determine dissociation constants. First, K D values derived by titrating the RNA target while holding the ligand concentration fixed are compared to K D values derived by titrating the ligand while holding the RNA target concentration fixed. Additionally, when the ligand is titrated, the effect of the RNA target concentration on the K D measurement is evaluated. Finally, the effect of solution conditions such as amount of organic solvent present in solution and the amount of salt present in solution on the K D measurement is investigated. The K D values measured with these approaches are compared with previously published values (20 –23). EXPERIMENTAL

Instrumentation. All experiments were performed on a Finnigan (San Jose, CA) LCQ quadrupole ion trap mass spectrometer equipped with an electrospray ionization source and operated in the negative ion mode. An analyte flow rate of 3 ␮L/min was used for all samples. The sheath and auxiliary gases were a 50/50 mixture of nitrogen and oxygen. Because the positively charged aminoglycosides adhere to fused silica capillaries, the standard fused silica capillary transfer line was replaced with a 127-␮m i.d. peek tubing transfer line to minimize sample carryover. To further minimize carryover, the transfer line was rinsed with 500 ␮L of 1 M glycine and 1 mL of deionized water between each sample and samples were run from low to high ligand/RNA concentrations. The heated metal capillary was maintained at 215°C for all experiments. Under these experimental conditions, no dissociation of the RNA–aminoglycoside complex is observed. To improve signal to noise, the mass spectra were collected over a narrow mass range (1600 –2000 m/z) and 50 spectra were averaged for each measurement. For each sample, three mass spectra were collected. In order to allow a direct comparison of ion abundances from samples with different ligand and/or RNA concentrations, the automatic gain control mode of operation was not employed. Instead, the ion injection time was held at 75 ms for low concentration solutions (ⱕ50 nM) and 25 ms for high concentration solutions (ⱖ500 nM). Chemicals. Tobramycin (MW ave ⫽ 467.5) and paromomycin (MW ave ⫽ 615.6) were purchased from Sigma (St. Louis, MO) and used without further purification

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(Fig. 1). A 27-mer nucleotide RNA (MW ave ⫽ 8639.3) containing the essential components of the 16S rRNA A-site (24), 16S, was purchased from Dharmacon (Boulder, CO) and deprotected immediately prior to use (Fig. 1). The RNA was dialyzed against 100 mM ammonium acetate using a Millipore membrane disk to reduce adduct ions of sodium and potassium cations. Unless otherwise specified, the RNA/aminoglycoside solutions were prepared in a 150 mM ammonium acetate/50% (v/v) isopropyl alcohol solution and allowed to equilibrate for 10 min before analysis. RESULTS AND DISCUSSION

Determination of RNA target and complex concentrations. Over the narrow mass range analyzed (1600 – 2000 m/z), only the 5⫺ charge state of the free RNA target and the RNA complexes are observed. Under the solution conditions used, 95% of the RNA signal comes from the 5⫺ charge state. Interestingly, the complexation of an aminogylcoside to the RNA does not change the observed charge state. Thus, contributions to the signal from other charge states were not taken into account for these analyses. For each mass spectrum, the peak areas for the free RNA target (plus up to five sodium cation adducts) and the RNA complexes (plus up to five sodium cation adducts) were calculated using standard numerical integration routines. In these studies, the mass of the ligand is small relative to that of the RNA target (i.e., ⬍10%) and, as previously observed by others, the binding of the ligand to the RNA target does not appear to alter the ionization efficiency of the complex (4, 14, 25). Therefore, for all calculations it was assumed that the free RNA target and the RNA complexes have similar ionization efficiencies and that a correction factor for different ionization efficiencies is not needed. Furthermore, it was assumed that the measured peak areas correlate with the solution concentrations; a reasonable assumption considering the relatively low concentrations of target and ligand employed in these studies (26). Since the initial concentration of the RNA target [R i] is always known and [R i] ⫽ [R] ⫹ [RL] ⫹ [RL 2], where R is the free RNA target, RL is the 1:1 ligand–RNA target complex, and RL 2 is the 2:1 ligand–RNA target complex, the equilibrium concentrations of the free RNA target and complexes can be calculated directly from the measured peak areas. For each sample, the calculated equilibrium concentrations from three different mass spectra are averaged together and used in the analysis described below. Determination of dissociation constants. Typically, dissociation constants are graphically determined from Scatchard analysis, which assumes that the binding sites are independent and equivalent (27). For Scatchard analysis, r/[L] is plotted against r where r is

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FIG. 1.

Structures of tobramycin, paromomycin and the 16S A-site of E. coli.

defined as the ratio of bound ligand to total amount of RNA target (Eq. [1]).

the case where there is only one ligand binding site, the dissociation constant is

r/关L兴 ⫽ p/K D ⫺ r/K D

K D ⫽ 关R兴关L兴/关RL兴

[1]

The K D is calculated from the slope, whereas p, the number of independent and equivalent binding sites, is calculated from the intercept. Scatchard analysis works well for solution phase binding assays were only the free ligand concentration or the concentration of all complexes is measured and the stoichiometry of the complexes is unknown. Using ESI-MS the stoichiometry and abundance of complexes, as well as the abundance of the free RNA target, can be simultaneously determined. Since the number of binding sites for a given ligand–RNA target combination are determined directly from the spectra, RNA targets with a single binding site and with two binding sites can be examined as individual cases. For

[2]

where R is the free RNA target, L is the free ligand, and RL is the 1:1 ligand–RNA target complex. The initial ligand concentration [L i] is: 关Li兴 ⫽ 关L兴 ⫹ 关RL兴.

[3]

Thus, substituting Eq. [3] into Eq. [2] yields: 关RL兴/关R兴 ⫽ 共1/K D兲共关Li兴 ⫺ 关RL兴兲.

[4]

A plot of [RL]/[R] vs [RL] yields a straight line with slope ⫺1/K D and intercept [L i]/K D. Conversely, a plot of [RL]/[R] vs [L i] produces a straight line with slope 1/K D

MEASURING DISSOCIATION CONSTANTS OF RNA AND AMINOGLYCOSIDE

and an intercept of zero since there is no complex formed at zero ligand concentration. When performing a linear least-squares fit of the data, the intercept can be set to zero giving an extra data point. The uncertainty in the slope derived from the linear leastsquares fit translates into an absolute uncertainty in the K D measurement. It is this uncertainty that we report in the K D measurements derived throughout this work. For the case where there are two binding sites that are not necessarily equivalent, the dissociation constants are K D1 ⫽ 关R兴关L兴/关RL兴

[5a]

K D2 ⫽ 关RL兴关L兴/关RL2 兴,

[5b]

where RL 2 is the 2:1 ligand–RNA target complex. Alternatively, the equilibrium constants below can also describe two nonequivalent binding sites K 1 ⫽ 关R兴关L兴/关RL兴

[6a]

K 2 ⫽ 关R兴关L兴 2 /关RL2 兴,

[6b]

where K 1 ⫽ K D1 and K 2 ⫽ K D1 * K D2. Using both sets of equations and following the derivation in van Holde’s Physical Biochemistry (28), the relationship below is derived: 共关R兴 ⫹ 关RL] ⫹ [RL2 兴兲/关R兴 ⫽ 关L兴 2 /共K D1K D2兲 ⫹ 关L兴/K D1 ⫹ 1.

[7]

The above equation does not depend on the mechanism by which the ligands bind to the RNA target. Unfortunately, this means that using Eq. [7] to analyze the data does not give any information about the binding mechanism. Equation [7] can be modified to include the case where two different ligands (L a and L b) bind to the same target by replacing the [L] 2 term with [L a][L b] and the [L] term with [L a]. Since the amount of bound ligand is small relative to the total amount of ligand, we assume that [L] ⫽ [L i], where [L i] is the initial concentration of ligand. Thus, K D1 and K D2 can be calculated from a binomial fit of ([R] ⫹ [RL] ⫹ [RL 2])/[R] vs [L i]. The intercept will be 1 at [L i] ⫽ 0 and can be used as an additional data point. Based on the above equations, there are several possible ways that can be considered for measurement of dissociation constants: (1) The concentration of the ligand can be held at or below the expected K D and the RNA target can be titrated through the K D; (2) the concentration of the RNA target can be held at or below the expected K D and the ligand titrated through the

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K D; (3) the concentration of the RNA target can be held above the expected K D and the ligand titrated through the K D. Background. There are several solution phase methods for determining dissociation constants. These methods are often laborious and may require large quantities of material. Typically, only the equilibrium concentration of either the free ligand or the complex(es) is experimentally measured (which in some cases may perturb the system equilibrium and skew the results) (16). Since the stoichiometry of the complexes formed cannot be easily determined by solution phase methods, a single dissociation constant that is an average of multiple dissociation constants may actually be calculated. Wong and co-workers used surface plasmon resonance to calculate the dissociation constants for several aminoglycoside antibiotics to the A-site of the 16S rRNA (20). The authors estimate the K D values to be accurate to within a factor of 3. By comparing dissociation constants for the 16S A-site with mutants under different salt conditions, it was demonstrated that specific binding increased with increasing salt concentration while nonspecific binding decreased. This behavior is consistent with nonspecific binding to RNA involving primarily charge– charge interactions while specific binding involves other interactions such as hydrogenbonding, base-specific interactions and ␲-stacking. Puglisi and co-workers determined a K D value for paromomycin with the 16S A-site using quantitative footprinting experiments with DMS (22). Rando and coworkers determined the K D values for several aminoglycosides using fluorescence anisotropy (21). In this work, we determine the dissociation constants for tobramycin and paromomycin with 16S A-site using ESI-MS and compare the results with previously published values. K D determination of tobramycin and 16S A-site. Using ESI-MS, the K D of tobramycin for 16S was measured by holding the tobramycin concentration at 500 nM and titrating the 16S from 200 nM to 1 ␮M. Measurements using 16S concentrations outside this range were complicated by signal to noise and carryover concerns making accurate determination of both the free 16S and complex difficult. Only a 1:1 complex was formed between tobramycin and 16S under these conditions (a negligible amount of the 2:1 complex is observed at 200 nM RNA). A linear least-squares fit of a plot of [RL]/[R] versus [RL] gives a straight line with the equation [RL]/[R] ⫽ 2.33–1.71 ⫻ 10 6[RL] (R 2 ⫽ 0.89); a K D of 674 nM (⫾164 nM) is calculated from ⫺1/slope while a K D of 226 nM (⫾16 nM) is calculated from [L i]/intercept (data not shown). The inconsistency between the K Ds calculated from the slope and intercept makes this method of K D determination unreliable

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in this case. The inconsistency may be due to the fact that the free ligand concentration is not monitored under the experimental conditions used. K D values for ligand–protein systems have been measured successfully with this approach (9). In an alternative approach, the K D of tobramycin for the 16S was measured by holding the 16S concentration at 500 nM and titrating the tobramycin concentration from 100 nM to 4 ␮M. At tobramycin concentrations ⱖ1 ␮M, a 2:1 complex of tobramycin and 16S was observed in addition to the 1:1 complex. The 5⫺ charge state of the free 16S, the 1:1 complex and 2:1 complex of tobramycin and 16S are shown in Fig. 2A. Figure 2B shows the plot of the data using Eq. [7]. A binomial fit of the data gives a correlation coefficient of 0.99; K D1 was calculated to be 352 nM and K D2 was calculated to be 9 ␮M. The data are consistent with there being two nonequivalent binding sites on the 16S A-site for tobramycin, with more affinity and specificity at the first binding site. The first binding site most likely corresponds to the A-site bulge where there are contributions to binding from electrostatic interactions, hydrogen bonding, and ␲ stacking (23). The second binding site in all likelihood involves nonspecific binding primarily due to electrostatic interactions. The above data can be also interpreted using Scatchard analysis (Fig. 2C). A linear least-squares fit of the data gives a correlation coefficient of 0.77, indicating that there is some curvature to the data. The K D calculated from the slope is 1.1 ␮M and the number of binding sites calculated from the intercept is 1.9. The K D calculated in this manner agrees quite well with the K D of 1.5 ␮M determined by Wong et al. and with the K D of 1.69 ␮M determined by Rando et al. (20, 21). It is not surprising that K D value from the Scatchard analysis agrees with that of Wong and co-workers since they also used Scatchard analysis to evaluate their data as well as holding the RNA concentration fixed in their method. However, Scatchard analysis assumes that the two binding sites are equivalent as well as independent. The binding sites in this case are clearly not equivalent, as evidenced by the observation of a 1:1 stoichiometry at low tobramycin concentration and both a 1:1 and 2:1 stoichiometry at higher tobramycin concentrations. Thus, these binding sites would be expected to have different dissociation constants as described using Eq. [7]. The effect of decreasing the salt concentration from 150 to 50 mM NH 4OAc on the K D measurement for tobramycin was evaluated. With 50 mM NH 4OAc/10% IPA solution conditions, the 16S concentration was held at 500 nM and tobramycin was titrated from 100 nM to 1 ␮M. In this case, the 2:1 tobramycin–16S complex was observed at tobramycin concentrations ⱖ600 nM. The data were plotted using Eq. [7] and K D1 was calculated to be 358 nM, while K D2 was calculated

FIG. 2. (A) ESI mass spectrum of 500 nM 16S A-site plus 1 ␮M tobramycin showing free 16S (16S), 1:1 tobramycin:16S complex (16S ⫹ Tob) and 2:1 tobramycin:16S complex (16S ⫹ 2 Tob). The respective sodium cation adducts are labeled with an *. (B) K D s for tobramycin and 16S were determined by holding the 16S concentration at 500 nM and titrating tobramycin from 100 nM to 4 ␮M. A binomial fit gives a correlation coefficient of 0.99. K D1 ⫽ 352 nM and K D2 ⫽ 9 ␮M. The solution conditions were 150 mM NH 4 OAc/ 50% IPA. (C) Using Scatchard analysis, the slope gives a K D of 1.1 ␮M and the intercept gives a p of 1.9. A linear least-squares fit gives a correlation coefficient of 0.77. Each data point is an average of the equilibrium concentrations from three mass spectra. The error bars on the data points represent ⫾ one standard deviation of the equilibrium concentrations from the individual mass spectra.

MEASURING DISSOCIATION CONSTANTS OF RNA AND AMINOGLYCOSIDE

FIG. 3. The K Ds for tobramycin and 16S A-site were determined by holding the 16S A-site concentration at 500nM and titrating tobramycin from 100 nM to 1 ␮M. A binomial fit gives a correlation coefficient of 0.99. K D1 ⫽ 358 nM and K D2 ⫽ 6 ␮M. The solutions conditions were 50 mM NH 4OAc/10% IPA/1% DMSO/10 mM EDTA. EDTA was added to help eliminate persistent copper adduct ions.

to be 6 ␮M (Fig. 3). Under both high and low salt conditions, the K D1s are the same, while the K D2s differ, again consistent with specific binding at the A-site bulge and a second nonspecific binding site primarily due to electrostatic interactions. K D2 is smaller at lower salt concentration, as expected for a binding site mediated by electrostatic interactions as there are fewer NH 4⫹ ions competing with tobramycin for binding to the 16S. These results are in accordance with the influence of ionic strength observed by Wong and co-workers (20). K D determination of paromomycin and 16S A-site. With 150 mM NH 4OAc/50% IPA solution, the K D of paromomycin was measured by holding the 16S concentration at 50 nM and titrating the paromomycin concentration through the expected K D. Only a 1:1 complex was observed at all concentrations of paromomycin. Using Eq. [4], [RL]/[R] was plotted versus [L i]; from the slope of the least-squares fit ([RL]/[R] ⫽ 1.92 ⫻ 10 7[L i]; R 2 ⫽ 0.97), a K D of 52 nM (⫾4 nM) was calculated (data not shown). The calculated K D is within a factor of 4 of the value (200 nM) determined with surface plasmon resonance and quantitative footprinting studies (20, 22). Additionally, this value is within a factor of 2 of the value previously reported using ESI–FTICR (23); in that work nonspecific ligand binding to the transfer line may have skewed the titration curved toward higher K D values. All of these K D values differ significantly from the value (1.85 ␮M) determined by fluorescence anisotropy (21). As noted under Experimental, all solutions contained 50% isopropyl alcohol by volume, which helps ESI sensitivity and desolvation. In general, organic solvents denature proteins and therefore cannot be

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used in ligand–protein binding studies. In contrast, organic solvents may help RNA fold correctly by stabilizing tertiary interactions (29). To verify that organic solvents do not interfere with ligand–RNA binding, the effect of decreasing the amount of isopropyl alcohol from 50 to 20% on the K D measurement for paromomycin was evaluated. It should be noted that no RNA precipitation is observed from 50% (v/v) isopropyl alcohol solutions while we observe complete RNA precipitation from solutions which are 75% (v/v) isopropyl alcohol (30). With 150 mM NH 4OAc/20% IPA solution conditions, the 16S concentration was held at 50 nM while paromomycin was titrated from 10 to 200 nM. Only a 1:1 paromomycin–16S complex was observed at all concentrations of paromomycin. Figure 4A shows the 5⫺ charge state for the free 16S and the 1:1 complex of paromomycin and 16S. Using Eq. [4], a K D of 53 nM (⫾2 nM) was calculated from the slope of a leastsquares fit (Fig. 4B). The dissociation constants calculated with 50% isopropyl alcohol and 20% isopropyl alcohol are the same, indicating that isopropyl alcohol,

FIG. 4. (A) ESI mass spectrum of 50 nM 16S plus 200 nM paromomycin showing free 16S (16S) and 1:1 paromomycin:16S complex (16S ⫹ PM). The location of a 2:1 paromomycin:16S complex (16S ⫹ 2 PM) is indicated by the arrow. Sodium adducts are labeled with an *. (B) The K D for paromomycin and 16S was determined by holding the 16S concentration at 50 nM and titrating paromomycin from 10 to 200 nM. A linear least-squares fit gives a correlation coefficient of 0.99. From the slope, the K D ⫽ 53 nM. The solutions conditions were 150 mM NH 4OAc/20% IPA.

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within the range evaluated, does not interfere with ligand binding to RNA. Measuring K Ds while holding the RNA at low nanomolar concentrations is experimentally difficult. Even with optimal instrumentation performance, there are problems with sensitivity, contamination, and sample carryover. Thus, the effect of holding the RNA concentration above the expected K D on the dissociation constant determination was examined. An attempt was made to measure the K D for paromomycin by holding the 16S concentration at 2 ␮M (which is 40-fold higher than the expected K D of 52 nM) and titrating paromomycin from 30 to 600 nM (which brackets the expected K D). Carryover contamination concerns prevent the use of higher concentrations of paromomycin. Again, only a 1:1 complex of paromomycin and 16S was observed under these conditions. Using Eq. [4], a K D of 617 nM (⫾79 nM) was calculated from the slope of a least-squares fit ([RL]/[R] ⫽ 1.62 ⫻ 10 6[L i]; R 2 ⫽ 0.92) (data not shown). The dissociation constants determined with holding the RNA concentration at 50 and 2 ␮M differ significantly. At the higher RNA concentration, the dissociation constant measured is probably a combination of the dissociation constant and the off rate of paromomycin from 16S. This indicates that K D measurements must be carried out with the RNA target concentration held below the expected K D, even though the ligand is titrated through the expected K D. CONCLUSIONS

In this work, the measurement of micromolar and nanomolar dissociation constants with electrospray ionization mass spectrometry has been demonstrated. Using ESI-MS, it is possible to simultaneously determine the abundance of the free RNA target and RNA complexes as well as determine the binding stoichiometries of the RNA complexes. The equilibrium concentrations of free RNA target and RNA complexes determined in the gas phase by mass spectrometric analysis reflect solution equilibrium concentrations. Since MS determines the binding stoichiometry of the RNA complexes, the appropriate equation can be used to analyze the data based on the number of binding sites. Equations can be derived which include nonequivalent binding sites. It is shown that holding the RNA target concentration fixed below the expected K D and titrating the ligand is the preferred method for determining dissociation constants using ESI-MS. If the expected K D value is unknown, several RNA target concentrations should be tried since it was demonstrated that holding the RNA target concentration above the K D adversely effects the K D measurement. For tobramycin, there are two nonequivalent binding sites and two K Ds have been calculated using Eq. [7]. For paromomycin, there

is only one binding site and one K D has been determined using Eq. [4]. The K Ds measured with this method agree with the literature values within a factor of 3. Decreasing the amount of salt in the solution from 150 mM NH 4OAc to 50 mM NH 4OAc affected the measurement of K D2 but not K D1 for tobramycin, consistent with the second binding site involving nonspecific electrostatic interactions. To minimize nonspecific binding due primarily to electrostatic interactions, the K D measurements should be carried out at a relatively high salt concentration. The K D measurement for paromomycin was not influenced by the amount of organic solvent utilized over the range of 20 –50% by volume. ACKNOWLEDGMENTS This work was supported in part by the Department of Defense through DARPA Grant BAA 98-25-544 and a NIST Advanced Technology Program Grant 97-01-0135 awarded to the Ibis Therapeutics Division of Isis Pharmaceuticals.

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