Spectral Differentiation and Immunoaffinity Capillary Electrophoresis

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Chem. Res. Toxicol. 2007, 20, 1192–1199

Spectral Differentiation and Immunoaffinity Capillary Electrophoresis Separation of Enantiomeric Benzo(a)pyrene Diol Epoxide-Derived DNA Adducts Beata Miksa,†,‡ Raja Chinnappan,†,‡ Nhan C. Dang,† Mike Reppert,† Brock Matter,§ Natalia Tretyakova,§ Nenad M. Grubor,| and Ryszard Jankowiak*,† Department of Chemistry, Kansas State UniVersity, Manhattan, Kansas 66502, Department of Medicinal Chemistry, UniVersity of Minnesota School of Pharmacy, Minneapolis, Minnesota 55455, and Bristol-Myers Squibb Co., East Syracuse, New York 13057 ReceiVed April 6, 2007

Antibody cross-reactivity makes separation and differentiation of enantiomeric analytes one of the most challenging problems in immunoanalytical research, particularly for the analysis of structurally related biological molecules [such as benzo(a)pyrene (BP) metabolites and BP-derived DNA adducts]. It has recently been shown that the interaction of enantiomers of BP tetrols (BPT) with a promiscuous anti-polycyclic aromatic hydrocabon (anti-PAH) monoclonal antibody (mAb) allowed for separation of all four enantiomeric isomers using immunoaffinity capillary electrophoresis [Grubor, N. M., Armstrong, D. W., and Jankowiak, R. (2006) Electrophoresis 27, 1078] and unambiguous spectral resolution using fluorescence line narrowing spectroscopy (FLNS) [Grubor, N. M., Liu, Y., Han, X., Armstrong, D.W., and Jankowiak, R. (2006) J. Am.Chem. Soc. 128, 6409]. Here, we expand the use of the above two methodologies to the group of biologically important molecules that are products of BP diol epoxide (BPDE)-induced DNA damage. Four diastereomeric anti-BPDE-derived deoxyguanosine (dG) adducts, that is, (+)- and (-)-anti-trans-BPDE–N2-dG and (+)- and (-)-anti-cis-BPDE–N2-dG, were electrophoretically separated and spectroscopically differentiated using 8E11 mAb raised against BP–DNA conjugates. In fluorescence line narrowing spectroscopy (FLNS) experiments, complexes of BPDE–dG adducts with mAb revealed differences in fluorecence origin band positions, bandwidths, and vibrational patterns for all four BPDE–N2-dG adducts. Narrow fluorescence origin bands observed for (-)-transBPDE–dG (70 cm-1) and (+)-trans-BPDE–N2-dG (80 cm-1) suggest spatial constraint within the mAb binding pocket. Broader origin bands observed for cis type adducts (∼120 cm-1) in 8E11 mAb suggest different binding geometries and/or conformational changes, as also indicated by changes in vibrational frequencies observed for the (+)-anti-cis and (-)-anti-cis adducts complexed with mAb. FLNS revealed that binding conformations and interactions within the mAb binding pocket are different for each adduct, enabling unambiguous positive identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides. Introduction Immunoaffinity modes of analytical separation and detection techniques are extraordinarily useful for the selection and identification of molecules of interest from very complex sample matrices such as biological fluids and tissue and cell extracts (1). This quality originates from the exceptional capability of antibodies (Ab 1) utilized in these techniques to selectively recognize and reversibly bind target molecules against which they were originally raised. Because of the chiral nature of proteins, it is possible for antibodies to interact with stereoiso* To whom correspondence should be addressed. Fax: 785-532-6666. E-mail: [email protected]. † Kansas State University. ‡ Both authors contributed equally to this work. § University of Minnesota School of Pharmacy. | Bristol-Myers Squibb Co. 1 Abbreviations: Ab, antibody; PAH, polycyclic aromatic hydrocarbon; mAb, monoclonal antibody; BP, benzo(a)pyrene; BPDE, benzo(a)pyrene diol epoxide; BPT, benzo(a)pyrene tetrol; CE, capillary electrophoresis; FTPFACE, flow-through partial-filling affinity capillary electrophoresis; LIF, laser-induced fluorescence; FLNS, fluorescence line narrowing spectroscopy; FWHM, full width at half maximum; NLN, nonline narrowed.

mers of chiral molecules in a qualitatively (i.e., different complex geometry) and/or quantitatively different fashion (2) (i.e., with different binding strength), thus allowing for chiral discrimination. Although Ab–antigen/hapten interaction is one of the most specific known molecular recognition phenomena, examples of nonspecific (cross-reactive) Abs are not uncommon (3, 4). Crossreactivity of Abs ranges from broad promiscuity (where an Ab binds molecules that are not structurally related, sometimes even with a several orders of magnitude in size difference) to socalled group specificity (where an Ab recognizes a family of molecules with similar structural features but ignores structurally unrelated molecules) (5–8). Ab cross-reactivity is especially common among structurally related biological molecules derived from a single parent precursor through various metabolic pathways. One particularly large and complex group of biologically important molecules is polycyclic aromatic hydrocarbons (PAH), their metabolites, and PAH-derived DNA and protein adducts. Among them, special attention has been paid to benzo(a)pyrene (BP) and its derivatives due to their ubiquitous potency toward cellular DNA

10.1021/tx7001096 CCC: $37.00  2007 American Chemical Society Published on Web 07/13/2007

FLNS and IACE of BPDE DNA Adducts

Chem. Res. Toxicol., Vol. 20, No. 8, 2007 1193

Figure 1. Structures of (+)- and (-)-anti-cis- and (+)- and (-)-anti-trans-BPDE–N2-dG adducts. Torsion angles χ, R′, and β′ are defined as follows: χ, O4′–C1′ –N9–C4; R′, N1–C2–N2–C10 (BP); and β′, C2–N2–C10 (BP).

damage and the subsequent carcinogenicity of these processes. Depending on the metabolic activation pathway that BP undergoes (four different pathways (9–11) have been proposed and/or demonstrated), various mechanisms of DNA damage and lesions can occur, leading to different metabolic and adduction products that can be identified through in vivo and in vitro experiments (12–14). Abs targeting particular molecules from this group have been developed, but selectivity has frequently been difficult to achieve. Therefore, the development of alternative methodologies that utilize the existing pool of Abs for highly specific analyses of target molecules is of considerable interest. Fluorescence line narrowing spectroscopy (FLNS) is a highresolution technique that can be used to differentiate between diastereomeric cis- and trans-isomers of various molecules and adducts (12, 15). However, in nonchiral environments, (+)- and (-)-enantiomers produce identical FLNS spectra, limiting the usefulness of this approach for the differentiation of enantiomeric analytes. To overcome this limitation, FLNS may be linked together with the immunoaffinity methodologies discussed above. In this technique, analytes are immunocomplexed with a group-specific monoclonal Ab (mAb); the chiral environment of the binding site leads to different binding geometries and qualitatively different interactions between the isomeric analytes and the Ab. As a result, different spectra are observed even for enantiomeric analytes, allowing them to be easily distinguished using a high-resolution spectroscopy, that is, FLNS (6, 20, 21). Similarly, various analytical separation methods have been used for efficient chiral separations. Many different modes of those separation techniques allow for the study of intermolecular interactions, including protein–ligand and protein–protein interactions (16). Capillary electrophoresis (CE) is of particular

interest due to its various operational modes (including immunoaffinity techniques) that enable versatile experimental design (17, 18). Our recent work has established the applicability of crossreactive Abs for highly specific analyses of complex stereoisomeric mixtures using FLNS (15, 19). It was shown that the highly promiscuous anti-PAH mAb can be used for capturing and analysis (detection, differentiation, and structural analysis) of not only structurally related PAHs (i.e., fluoranthene, pyrene, and BP) (6) but also their metabolic derivatives [i.e., diastereomeric BP tetrols (BPTs)] (20). We have also demonstrated that efficient and versatile immunoaffinity electrophoretic chiral separation of closely related stereoisomers can be achieved by using minuscule amounts of the group-specific Ab as a chiral selector (21). Here, we report on the expansion of the utility of these methodologies, by examining another Ab from the pool of existing (cross-reactive) mAbs targeting the group of biologically important BP-derived molecules. Four diastereomeric BP diol epoxide (BPDE) deoxyguanosine (dG) adducts—(+)-antitrans-BPDE–N2-dG, (-)-anti-trans-BPDE–N2-dG, (+)-anti-cisBPDE–N2-dG, and (-)-anti-cis-BPDE–N2-dG)—are formed from the BP precursor undergoing a monooxygenation metabolic pathway (9, 10, 22, 23). The structures of the anti-BPDE-derived dG adducts are shown in Figure 1. As described above, FLNS can be used to distinguish between the diastereomeric cis- and trans-BPDE–dG adducts, but in nonchiral environments, the (+)- and (-)-enantiomeric adducts have identical FLN spectra. This is because the (+)- and (-)-mononucleoside adduct pairs [both (+)- or (-)-cis- and (+)- or (-)-trans-BPDE–dG] have mirror image symmetry broken only by the sugar and C4′–C5′ group at the nucleoside level (24). These structural differences do not lead to any appreciable differences in FLN spectra. Identification, isolation, and structural analysis of these adducts

1194 Chem. Res. Toxicol., Vol. 20, No. 8, 2007

are important from the perspective of the analysis of DNA damage, that is, cancer risk assessment, as well as for studying the mechanisms and structure–activity relationships of DNA adduction. Here, diastereomers of BPDE–dG and their complexes with 8E11 mAb were subjected to FLNS analysis for their spectroscopic chiral resolution, as well as to immunoaffinity CE separation in a flow-through mode for their analytical separation and identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides. Although the CE experiments reported in this manuscript were conducted using absorbance detection (with a detection limit in the picomolar range), the technique could just as easily be conducted using fluorescence detection, lowering the detection limit into the sub-femtomolar range (near the detection limit of the FLNS techniques). With this caveat, both of the methodologies employed in this study could be used for analysis of hydrolyzed DNA samples from various in vitro and in vivo experiments. The results are discussed in terms of interactions between the ligands and the mAb and conformational preferences.

Materials and Methods Caution: BPDE is carcinogenic and should be handled with extreme caution. Synthesis and Characterization of BPDE–dG Diastereomers. BPDE–dG diastereomers were prepared by treating calf thymus DNA with (()-anti-BPDE (1). Calf thymus DNA (10 mg) was dissolved in 6 mL of 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7, and (()-anti-BPDE (1 mg in 500 µL THF) was added. Following incubation for 10 h at 37 °C, unreacted BPDE was extracted with ethyl acetate, and the DNA was precipitated with NaCl and cold ethanol. Adducted DNA was dissolved in 3 mL of 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7, and hydrolyzed to 2′-deoxyribonucleosides with a mixture of deoxyribonuclease I (2800 U), phosphodiesterase I (17 U), phosphodiesterase II (7 U), and alkaline phosphatase (700 U). BPDE–dG was isolated by solidphase extraction on C18 Sep-Pak cartridges (Waters Associates, Milford, MA), and individual diastereomers were purified by reversed-phase HPLC as described below. BPDE–dG diastereomers were separated by HPLC with an Agilent Technologies HPLC system (model 1100) incorporating a diode array UV detector. An Extend-C18 column (4.6 mm × 150 mm, 5 µm, Agilent Technologies) was eluted at a flow rate of 1 mL/min. The buffer system was composed of 33% methanol in 150 mM ammonium acetate (A) and 25% buffer A in acetonitrile (B). The solvent composition was changed from 13 to 15% B in 24.5 min and then further to 30% B by 30 min. BPDE–dG adducts were detected by UV absorption at 345 nm. At these conditions, HPLC retention times for the individual diastereomers were as follows: (-)-transBPDE–dG (tR, 20.3 min), (+)-cis-BPDE–dG (tR, 21.1 min), (-)cis-BPDE–dG (tR, 22.8 min), and (+)-trans-BPDE–dG (tR, 24.6 min). The HPLC peaks corresponding to individual BPDE–dG diastereomers were collected and thoroughly dried under reduced pressure to remove most of the ammonium acetate and then dissolved in 33% methanol/water. BPDE–dG stock solution concentrations were determined by UV spectrophotometry (ε345 ) 34293). CD spectra were obtained with a Jasco Spectrapolarimeter (model J-710). Samples were scanned from 200 to 400 nm, with a resolution of 0.1 nm. Circular dichroism (CD) spectra were used to test the purity of our preparation; as expected, CD spectra for (+)- and (-)-enantiomeric pairs exhibit nearly perfect mirror symmetry, indicating high enantiomeric purity (23) (data not shown). The concentrations of adducts used in immunoaffinity CE and FLNS experiments were ∼10-6 and ∼2–5 × 10-7 M, respectively. Flow-Through Partial-Filling Affinity Capillary Electrophoresis (FTPFACE) Separation of Diastereomeric anti-

Miksa et al. BPDE–dG Adducts. For separations, phosphate saline buffer, pH 7.2 (PBS saline), was purchased from Pierce (United States). This buffer included 0.2 M sodium phosphate and 0.3 M sodium chlorate in 1 L of distilled water, and it was adjusted to pH 8.8 using a 1 N solution of NaOH. PBS saline buffer (with an ionic strength of 1.0) was used in CE experiments as running buffer and was filtered through a 0.22 µm nonpyrogenic filter (Millex, United States). All buffers and samples were prepared using water purified by a Bransted Ultrapure Water System (Dubuque, IA). mAb 8E11 (a kind gift of Dr. R. M. Santella of Columbia University, New York) was used for the separation of (+)- and (-)-trans- and (+)- and (-)-cis-BPDE–dG enantiomers. Pure enantiomers of (-)-cisBPDE–dG, (+)-cis-BPDE–dG, (-)-trans-BPDE–dG, and (+)trans-BPDE–dG were dissolved in 33% methanol in water. The amount of each of enantiomer was 2 nmol in 100 µL of solution. Protein–ligand interactions were studied by using the FTPFACE method (25, 26) using a normal polarity mode (that is, the sample was introduced into the capillary at the anode side). UV detection was performed at 280 nm (the absorption maximum of mAb 8E11). In our previous work, for neutral ligands (e.g., PAH) and charged mAbs (18), the capillary was first filled with a plug of Ab followed by a plug of a sample containing ligands. This approach does not work for the enantiomeric BPDE–dG adducts since at pH 8.8 both mAbs (pKa values are 7.5–8.3 (27)) and adducts (28, 29) are negatively charged. Under these conditions, the charge to mass ratio of ligands is larger than that of the mAb; as a result, the electrophoretic mobility of adducts (toward the positively charged anode) is greater than that of the mAb. In contrast, electroosmotic flow is directed towards the negatively charged cathode; as a result, buffer flows through the capillary from the inlet to the outlet vial and drives all analytes towards the detection window on the cathode side. The net result is that ligands are carried to the cathode at a lower velocity than the mAb. Therefore, in our experiments, the order of injection has been reversed relative to our previously published data; that is, the capillary was first filled with a plug of sample (i.e., BPDE–dG adducts) followed by a plug of mAb. Because of the different migration rates induced by the differences in electrophoretic mobilities of mAbs and ligands, upon application of a voltage gradient (20 kV), the ligand and mAb zones temporarily overlap during the electrophoretic run. By injecting the ligand plug 0.5–1.0 min prior to the mAb plug, the Ab zone penetrates the ligand zone during the course of the experiment. Final resolution depends on experimental conditions. Samples were hydrodynamically injected as follows: (i) ligand: p ) 35 mba, t ) 0.3 min; (ii) buffer: p ) 15 mba, t ) 0.5-1 min; and (iii) 8E11 Ab: p ) 35 mba, t ) 0.2 min. All separations were carried out at 20 kV in running buffer (PBS saline, pH 8.8). UV transparent capillaries (74 µm i.d. and 366 µm o.d.) were obtained from Polymicro Technologies and used with a length of 80 cm (48 cm to the detection window). Prior to use, the capillary was conditioned for 30 min each with 0.1 M sodium hydroxide, water, and running buffer. Low Temperature Fluorescence Spectroscopy. For laserinduced fluorescence (LIF)-based spectroscopic measurements [under nonline narrowing (NLN) conditions], laser excitation at 308 nm was provided by a Lambda Physik Lextra 100 XeCl excimer laser. For FLNS measurements, the same laser was used as a pump source for a Lambda Physik FL 2002 Scanmate tunable dye laser system (10 Hz). A 1 m McPherson monochromator (model 2601) and a Princeton Instruments photodiode array were used for dispersion and detection of fluorescence. A Princeton Instruments FG-100 pulse generator was used for time-resolved spectroscopy with detector delay times from 0 to 160 ns and a gate width of 200 ns. For all spectroscopic measurements, 30 µL volumes of sample were placed in quartz tubes and immersed in a helium cryostat with quartz optical windows. To ensure quantitative binding of haptens, immunocomplexes were formed by incubation of a 10-fold excess of Abs relative to the concentration of haptens (2–5 × 10-7 M) in PBS buffer. All fluorescence spectra are the average of ten 1 s acquisitions unless stated differently. The resolutions for FLN and NLN spectra were 0.05 and 0.2 nm, respectively. Thus, the


Figure 2. CE electropherogram obtained for a mixture of four BPDE–dG enantiomers. The peaks labeled 1 and 2 correspond to (+)and (-)-cis- and (+)- and (-)-trans-enantiomeric mixtures, respectively (see the text for details).


Figure 3. CE electropherogram obtained for a racemic mixture of (()trans-BPDE–dG and (()-cis-BPDE–dG enantiomers and a plug of 8E11 mAb. The ligand was injected first, followed by mAb (see the text for details).

corresponding accuracy in vibrational frequencies for FLNS measurements is about (3 cm–1.

Results and Discussion FTPFACE Separation of Diastereomeric BPDE–dG Adducts. Although the (+)- and (-)-enantiomers studied here are not separable by conventional CE (i.e., in a nonchiral environment), the trans- and cis-diastereomers of BPDE–dG adducts are conformationally distinct and can be separated, as shown in the electropherogram in Figure 2. This figure presents the result of a conventional CE separation (PBS buffer, pH 8.8) of a mixture of all four (+)- and (-)-trans- and (+)- and (-)cis-adducts. Two distinct peaks (labeled as 1 and 2) were observed, corresponding to enantiomeric mixtures of (+)- and (-)-cis- and (+)- and (-)-trans-adducts, respectively. (Peaks were assigned based on migration times and concentration spiking experiments; data not shown.) Because the four isomers have the same net charge, it appears that the (+)- and (-)-cisenantiomers have a different hydrodynamic radius than the (+)and (-)-trans-enantiomers, resulting in the higher migration rate of the cis-adducts observed experimentally. Thus, under nonchiral conditions, trans type adducts were easily separated from cis type adducts primarily due to different conformations (22), but separation of (+)- and (-)-enantiomers was not possible. In addition, neither separation nor FLNS identification of the four BPDE–dG diastereoisomers could be accomplished by using the anti-PAH mAb (vide infra). However, separation of all four diastereomers is possible using 8E11 mAb in an FTPFACE regime as shown in the electropherogram in Figure 3. For this electrophoretic run, a mixture of the four isomeric BPDE–dG adducts was hydrodynamically injected first, followed by a 1 min-delayed injection of a plug of 8E11 mAb. As discussed in the Materials and Methods section, the higher migration rate of the mAb causes the Ab plug to pass through the mixture of BPDE–dG adducts. Because of the quantitatively different interactions (different binding constants (30)) of the four isomeric adducts with the mAb, the migration rate of each isomer is affected differently by the passage of mAb through the ligand band, resulting in four separate elution peaks. Standard spiking procedures established that the (+)-cis-enantiomer elutes first, followed by the (-)-cis-, (+)-trans-, and (-)-trans-adducts, respectively. Apparently, the interactions of the (+)-enantiomers with the Ab are somewhat stronger than those of the (-)-enantiomers

Figure 4. Normalized fluorescence emission spectra of free (+)-transBPDE–dG (a) and (+)-trans-BPDE–dG/mAb complex (b) in PBS/ glycerol; λex ) 308 nm; delay time ) 40 ns, and T ∼ 4.2 K.

(in each enantiomeric pair), causing changes in migration rates. These results are in agreement with inhibition values published earlier (30) where it was shown that the 50% inhibition rates for (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-adducts formed in oligonucleotides are 450 fmol, 680 fmol, 5.5 pmol, and 11 pmol, respectively (30). Low-Resolution Fluorescence Spectra of BPDE–dG Adducts Complexed with 8E11 mAb. Figure 4 shows, as an example, the 4.2 K NLN spectra obtained for (+)-transBPDE–dG and the (+)-trans-BPDE–dG/8E11 mAb complex to illustrate the substantial narrowing of the fluorescence origin band (and a relatively small red shift) induced by complexation with mAb. Results of NLN fluorescence experiments obtained for all complexed and uncomplexed BPDE–dG adducts are summarized in Table 1. In summary, identical spectra are obtained for the uncomplexed (+)-trans- and (-)-trans-adducts (with fluorescence maximum at 376.1 nm), as well as for the (+)-cis- and (-)-cis-adducts (with fluorescence maximum at 376.4 nm), as expected. Upon complexation of adducts with the 8E11 mAb, however, unique spectral shifts and changes in the (0,0) band widths are observed for each adduct, due to their specific interactions with the Ab binding pocket. The fluorescence maximum of the (+)-trans-adduct, for example, shifts from 376.1 to 376.7 nm, while that of the (-)-trans-adduct shifts to 377.2 nm. Similarly, on complexation, the fluorescence maximum of the (+)-cis-adduct shifts from 376.4 to 377.8 nm,


Miksa et al.

Table 1. Spectral Chracteristics of BPDE–dG Adducts and BPDE–dG Adduct/8E11 mAb Complexes in PBS/Glycerol (1:1) Matrix at ∼4.2 K adduct BPDE-dG Adducts (+)-anti-trans (-)-anti-trans (+)-anti-cis (-)-anti-cis

adduct/8E11 mAb complex

(0,0)a ( 0.2 nm

Γb ( 5 cm-1

(0,0)a ( 0.2 nm

Γb ( 5 cm-1

376.1 376.1 376.4 376.4

140 140 145 145

376.7 377.2 377.8 377.1

70 80 125 115

a The maximum of fluorescence (0,0) origin band (in nm). high energy side of the band (in cm-1).

b

Γ is the FWHM obtained by doubling the half width at half maximum measured at the

while that of the (-)-cis-adduct shifts to 377.1 nm. In addition to these spectral shifts, distinct changes in the bandwidth of the (0,0) fluorescence band were observed for each complex. The bandwidth of the uncomplexed (+)- and (-)-trans-adducts [measured as the full width at half maximum (FWHM) obtained by doubling the half width at half maximum measured at the high energy side of the band] is 140 cm-1; complexation with the 8E11 mAb results in a narrowing of the band to 70 and 80 cm-1 for the (+)- and (-)-trans-adducts, respectively. This significant reduction in spectral bandwidth suggests that these two adducts are bound tightly in well-defined conformations within the binding pocket, so that molecular motion is restricted (as indicated by the reduced inhomogeneous broadening). In contrast, although some narrowing of the spectral bandwidth is observed for the (+)- and (-)-cis-adducts (from 145 to 125 and 115 cm-1 for the (+)- and (-)-cis-adducts, respectively), the narrowing effect is much less pronounced, suggesting large differences in conformational flexibility of these adducts, in agreement with modeling studies (24). Note that both transadducts have significantly higher affinity than the cis-adducts towards this particular mAb (30). That is, if affinity for (+)trans is normalized to 100%, the affinity of (-)-trans, (+)-cis, and (-)-cis are 66, 8.2, and 4.1%, respectively. It has previously been demonstrated (20) that low temperature fluorescence spectroscopy enables enantiospecific differentiation between BPTs immunocomplexed with anti-PAH mAb. We tested whether or not this mAb has any affinity towards the PAH part of the studied adducts. Results have shown that neither low-resolution (NLN) nor high-resolution (FLN) fluorescence spectra of (+)- or (-)-trans- and (+)- or (-)-cis-BPDE–dG adducts revealed any spectral changes upon incubation with this mAb, indicating that anti-PAH either does not bind to or does not differentiate between BPDE enantiomers. The binding site of the anti-PAH mAb (raised against fluoranthene) might be too small or sterically hindered to accommodate the bulky antiBPDE–dG adducts that possess three flexible torsion angles governing the orientation of the base (χ) and its covalently linked BP residue (R′,β′); see Figure 1 (24). The results of this control experiment demonstrate that both the spectral and the electrophoretic characteristics of BPDE adducts interacting with 8E11 mAb do indeed arise from the preferential (group-specific) interactions of adducts with the Ab binding sites and not from their random interaction with the protein surface. High-Resolution Differentiation of the Stereoisomeric BPDE–dG Adducts Complexed with 8E11 mAb. Molecular electronic spectra carry information on inherent molecular structure and can also be used for molecular identification, as well as elucidation of the local environment of molecules (31–33). FLN spectroscopy is used here to directly probe the four intact diastereomers of BPDE–dG adducts as well as their complexes with the 8E11 mAb. Several excitation wavelengths within the 355–371 nm range were used to obtain corresponding high-resolution spectra revealing the whole range of characteristic vibrational frequencies for BPDE-dG adducts ranging from

Figure 5. FLN spectra (4.2 K) of BPDE–dG adducts and their mAb complexes in PBS/glycerol (1:1) excited at 369 nm; delay ) 40 ns and T ) 4.2 K. (A) Spectrum a was obtained for the (+)-transBPDE–dG adduct [a nearly identical spectrum was obtained for the (-)-trans-adduct and is not shown]. Spectra b and c correspond to (+)trans-BPDE–dG mAb/ and (-)-trans-BPDE–dG/mAb complexes, respectively. (B) Spectrum a was obtained for the (+)-cis-BPDE–dG adduct [a nearly identical spectrum was obtained for the (-)-cis-adduct and is not shown]. Spectra b and c correspond to (+)-cis-BPDE–dG/ mAb and (-)-cis-BPDE–dG/mAb complexes, respectively.

400 to 1700 cm-1. Figure 5 shows FLN spectra obtained with λexc ) 369.0 nm that reveals vibrational frequencies in the range between 450 and 608 cm-1. Although the FLN spectra of diasteromeric cis- and transBPDE–dG adducts were distinct from each other, virtually identical spectra were obtained for the respective (+)- and (-)enantiomers of both the cis- and the trans-adducts. Spectrum a in panel A of Figure 5 is the FLN spectrum of the uncomplexed (+)-anti-trans-BPDE–dG adduct; an identical spectrum was obtained for the uncomplexed (-)-anti-trans-BPDE–dG adduct (not shown). Similarly, spectrum a of panel B shows the FLN spectra obtained for the uncomplexed (+)-cis-BPDE–dG enantiomers, which is identical to that obtained for the uncomplexed (-)-cis-BPDE–dG enantiomer (not shown). Comparison of high-resolution fluorescence spectra of uncomplexed (+)- or (-)-anti-trans-BPDE–dG and (+)- or (-)anti-cis-BPDE–dG obtained for the same excitation wavelength reveals apparent differences in their vibrational frequency activity and enables a clear distinction between the two configurational isomers. For example, one observes a distinct 450, 463, and 472 cm-1 triplet, as well as a distinct 577 cm-1 frequency for the trans-isomers; the cis-isomers display a 466, 480, and 495 cm-1 triplet, as well as 539, 550, and 569 cm-1 lines. As described above, (+)- and (-)-enantiomeric pairs were not distinguishable even under high-resolution conditions. However, upon immunocomplexation of enantiomeric BPDE–dG diastereomers with 8E11 mAb, significant changes were observed in the distribution, abundance, and relative intensity


of their vibrational frequencies. The (+)-trans-BPDE–dG/mAb complex, due to the red shift of its (0,0) band, does not reveal 450–500 cm-1 ZPLs for 369.0 nm excitation; instead, the 548 and 577 cm-1 ZPL lines are clearly observed, with 548 cm-1 intensity increasing relative to 577 cm-1 intensity (spectrum b, Figure 5A). On the other hand, (-)-trans-BPDE–dG, upon incubation with the mAb, reveals a highly distinct 577 cm-1 ZPL, while other low frequency modes are absent, with the exception of a very weak 548 cm-1 line (spectrum c in Figure 5A). This clearly suggests that both (+)- and (-)-enantiomers of anti-trans-BPDE–dG adducts undergo red shifts with significant narrowing of the origin bands, in agreement with the NLN spectra (see, for example, Figure 4) and other data summarized in Table 1. Similarly, the interaction of (+)-cis-BPDE–dG with the mAb leads to the loss of a highly characteristic triplet (466/480/495 cm-1) clearly observed in the FLN spectrum of the uncomplexed molecule (see spectra b and a of Figure 5B). This loss is accompanied by significant changes in the relative intensities and frequencies of higher energy vibrational modes, in particular, the relative intensity of the modes at 593 and 608 cm-1 for (-)-cis-BPDE–dG adducts (see dashed arrows in Figure 5B). This is consistent with a relatively large (∼1.4 nm) spectral shift of the (0,0) band of the (+)-cis-BPDE–dG adduct as shown in Table 1. Finally, Ab complex formation with (-)-cisBPDE–dG (curve c in Figure 5) also changes the intensity of the vibrational modes in the 466–495 cm-1 region [again consistent with a smaller 0.7 nm shift of the (0,0) band; see Table 1], while altering the mode structure and mode frequencies in the higher vibrational frequency region. (Note that the 593 and 608 cm-1 lines have similar intensities and are clearly revealed.) In fact, the modes at 558, 576, and 593 cm-1 observed in spectrum c of Figure 5B are not observed in spectra a and b in the same frame. The changes in the vibrational frequencies observed for the (+)-anti-cis- and (-)-anti-cis-adducts complexed with mAb, when compared with those obtained for the isolated (+)- and (-)-anti-cis-BPDE–dG adducts, indicate that different conformational domains (and/or domain mixtures) are favored for cisadducts within the mAb binding pocket. Although cis-adducts are less conformationally flexible than trans type adducts, they can still exist in two low-energy domains with similar energies (22). Thus, it is feasible that binding to the 8E11 mAb induces a conformational change. However, at this point, it is not clear whether the cis-adducts exist as single conformers or as mixtures of two distinct conformations trapped at low temperatures. The latter possibility is not unreasonable since two conformations were already observed for these adducts in anti-BPDE-modified duplexes 5′-d(CCATCGCTACC) (GGTAGCGATGG), where G denotes the lesion site from trans- or cis-addition (34). That is, both (-)- and (+)-cis-BPDE–dG adducts could adopt external (minor) and intercalated (major) conformations. Interestingly, both external and intercalated BPDE–DNA adduct conformations revealed significantly larger spectral bandwidth (from ∼160 to 300 cm-1) than those observed for BPDE–dG adducts in 8E11 mAb. Another example of high-resolution spectra, obtained for λex ) 356.3 nm vibronic excitation, is shown in Figure 6. In contrast to the data shown in Figure 5, higher energy excitation reveals higher energy vibrational modes (in the 1300–1650 cm-1 region; i.e., small amplitude motion), which are less susceptible to change due to conformational motion (12, 15, 33). As a result, only changes in the relative intensity of the vibrational modes (due to different red shifts and bandwidths of the origin band)


Figure 6. FLN spectra of diastereomeric BPDE–dG adducts and their mAb complexes in PBS/glycerol (1:1) excited at 356.3 nm; delay ) 40 ns and T ) 4.2 K. (A) Spectrum a was obtained for the (+)-transBPDE–dG adduct [a nearly identical spectrum was obtained for (-)trans-BPDE–dG and is not shown]. Spectra b and c correspond to (+)trans-BPDE–dG/mAb and (-)-trans-BPDE–dG/mAb complexes, respectively. (B) Spectrum a was obtained for the (+)-cis-BPDE–dG adduct [a nearly identical spectrum was obtained for (-)-cis-BPDE–dG and is not shown]. Spectra b and c correspond to (+)-cis-BPDE–dG/ mAb and (-)-cis-BPDE–dG/mAb complexes, respectively.

are observed, with the exception of the mode near 1562/1564 cm-1. Nevertheless, as shown in Figure 6, all four stereoisomers can also be distinguished by excitation at this (λex ) 356.3 nm) and several other wavelengths (data not shown). The causes underlying alterations in high-resolution spectra of a chromophoric ligand following its complexation with a macromolecule have been thoroughly studied (6, 17, 35). For this discussion, it suffices to say that complexation of a massive Ab molecule with a small chromophoric ligand may involve various interactions, such as hydrogen bonds, electrostatic, hydrophobic, π–π, and π–cation interactions. The type of interaction depends on the nature of the target molecule and the Ab clone selection. In addition, steric features, Ab binding site topology, and target molecule conformational or structural flexibility each play an important role in recognition and binding. Because of these interactions, the binding of a small molecule to an Ab usually constitutes a significant change in the local environment of the bound molecule, that is, seclusion from solvent effects, etc. As a result, the binding of a small molecule is almost always accompanied by changes in spectral features. The most commonly observed spectral changes induced by the changes in immediate molecular environment are spectral band shifts toward either higher (blue shift) or lower (red shift) energies (31) as observed in Table 1 and data shown in Figures 5 and 6. These shifts are a result of a decreasing (red shift) or increasing (blue shift) energy gap between the ground and the first excited electronic state of a particular molecule. The relatively narrow fluorescence origin bands and small red band shifts (see Table 1) suggest that there is limited possibility for strong π–π interaction between the BPDE–dG adduct and the mAb binding pocket. Strong π–π interaction was observed for various PAHs (e.g., BP) immunocomplexed with anti-PAH mAb (6). In this case, however, the spectral shift for the major conformation of BP in anti-PAH mAb was larger by at least a factor of 2, in agreement with spectral shifts observed for BPDE–DNA adducts intercalated into DNA (32–34), where the typical (0,0) band was observed near 380–381 nm. Because the shifts presented in this study for diastereomeric BPDE–dG adducts complexed with 8E11 mAb are smaller and the (0,0) bands are more significantly narrowed, we suggest that the large


π-plane of the pyrene ring not only enhances the complexation ability but also maintains the orientation of the aromatic ring with respect to, for example, arginine and/or lysine, resulting in the observed narrowing of the spectral bandwidth. Such a π–cation interaction was demonstrated in modeling studies for BP in various mAbs (36) and occurs if the protons of arginine (or lysine) are close to the pyrenyl moiety, stabilizing its relative orientation. This type of interaction using several different mAbs will be discussed elsewhere (manuscript in preparation).

Conclusions This study demonstrates the utility of the 8E11 mAb for FTPFACE separation and spectroscopic differentiation of the four (+)- and (-)-anti-trans- and (+)- and (-)-anti-cisBPDE–N2-dG adducts. The approach previously used for immunoaffinity capillary separation of isomeric BP tetrols using anti-PAH mAb (18) was modified for separation and spectroscopic differentiation of the four diastereoisomeric BPDE adducts using 8E11 mAb. It has been shown that separation of all four investigated BPDE–dG adducts can be achieved, demonstrating different ligand–Ab interactions for the four adducts. Both NLN and FLN spectra revealed differences in the energy of the (0,0) bands and the extent of spectral inhomogeneous broadening for trans and cis type adducts. Both sets of data confirm that all four isomeric ligands interact in a qualitatively different manner with the binding pocket of the 8E11 mAb, allowing for unambigous CE separation and spectral differentiation. Differences were observed in the positions of the fluorecence origin bands, bandwidths, and (as a result) vibrational patterns for all four BPDE–dG/mAb complexes. The changes in vibrational frequencies observed for the (+)-anticis- and (-)-anti-cis-adducts complexed with mAb indicate that different conformational domains (or domain mixtures) are favored for cis-adducts within the mAb binding pocket. Significantly smaller adduct heterogeneity (i.e., small inhomogeneity) was observed for trans-adducts. We suggest that at least for the (+)- and (-)-trans-BPDE–dG adducts complexed with 8E11 mAb, the narrowing effect on the (0,0) band is caused by π–cation interaction that stabilizes the binding of trans-adducts within the mAb binding pocket. This effect is clearly observed for trans type adducts, since they appear to bind in a single confirmation. This type of stabilization was predicted theoretically in modeling studies for BP in 4D5 mAb (36). Acknowledgment. This work was supported by a grant from the NIH COBRE award 1 P20 R15563 and matching support from the state of Kansas. Our thanks are due to Professor Regina M. Santella (Columbia University, New York) for providing 8E11 mAbs. Supporting Information Available: Scheme of the formation of diastereomeric N2-BPDE–dG (()-anti-BPDE, synthesis of BPDE–dG diastereomers, HPLC separation of BPDE–dG diastereomers and their CD spectra, figure of UV spectra, and figure of induced CD spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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TX7001096

Spectral Differentiation and Immunoaffinity Capillary Electrophoresis

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