Photochemutry and Phofobiofogy, Vol. 58, No. 5, pp 637-642, 1993 Printed in the United States. All rights reserved

003 1-8655193 $05.00+0.00 0 1993 American Society for Photobiology

FLUORESCENCE LINE-NARROWING STUDIES OF ANTIBODY- BENZO[U]PYRENE TETROL

COMPLEXES KULDIP SINGHI, PAUL L. SKIPPER*, STEVEN R. TANNENBAUML3 and RAMACHANDRA R. DASARI*’

IGeorge R. Hamson Spectroscopy Laboratory (Room 6-0 l4), *Division of Toxicology and

’Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 021 39, USA

(Received 14 December 1992; accepted 2 1 May 1993)

Abstract-Benzo[a]pyrene tetrol (BPT) was used as a fluorescent probe to investigate the nature of antigen binding by two different monoclonal antibodies (MAb) that recognize a variety of derivatives of antZ-7,8-dihydroxy-9,10- epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrenes (BPDE). Fluorescence line-narrowed spectra of the physical complexes of BPT formed with antibodies 8El1 and 3C3 were recorded at 4 K by employing vibronic excitation into the S, electronic state. The frequencies of the vibrational modes of the S, state were only marginally affected, though changes in relative intensities of some bands were observed. Fluorescence spectra recorded at 77 K by excitation into the Sz state showed that the (0,O) fluorescence emission of BPT was shifted to red on complex formation. Intensity ratios of the (0,O) band and the main vibrational band at 1300 cm-I were used to assess the degree of interior binding of the chromophore. Quenching studies with acrylamide were employed to designate the complexes as type I, solvent inaccessible, or type 11, solvent accessible. These studies also indicated that antibody 3C3 complexes tend to be more heterogeneous compared to the 8E11 complex. Deuterated BPT-d- 12 also formed complexes with both antibodies, however, with different quenching behavior.

INTRODUCTION

Fluorescence line-narrowing (FLN)? spectroscopy, which has also been termed site selection spectroscopy, has been applied extensively to the study of natural biological systems. It has also been applied to biological systems into which an exog- enous chromophore has been introduced, such as by reaction with a chemical carcinogen.‘-’ Most chemical carcinogens require metabolic activation to electrophilic species before they can react with D N A and proteins to form so-called adducts4 The metabolic process may give rise to a variety of intermediates, with differences as minor as which of the two enantiomers predominates. As a result, several types of adducts can be formed from one carcinogen, and these ad- ducts may be only slightly different from one another.* The sensitivity of FLN spectra to such subtle differences has made FLN spectroscopy particularly well suited to the analysis of certain carcinogen adducts.

Among the numerous carcinogenic polycyclic aromatic hy- drocarbons (PAH), benzo[a]pyrene has been subjected to extensive study. Benzo[a]pyrene is metabolized t o several isomeric 7,8-dihydroxy-9,1 O-epoxy-7,8,9,1 O-tetrahydroben- zo[a]pyrene (BPDE) chromophores, making them ideally suited for fluorescence studies.’ T h e BPDE react with DNA and protein to form a covalent adduct. This reaction is con- sidered to be an important step in carcinogenesis and tu- morigenesis. Recently, protein adducts formed by reacting

~~ ~

*To whom correspondence should be addressed. tilbhreviations: ACR, acrylamide; anti-BPDE, r-7,t-8-dihydroxy-

t-9,t- 1 O-epoxy-7,8,9,1O-tetrahydrobenzo[a]pyrene; BPDE, ben- zo[a]pyrene diol epoxide; BPT, benzo[a]pyrene tetrol; FLN, flu- orescence line narrowing; FWHM, full width at half maximum; HPLC, high-performance liquid chromatography; MAb, mono- clonal antibodies; PAH, polycyclic aromatic hydrocarbons; R, ra- tio of fluorescence intensity of(0,O) band relative to the vibrational band at 1300 cm ~ I; ZPL, zero phonon lines.

anti-BPDE with amino acids, human hemoglobin6 and hu- man serum albumin’ were studied, and it was shown that their FLN spectra were sensitive to changes in the substituent a t C( lo), the reactive center of BPDE. The covalent adducts on hydrolysis produce tetrols. It was also observed that the FLN spectra of benzo[a]pyrene tetrols (BPT) were influenced by noncovalent binding t o the protein globin.

Monoclonal antibodies (MAb) are proteins that have a highly specific binding site for the antigen against which they were raised. The highly specific nature of MAb-antigen bind- ing makes MAb potentially useful reagents for quantitative analysis of their antigens. It was thus of interest to determine the effect on the BPT fluorescence spectra of association of BPT with M A b that recognize them. We have two MAb, designated 8E11 and 3C3, that bind BPT. They were pro- duced with different antigens made from guanosine and thioether adducts, respectively, of anti-BPDE. They have similar affinity constants, but potentially different structural properties of their binding sites. In this report we describe FLN spectroscopic studies of the complexes formed by the two antibodies with BPT. In the interest of generating ad- ditional information concerning the nature o f the interactions involved in MAb-antigen binding, we also investigated flu- orescence quenching by acrylamide (ACR) and the effect of deuterium substitution in the BPT.

MATERIALS AND METHODS

Anti-BPDE was purchased from the National Cancer Institute chemical carcinogen repository maintained by the Midwest Research Institute (Kansas City, MO). The BPT were produced from the anfi- BPDE by hydrolysis and purified by high-performance liquid chro- matography (HPLC) as described previously. Benzo[a]pyrene-d- 12 was purchased from MSD isotopes (St. Louis, MO). It was subjected to microsomal oxidation by procedures similar to those described previously,8 and deuterated BPT were isolated by HPLC. Only the major isomer (trans, cis, trans) of both normal and deuterated BPT

637

638 KULDIP SINGH et al.

X E ~ = 357.5nm

I I I I I I I I I

374 378 382

Wavelength (nm) Figure 1. Fluorescence line-narrowed spectra of BPT and its com- plcxcs with antibodies 8El l and 3C3 at 357.5 nm excitation, T = 4.2 K. Relative intensities of the spectra for BPT, 8E11 and 3C3

complcx were 1 , 0.52 and 0.12, respectively.

was used in the studies. Acrylamide was an electrophoresis purity reagent obtained from Bio-Rad Laboratories (Richmond, CA). Monoclonal antibody 8E1 l Y was a generous gift from R. M. Santella of' ( 'olumbia University. Monoclonal antibody 3C3 was produced by us in collaboration with G. N. Wogan of Massachusetts Institute of' Technology (unpublished rcsults).

In these experiments, a frequency-doubled Nd-YAG laser-pumped dye laser was employed whose output was doubled by using a KDP crystal. Typical pulse energies were in the range of 100 pJ to 2 mJ/ pulse at 10 H L , and the pulse width was 5 ns. Samples were dissolved in glycerol-water-ethanol (5:4: I ) solvent. The BPT was mixed with an excess of antibody to form the complexes. Typical concentration of' BPT and antibody in a mixture was 0.5 &f and 50 &f, respec- tively. Long-standing samples stored in a freezer showed spectra similar to that of freshly prepared samples. Samples were sonicated to remove dissolved air and taken in a 2 mm inner diameter, 3 mm outer diameter quartz tube. Sample holder was slowly lowered into the liquid helium cryostat to control the cooling rate. A gated (100 ns to 10 ms), blue-intensified photodiode array mounted on a 0.32 m spectrograph was used to record the spectra. We used 3600 gr/ mm grating for recording high-resolution spectra at 4 K and 1800 gr/nim grating for recording the spectra at 77 K. The detector was interfaced to an IBM-compatible PC, and EG&G PAR OMA88

Table 1 . Vibrational frequencies of the first electronic state S, for BPT and antibody-BPT complexes (cm- I)*

8El l - 3C3- BPT 8E 1 1-BPT 3C3-BPT BPT-d- 12 BPT-d- 12 BPT-d- 12

75 1 82 1 949

101 1 1038 1 I03 1145

1209 1245 1285 1323 I355 1376 1398 1433 1494 1512 1555

75 1 756 82 I 820 948 949

1015 1008 1035 1036 1102 1103 1105 1136 I I36 I146 1150 1152 1203 I205 I187 1242 I243 1236

100 I I03 144 1145

I89 I189 235 1235

1286 1287 1271 1273 1273 1319 I323 1314 131 1 1317 1348 1354 1350 1347 1350 1375 I379 1396 1395 1429 1433 1426 1426 1428 1495 1495 1509 1513 1555 1558

*The frequencies given in the table do not represent a complete list and are given for comparison only. The vibrational frequencies were obtained from the spectra recorded at different laser exci- tations from 355 to 365 nm at 0.5 nm intervals.

software was used to record the spectra. The detector was gated using delays of 60-1 20 ns to reduce the background scatter from the laser excitation. This delayed detection only marginally affected the flu- orescence intensity as most of the fluorescence from BPT or com- plexes had decay times on the order of 200 ns.

RESULTS AND DISCUSSION

The FLN spectra of BPT mixed with an excess of M A b were recorded at 4 K at different laser excitations ranging from 355 to 365 nm. The laser excitation was into the vi- brational manifold of the upper electronic state, S, . The min- imum width (full width a t half maximum [FWHM]) of the /ero phonon lines (ZPL) observed was 15 c m - ' . Figure 1 shows the spectra of free BPT and its complexes with two antibodies 8E11 and 3C3 at 357.5 n m excitation. Different FLN spectra obtained for antibody complexes showed that BPT was bound to the antibody. The frequencies labeled in the figure are the upper state vibrational frequencies. As seen in the figure, for a fixed laser excitation, higher vibrational modes of the S, state were excited for MAb 8E11 and 3C3, indicating that the S, state in the antibodies was shifted t o lower energies. The (0,O) transition recorded at 77 K supports this observation (Fig. 3). The same vibrational modes could be excited in BPT and its complexes by appropriate change in the excitation laser wavelength.

Table 1 gives the upper state vibrational frequencies of the S, electronic state for the BPT and antibody-BPT complexes, which werc obtained from the spectra recorded at different wavelength excitations from 355 to 365 nm at 0.5 n m in- tervals. As the laser was tuned, ZPL of vibrational modes appear a t different places within a band width of 250 cm-I, the inhomogeneous width of the transition. As seen in Table 1, the antibody-BPI' complex vibration frequencies are al- most the same as those of BPT within the experimental ac-

Benzo[a]pyrene fluorescence 639

Table 2. Fluorescence emission Characteristics of antibody-BPT complexes at 77 K, excitation wavelength 346 nm

Quench-

with

Fluorescence emission ing

origin FWHM ACR (nm) (cm-I) R ratio (%) Site type

BPT 376.1 153 1.62 8EI I--BPT 377.2 129 1.54 70 I1 3C3-HPT 377.1 182 0.85 o r BPT-ti- I2 375.4 140 1.71 8Ell-BPT-d-12 376.4 116 1.47 33 I1 3C3-DPT-d-12 377.0 242 0.84 69 I and I1

curacies of +-2 cm-I. The only exception was the vibrational mode at 1 145 cm-I, which appeared broad in BPT and was split into two (1 136 cm - I and 1 I50 cni-I) in the complexes. Shifts in some of the vibrational frequencies by 5-10 cm-’ have been reported in the BPDE-DNA adducts. In these cases the BPDE forms a covalent bond with DNA/protein at thc C-10 position.2 Observation of the same vibrational frequencies indicates that the antibody formed a physical complex with BPT. One does notice that the relative inten- sities of different vibrations are not the same between BPT and the complexes suggesting that these antibodies form dif- ferent types of physical complexes.

In order to understand the role of the OH groups and the rest of the molecule in the complex formation with the an- tibody, deuterated BPT-d- 12 was used. Deuterated BPT had only the 12-CH hydrogens replaced with deuterium. The idea was to see ifthese hydrogens play any role in physical binding of thc molecule to the protein. The FLN spectra at 4 K were recorded for deuterated BPT and its antibody complexes (Fig. 2), which showed the formation of a complex. It was seen that changing the 12-CH hydrogens changed the fluorescence quenching characteristics of the 3C3-BPT-d- 12 complex, which showed that these hydrogens affect the strength of interactions. Deuteration also shifted the (0,O) fluorescence emission to blue (Table 2) compared to normal BPT as ex- pected. Vibrational frequencies of BPT-d- 12 included in Ta- ble 1 were the same as its antibody complexes within ex- perimental errors.

Qucriching studies with ACR

Fluorescence of free BPT is known to be quenched by ACR’O and other quenchers.” Previous studies of covalent adducts of DNA with BPDE used fluorescence quenching techniques to classify the adducts as type I, when the chro- mophore was solvent inaccessible, or type 11, when the chro- rnophore was solvent accessible.12,13 In other words, type I complexes reside in a more hydrophobic environment com- pared to type I1 adducts. The quenching with ACR was mea- sured by measuring the intensity of the origin fluorescence band of the samples with and without 1 M ACR. All other experimental conditions were kept constant. In earlier tests it was shown that such measurements could give quantitative results with an accuracy o f f 10%. Quenchingstudies of8El1- BPT and 3C3-BPT showed that the fluorescence ofthe 8E11 complex was quenched by ACR, whereas the 3C3 complex

N n ... - A

h =357.5nm Ex

n t

n

374 378 382 Wavelength (nm)

Figure 2. Fluorescence line-narrowed spectra of deuterated BPT-d- 12 and its complexes with antibodics 8El1 and 3C3 at 357.5 nm excitation, T = 4.2 K. Relative intensities of the spectra for BPT- d-12, 8E1 I and 3C3 complex were 1, 0.56 and 0.15, respectively.

was not quenched (see Table 2). It may be concluded from these studies that in the case of the 3C3-BPT complex, BPT is located in the interior regions of the protein and is not solvent accessible. Surprisingly, quenching studies ofthe BPT- d- 12 complexes ofthese MAb showed different behavior than for the normal BPT complexes. Both 8E11 and 3C3 com- plexes were quenched with acrylamide in this case. The 3C3 complex was quenched more strongly (70°/o), whereas the 8E11 complex was only weakly quenched (33%), suggesting that the 8E11 complex was bound relatively strongly to the deuterated BPT unlike 3C3. Both these complexes were clas- sified as external complexes.

Fluorescence spectra at 77 K

Fluorescence studies at 77 K employing S2 - So excitation in combination with quenching studies have proven to be very useful in adduct analysis. In S, excitation, the fluores- cence emission intensity increases due to a higher absorption coeficient. However, the spectrum becomes broad mainly

640 KULDIP SINGH rt al

t I I I I I I

575 385 395 405

Wavelength (nm) Figure 3. Fluorescence spectra of BPT (A), 8EI 1-BPT (B) and 3C3- BPI‘ (C) at 77 K, excitation wavelength 346 nm. Relative intensities

of A, B and C were 1 , 0.5 and 0.3.

for two reasons, reduced selectivity in exciting sites due to increased density of states and higher homogeneous broad- cning at 77 K. The position of the (0,O) emission band whose width is of the order of 300 cm- is characteristic of the adduct excited. In a mixture of different adducts, quenching the fluorescence of one adduct relative to the other using direrent gate delays will help to assess the type of adduct present.

In the case of pyrenyl chroniophores the S, + So transition is weakly allowed and the vibrational structure appears strong due to the breaking of pairing symmetry by the ~o lven t . ’~ . ’~ Therefore, the ratio of the fluoresccnce intensity of the origin (0,O) band relative to the vibrational bands is used as an indcx for solvent cage polarity around the chrornoph~re . ’~ . ’~ I n our studies we have used both fluorescence quenching and R values to classify the antibody-BPT complexes as type I or type 11. The R value is the ratio ofthe fluorescence intensity of the origin band to the intensity of the main vibrational band at 1300 cm-I. The R values were calculated from the

spectra recorded at 77 K for the normal and deuterated BPT complexes and are given in Table 2.

Figure 3 shows spectra of BPT and its complexes with antibodies 8E1 I and 3C3 recorded at 77 K by exciting at 346 nm into the S, electronic state. The (0,O) origin peak at 376.14 nm in BPT is shifted to lower energies on complex formation. The peak positions and band widths (FWHM) of the (0,O) emission along with R values are given in Table 2 for BPT and its antibody complexes. In general, the decrease in R value indicates decreased polarity of the binding sites (more hydrophobic), stronger transition electron phonon coupling and weaker ZPL.” Thus, in our case the R value of 0.85 for the 3C3-BPT complex indicates decreased po- larity (more hydrophobicity) of the binding positions in the antibody molecule. These results were in agreement with the quenching behavior of the 3C3-BPT complex discussed ear- lier. This is further supported by the FLN spectra recorded at 4 K (Fig. 1) for this complex, which showed weaker in- tensity for the ZPL compared to the intensities of the 8E11 complex and free BPT. Zero phonon line intensities of 3C3- BPT, 8E1 I-BPT and free BPT were in the ratio ofO. 12:0.52: I , respectively. On the other hand, fluorescence of the 8E11 complex having an R value of 1.54 (about the same as for free BPT of 1.62) was quenched with ACR, and the FLN spectra at 4 K showcd stronger ZPL compared to 3C3 but weaker than BPT. On the basis of these studies, the 8E11 complex was assigned as type I1 and 3C3 complex as type 1. The shift towards the red of the S, state has also been cor- related with the increased interaction of the BPT molecule toward protein leading to strong electron phonon coupling.’R The line position and shape of the band changes slightly with the excitation used. In Table 2 the 3C3-BPT appears slightly blue shifted compared to the 8E1 I-BPT complex. The (0,O) band of 3C3-BPT is very asymmetric in shape and broad, and we believe that this is due to a mixture of external and internal complexes. Depending upon the laser excitation used, the complexes in certain sites get more excited compared to others. At a different excitation (352.5 nm) used it was seen that the origin band of 3C3-BPT was actually red shifted compared to the 8E 1 1-BPT complex.

Table 2 also gives R ratios for the dcuterated BPT and its complexes with 8E11 and 3C3 antibody. The 8E11 complex has an R ratio of 1.47 (Table 2) compared to the R value of 1.7 1 for the deuterated BPT, and the fluorescence was weakly quenched in 1 M ACR. Fluorescence of antibody 3C3-BPT complex was strongly quenched, and its R value of 0.84 was not consistent with the quenching behavior. As seen in Table 2, the band width of the BPT (0,O) fluorescence emission decreased from 140 cm - I to 116 cm I in the 8E11 complex but increased to 242 cm - I for the 3C3 complex. The FLN spectra at 4 K also showed greater width of the spectrum comprising active vibrational modes. This indicates that the 3C3 complex had a large number of different microenviron- ments compared to 8E 1 1. Quenching behavior of the 3C3- BPT-d- 12 complex at 77 K indicates the presence of at least two complexes (see Fig. 4). Spectra B in Fig. 4 having S, state energy at 376.13 nm represents the unquenched complex in 1 M ACR. Subtracting spectrum B from spectrum A gives the quenched complex with 377.54 nm as the S, state energy. This coupled with inferences on quenching studies, the 8E11 antibody complexes with deuterated BPT were classified as

Benzo[a]pyrene fluorescence 64 1

X E ~ = 346nm

A = 3C3-BPT-d-12

B = A t 1 M ACR ID

Lo ? C = A - B

M

-' A \

\ I

-I I I I I I I

375 385 395 405

Wavelength (nm) Figure 4. Fluorescence spectra of the 3C3-BPT-d-12 complex at 77 K (A), 3C3-BPT-d- 12 in 1 M ACR (B) and difference of A - B (C);

excitation wavelength 346 nm.

type 11, whereas 3C3-BPT-d- 12 were classified as type 1 and 11.

Analysis of the relative intensities of BPT and antibody- BPT complexes was carried out at different wavelengths for laser excitation into the S, and S2 electronic states. The dif- ferences in relative intensities give information about the type of interaction and are also important for quantitative estimation of BPT in low levels of concentration. In general, for S, excitation, intensitiesofBPT, 8E11 and 3C3 complexes were in the ratio 1:0.5:0.1, respectively. In the S2 excitation, the maximum fluorescence intensity was observed for laser excitation at 345 nm and 348 nm for the BPT and MAb complexes, respectively. However, for laser excitation in the range of 349-353 nm excitation an anomaly of relative in- tensities was seen (see Fig. 5 ) where the intensity of the 8EI 1 complex was an order of magnitude greater than the BPT and 3C3 complex, and the line width of the emission band

B PT,

374 378 382

Wavelength (nm) Figure 5. Comparison of fluorescence intensities of BPT, 8E11-

BPT and 3C3-BPT at 35 1 nm excitation, T = 4.2 K.

was very narrow. This was attributed to the homogeneous distribution of the 8E1 1 complexes.

Conclusion

Benzo[a]pyrene tetrol forms stable complexes with anti- bodies 8E11 and 3C3. The FLN spectra of the complexes were Characteristic of the antibody used. These studies showed that relative intensities of the ZPL act as a sensitive probe of the physical interactions of BPT with antibody. Fluores- cence quenching studies with ACR and spectra recorded at 77 K by S2 excitation were used to extract information about the degree of interior binding of the BPT molecule with an- tibodies. From the quenching studies, one may infer that antibody 8E11 forms external complexes that were solvent accessible, whereas antibody 3C3 forms internal complexes, The K values calculated from the spectra at 77 K predicted the degree of interior binding, which was in agreement with the quenching behavior of the complexes with ACR. Analysis of relative fluorescence intensities showed that ZPL of the antibody complexes were weaker compared to BPT. Deu-

642 KULDIP SINGH et ul.

tcrated BPT also formed similar physical complexes with 6. Jankowiak, R., B. W. Day, Pei-qi Lu, M. M. Doxtader, P. L. . -

anti bodies but with different quenching behavior. In contrast to normal BPT, complexes o f the antibodies were quenched with ACR. Antibody 3C3 formed more heterogeneous dis- tribution o f complexes.

O u r results indicate that the binding forces a n d stereo- chernical features o f the MAb-BPT complexes can differ sig- nificantly between different M A b with consequences for the fluorescence properties o f the BPT. Th i s observation pre- sumably extends t o complexes formed between other MAb and their antigens. In the present instance, i t m a y be o f c o n - siderable value for confirmation of BPT identity a t trace levels that when BPT is bound to one MAb its fluorescence can be quenched by ACR, while binding t o the other MAb protects it f rom quenching. T h e differential effects o f M A b binding on the fluorescence intensity following S2 excitation may be similarly useful.

,4dnowledgement- We thank Dr. Richard P. Rava (Spectroscopy Laboratory, Massachusetts Institute of Technology*) for many stim- ulating discussions and reviewing this paper. This research was sup- ported by NIH grants, Nos. ES04675, ES02109, and P41-RR02594.

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