Immunocheml~try, 1975, Vol 12. pp 291 296 Pergamon Press
Printed in Great Britain
FLUORESCENCE QUENCHING OF ADRIAMYCIN BY SPECIFIC ANTIBODIES* Y U E H - H S I U C H I E N t and L A W R E N C E LEVINE~ Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154, U.S.A. (Received 15 July 1974)
Abstract--The interaction of adriamycin, daunomycin, N-acetyldaunomycin, daunomycin benzoylhydrazone and adriamycinone with rabbit and goat antibodies to adriamycin results in quenching of the fluorescence intensity of these haptens. In solvents of low dielectric constant, the fluorescence of the hgands is enhanced and a peak at longer wavelength appears. With solutions of acidtc, basic and neutral amino acids, the fluorescence of the haptens is increased. With aromatic amino acids however, a concentration dependent quenching effect is observed. These results, in conjunction with data obtained when amino and carboxyl analogs of the aromatic amino acids are used, suggest that aromatic amino acid residues and possibly carboxyl groups as well are near the antibody combining sites.
INTRODUCTION Anthracycline antibiotics, such as adriamycin and daunomycin, have been shown to have antitumor activities (DiMarco et al., 1963). Van Vunakis et al. (1974) have successfully produced antibodies against adriamycin by immunizing rabbits, a goat and monkeys with adriamycin-protein conjugates. The antibodies in these antisera, as determined by radioimmunoassay, recognize the anthracycline structures of adriamycin, daunomycin and their structural analogues, adriamycinone, N-acetyldaunomyein, and daunomycin benzoylhydrazone. The anthracycline moiety of those molecules imparts unique fluorescence characteristics which have been used to determine the levels of daunomycin and its metabolites in serum and urine (Bachur et al., 1970). This haptenic anthracycline chromophore also enabled us to study the change in its fluorescence intensity when it binds to the antibody and to study the strength and properties of the antibody combining site. Huorescence dyes have provided special advantages as molecular probes to study protein small molecule interactions (Weber, 1952). Such studies have also been applied to antigen-antibody systems (Parker et al., 1967; Parker and Osterland, 1970; Velick et al., 1960; Eisen, 1964; Lopatin and Voss, Jr., 1971). We have found that the fluorescence of adriamycin and the anthracycline analogues is quenched when bound to specific antibodies. This quenching effect may represent interactions between the hapten and aromatic amino acids and carboxyl groups on residues at the antibody combining site. MATERIALS A N D M E T H O D S
Adrlamycln was obtained from the National Cancer Institute. Adriamycinone, the aglycone of adriamycin, was * Publication No. 996 from the Graduate Department of Biochemistry, Brandeis University. Supported in part by Grant IC-IOM from the American Cancer Society. t Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California. :~American Cancer Society Professor of Biochemistry (Award N. PRP-2I).
prepared by Dr. John J Langone, according to the procedure used to prepare daunomycinone from daunomycin (Arcramone et al, 1964). Daunomycin, N-acetyldaunomyctn, and daunomycin benzoylhydrazone were generous gifts from Dr. Nicholas R. Bachur of the Baltimore Cancer Research Center, NCI, Baltimore, Md. Some of the structures are shown in Table 1. The L-amino acids and tyramlne were purchased from Mann Research Laboratory while phenylacetlc acid, tryptamine and hydroxyphenylacetlc acid were purchased from Aldrich Chemical Company. Phenylethylamine was obtained from Eastman Company and 1-ethyl-3-(dlmethylammopropyl)-carbodlimlde from Ott Chemical Co. All the common salts were commercially available reagent grade chemicals used without purification. Goat and rabbit antisera were prepared according to the methods of Van Vunakls et al. (1974). Antibodies were purified by affinity chromatography. Adrlamycin was covalently linked to affinose 202 resin with water soluble 1-ethyl-3(dlmethylamlnopropyl)-carboditmide The resin conjugate (5 ml) and 5-0 ml of undiluted goat anti-adriamycin were incubated within the column overnight at 2-4°C. The resin column was then washed exhausttvely and the antibodies were eluted by allowing 1.0 ml of adrlamycin (10 mg/ml) to penetrate the resm bed, at which time the flow rate was adjusted to 1 ml per hour. The individual fractions (1 ml) were exhaustively dialyzed to remove the free adriamycin. They were assayed for protein by absorbance at 280 nm and antibody activity by radtoimmunoassay (Van Vunakls et al., 1974). Only small quantities of purified adnamycin antibodies were prepared because of the large amounts of adriamycin required for elution. No experiment was performed to test for binding of normal immunoglobulins to the adrlamycm-affinose resin. Absorption spectra were obtained on a Cary 14 spectrophotometer. A Zeiss spectrophotometer was used for absorbance measurements. Fluorescence measurements were made using a Perkin-Elmer MPF-3 fluorescence spectrophotometer. Ratios of fluorescence intensity relative to exciting light intensity were measured so as to correct for source intensity fluctuation. All experiments were carried out at room temperature. In binding expertments, the sample was excited at 467 nm, and the wavelength of emission was 555 nm. All the experiments were carried out in isotris buffer (0.01 m Tns-HC1, pH 7-4, 0.147 M NaC1 solution) unless otherwise stated. Immunoglobuhns from normal rabbit and goat sera and from immune goat and rabbit antisera were precipitated with 40% (NH4)2SO4. The precipitates were dissolved in
291
292
YUEH-HSIU C H I E N and LAWRENCE LEVINE Table 1 Average intrinsic association constants ( M Llgands
Rabbit 895C-7
Goat 7
44 x 104 5.0 x 10"* 44 x I()4
29 × It) ~' 33 ~ l0 ~ 23 , l(J"
2-4 x I()4 24 x 10"t
12 ~ 10 I 2 ,- 10 ~
Adriamycin Daunomycin N-Acetyldaunomycm Daunomycin benzoylhydrazone ~ Adriamycinone b 0 ~
OH ~L. / ~
1~ 14 /COCH3
0 ~J.
Y
OCH3 0
OH ~
13 14 /COCH20H
~
---f,-;~:.^ OH H u "
I
HO ~ NH 2
H
NH z
1)
Daunomycln
"H H
Adrlarnyctn
C-13 benzoylhydrazone h Adrlamycln aglycone As measured by equilibrium dialysis with an [~25i]_adriamycl n derivative, the /k+, of the goat antiserum was also found to be low: in the order of 10 ~ M - ~. "
A
380
420
460
500
540
580
Wavelength (nm)
a
iE
520
the equation X~ = (1, - I~I/I, Ao, where Xo is the total hgand added, I, is the fluorescence intensity of the control immunoglobulin; and I~ is the fluorescence intensity of the antiserum solution Free ligand concentration Xj was then calculated from the equation X / = X o - Xb The reciprocal values of Xb were plotted against the reciprocal values of X0, data were fitted into a straight line The reciprocal value of the intercept at l/X0 = 0 was taken as the total hgand binding sites, L, in the solution. Assuming all of the lmmunoglobulins are divalent, L/2 was taken as the antibody concentration in the solution Binding data were expressed in terms of r and c, were t is moles of hgand bound per mole of antibody and c is the free ligand concentration, r/c was plotted as a function of r (Scatchard, 1949)and the average lntrmsic association constant Ko was taken as the value of r/c at r = I
RESULTS
/
\
iI/ 550
580
610
640
Wavelength (nm)
Fig. IA. Absorption spectra of adriamycm. Adriamycm in 0'01 M Tris-HCL pH 7.4 buffer [NaC1] = 0.147 M. lB. Fluorescence spectra of adriamycin (479 x 1 0 - 6 M ) i n 0-01 M Tris-HCl, pH 7-4 ( - - ) , in the presence of 3-3 × 10-TM purified anti-adriamycIn antibody ( ), and in the presence of 3.5 mg/ml bovine serum albumin ( --). .
.
.
.
.
lsotrls buffer and dialysed before they were used for binding experiments. Protein concentration was determined by the Lowry method (Lowry, 1951). Binding experiments were carried out as follows: Three ml of the immunoglobulin solutions were titrated. Concentrated hgand solutions (0-8 mg/ml) were added from a mlcrosyringe, the contents were mixed and the fluorescence intensity was recorded after each addition. Titration of the immune lmmunoglobulins was always accompanied by a titration of nonimmune immunoglobulins (from the same species) at the same protein concentration. For each addition of the hgand solution the number of moles of ligand bound was calculated from
The absorption and emission spectra of adriamycin in isotris buffer are given in Fig. IA and B. The anthracyclic moiety exhibits a fluorescence maximum at 555 nm and a shoulder at 578 nm. Daunomycin, Nacetyldaunomycin, daunomycin benzoylhydrazone and adriamycinone all have the same absorption and emission spectra. Addition of purified antibodies to the adriamycin solution resulted in a decrease in the fluorescence yield which was not accompanied by a shift m the emission spectra (Fig. I B). This suggested that a quantitative assay for adriamycin anti-adrlamycin interaction based on the quenching of the fluorescence of adriamycin when it was bound to antibody was feasible. To test this, increments of purified antibodies were added to 4.79 × 10 -6 M adriamycin. The amount of quenching was proportional to the purified antibody added. Immunoglobulin solutions obtained from normal goat or rabbit serum did not quench the fluorescence of the antibiotic;they increased its fluorescence intensity without changing its emission spectra. Similar increases in fluorescence were observed when bovine serum albumin was added to adriamycin solutions (Fig. 1B). Since immunoglobulins from normal serum
Fluorescence Quenching of Adrtamycin by Specific Antibodies
293
5 -A 5
4 4 O
3 3
2 2
1 I
0
i
I
04
08
I
I
12
16
I
20
r
0
I 04
018
I
i 16
12
I 20
r
Fig. 2A. Binding of adnamycin by goat antiserum. Total protein concentration 5.4 mg/ml, antibody concentration 3.03 × 1 0 - 6 M. 2B. Binding of daunomycin benzoylhydrazone by goat antiserum. Total protein concentration of the serum 5.4mg/ml. Antibody concentration 3.03 x 1 0 - 6 M. Experiments were carried out in isotrls buffer. increased the fluorescence intensity of adrlamycin, although slightly, titration of the immune globulins was always accompanied by a parallel titration using nonimmune globulins at the same protein concentrations. The association constants of goat and rabbit anti-adriamycin antisera and adriamycin, daunomycin, daunomycin benzoylhydrazone, N-acetyldaunomycin and adriamycinone were determined. These data are shown in Table 1. Two typical Scatchard plots are shown in Figs. 2A and B. In order to elucidate the nature of fluorescence quenching when these anthracycline ligands are bound to specific antibodies, fluorescence emission of adriamycin was measured in dioxane-isotris buffer solutions. With increasing percentages of dioxane in the medium (and corresponding decreases in the dielectric constant) the fluorescence yield increases and a 578 nm emission peak appears (Fig. 3). Daunomycin, daunomycin benzoylhydrazone, N-acetyl daunomycin and adriamycinone all showed similar effects. The effects of different amino acids on the fluorescence spectra of adriamycin and its structural analogues were also examined. The amino acids were dissolved in isotris buffer, pH 7.4. When necessary, the pH of acidic and basic amino acid solutions were adjusted back to pH 7.4 by addition of NaOH or HC1. Tyramine was used instead of tyrosine, because of tyrosine's low solubility. Glycine, leucine, glutamlc acid and arginine increased the fluorescence intensity of the system. However, tyramine and tryptophan quenched the fluorescence intensity of all five ligands. Phenylalanine also quenched the fluorescence intensity of the adriamycin, daunomycin and daunomycin benzoylhydrazone systems. However, phenylalanine (2 × 10--" MI did not decrease the fluorescence intensity of the Nacetyldaunomycin and the adriamycinone solution to below their reference points, which is the fluorescence intensity of corresponding ligands in lsotris buffer. Nevertheless, with increasing concentrations of phenylalanine the quenching trend is similar to those of tryamine and tryptophan. The results of amino acid effects are presented in Figs. 4A and B and Figs. 5A, B, and C. Although not shown in Figs. 4 and 5, amino acid
effects at concentrations lower than 1 x 10 -3 M were also carried out but with one third of the dye concentrations used previously. In the adriamycin, daunomycin and daunomycin benzoylhydrazone systems, only tryptaphan showed an observable concentrationdependent quenching effect at concentrations between 1 x 10 -4 and 1 x 10 -3 M. Glutamic acid, arginine and leucine enhanced the fluorescence intensity of Nacetyldaunomycin but only at concentrations higher than 5 x 10-4M. Between 1 x 10 -4 and 1 x 10-3M, the fluorescence intensity of aglycone increased as the concentration of amino acids increased. In order to reveal the nature of the quenching by these aromatic amino acids, the effects of several organic compounds on the fluorescence intensity of adriamycin and adriamycinone were studied. These compounds were acetate, tryptamine and indoleacetic acid which have indole groups in common with tryptophan, hydroxyphenyl acetic acid which has the hydroxyphenyl group in common with tyrosine and tyramine, and phenylacetic acid and phenylethylamine which have the phenyl group in common with phenylalanine. These organic acids and amines were dis-
>,
ca
8
520
5~o
5~o Wavelength
61o
6~o
(nrn)
Fig. 3. Fluorescence emission spectra of adriamycm m: (1) dioxane solution; (2) dioxane-isotris buffer (80:20); (3) dioxane-lsotris buffer (60:40); (4) dioxane-isotris buffer 140:60); (5) dioxan~isotris buffer (20:80); and (6) isotris buffer
294
Y U E H - H S I U C H I E N and L A W R E N C E LEVINF 25
(B)
20
15
g o g
IO 05
13::
"'o rio
0
20I
[A mlrlO Clcld] ~
mM
llO lAmmo acldl,
2JO mM
Fig. 4A. Effect of a m i n o acids on the fluorescence emission of the adriamycm system The effects of glycme(- - • •-4,1eucine(--II--II--),glutam]cacld ( A--• ), arginme [- [] [] L phenylalanme (- A ~ ), tyramme ( - - O - Q - - J , tryptophan ( O O ) ~vere studied. The system contained 2-4 × 10-" M adriamycm in 0.01 M Tns-HC1, pH 7.4, 0 147 M NaCI solution. 4B. Adriamycmone system. The effectsofglycinel • - • ),leucme(--I--II--),glutamicacld( • - ~ ),argmme ( ~ [~ t, phenylalanme (- A z5 k tyramine ( - - ~ O -~ ), and tryptophan ( O --O--I were studied The system contained 3.5 × i0 -~ M a d n a m y c m o n e in 0-01 M T n s HC1, pH 7.4, 0-147 NaC1 soluuon I 5 -(C)
I 5f(B)
I0
~
!
g g 8
05
to
c
_s
*6
I'0 [Amino OCld],
_s
20 mM
rO [Amino
OCld],
2'0 mM
,", - - ~ - - ~ O [Amino ocld],
20 mM
Fig. 5A. Effect of amino acids on the fluorescence emission of the d a u n o m y c m system The effects of glycme ( V - - • 1, leucme ( - - I - - I I - - ) , glutam]c acid ( A - • I, arglnme ( U~ D L phenylalanlnc ( A A J, tyramme ( O - - O - - ) , and tr)optophan ( O - O ~ ) were ~tudled The system contained 2 5 × I()-~'M daunomycin m 0"01M Tris HCI, pH 74 buffer, 0-147M NaCI solution 5B Effect of amino acids on the fluorescence emxss~on of N-acetyldaunomycm s~stem The effects of glycme ( - - • • - ) , leucme ( - - I - - I I - - I , glutamlc acid ( - • •---), argimne ( ~ W ), phenylalanlne ( A ~ ), tyramme ( - - ~ - I and tryptophan ( O © ) were studied The system contained 2-3 × I 0 - 6 M N-acetyldaunomycm m 0.01 M Tris HCI, pH 7 4 buffer, 0 I47M NaC1 soluUon. 5C. Effect of amino acids on the fluorescence emission of d a u n o m y c m benzoylhydrazone system The effect ofglycine ( - - • • ), leucme (--I1--11--), glutamic acid ( - A m • I, argmme ( [] r2 I, phenylalanine ( - - A A--), tyramine ( - - ~ - O - - ) , and tryptophan ( O O -t v~ere studied. The system contained 2.1 × 10-~'M daunomycin benzoylhydrazone in 0.01 M THs-HCI pH 7,4 q 147 '~I Na('l solution
Fluorescence Quenching of Adriamycin by Specific Antlbo&es
295
Table 2. Relative fluorescenceintensity Concentranon Isotns buffer Indoleacetlc acid Tryptamme Hydroxyphenylacetic acid Tyramine Phenylacetlc acid Phenylethylamlne Acetate
1× 2× 1× 2x 1× 2× 1× 2x 1x 2× 2× 1x 2x
10-3 10 - 3 10 - 3
10 3 t0- 3 10 -' 10 - 3
10-2 10-3 10-z 10- z 10-3 10-2
Adriamycin
Adriamycinone
1.00 0.93 0'33 0.95 0.36 0.98 0-57 1"00 0'75 0.81 0-78 0.84 0-96 0.76
1.00 0-62 0.31 0.62 0.31 0.65 0.32 0.97 066 0-62 0.46 1.00 0.92 0-72
Experiments were carried out in isotris buffer. [Adrlamycin] = 2.4 x 10-6M [Adriamycinone] = 3.5 x 10 - 6 M. solved in isotris buffer and the pH of the solutions was adjusted to 7.4 by adding NaOH or HC1. The results are shown in Table 2. DISCUSSION
When adriamycin and its structural analogues, daunomycin, daunomycin benzoylhydrazone. Nacetyldaunomycin and adriamycinone are dissolved in dioxane solution, the fluorescence intensity of the system increases. Similar increases were also observed when daunomycin was dissolved in alcohol solutions (Bachur et al., 1970). These increases in solvents of low dielectric constant suggest that the small increases in fluorescence intensity when the ligands bind nonspecifically to immunoglobulinsprepared from normal goat and rabbit sera are due to factors related to the low polarity of the environment. Similar increases in fluorescence intensity were observed when bovine serum albumin which has been shown to have hydrophobic binding sites for organic dye molecules was added to the system. The excitation (467nm) and emission (555nm) maximum of these anthracyclic molecules are quite different from the excitation (280-295 rim) and emission (305-350 nm) spectra of proteins. Therefore, it is unlikely that the reduction of fluorescence of these molecules when they bind to specific antibody combining sites could be caused by energy transfer between the ligands and the protein molecules. Therefore, fluorescence quenching of the ligands could be used to determine the binding strength of the antibodies without the use of purified antibodies. Scatchard plots show that these antibodies are heterogeneous. Nevertheless, the average intrinsic association constants can give good approximations of their binding strength. Adriamycin, daunomycin, two derivatives of daunomycin, N-acetyldaunomycln and daunomycin benzoylhydrazone and adriamyclnone all have the similar binding strengths with antibodies directed toward adriamycin. This finding is consistent with the inhibition studies by Van Vunakis et al. (1974), who found that the anti-adriamycin antibodies recognized the anthracycline structure. Both by radioimmunoassay and by fluorescence quenching the relatively bulky benzoylhydrazone on the C-13 keto group has little effect on the binding strength of the I\l~l 12 4
(
system and indicates that the area of the agylcone complementary to the antibody receptor site is distant from the C-13 and C-14 group. Anthracyclic chromophores have two phenolic hydroxy groups at positions 5 and 12 and two keto groups at positions 6 and l l. Studies have shown that a phenolic hydroxy group is more aci&c in the excited state than in the ground state, and that the ionized species is nonfluorescent (Van Durren, 1963). According to Bachur et al. (1970), when daunomycin was dissolved in alcohol or 0'3 M HC1 solutions, the fluorescence yield increases and the longer wavelength peak appears. At alkaline pH values, the anthracyclic compounds no longer fluoresce. This decrease of fluorescence in alkaline solution follows closely the titration curve of the dye. The increase in fluorescence of adriamycin and its structural analogues in dioxane which not only is of lower dielectric constant but also is less of a proton acceptor than water, may be explained by the fact that the dioxane molecule has lower affinity for the phenolic proton: in addition, the less polar environment may stabilize the non-ionized phenol group more. Thus, the non-ionized fluorescent form would be favored. The effects of different amino acids on the fluorescence intensity suggest that these interactions are specific since they are observable even at 1 × 10-3 M. Acidic, basic or neutral amino acids all increase the fluorescence intensity of the system. At low concentrations of phenylalanine and tryptophan, the fluorescence intensity also increases. However, tyramine, tryptamine, indole-acetic acid, hydroxyphenylacetic acid, phenylacetlc acid and phenylethylamine fail to show similar effects, suggesting that the zwitter ion contributed by both the amino and carboxy groups on the amino acid backbone is required. However, due to the complexity of the chromophore, further studies are necessary in order to elucidate the mechanism of this fluorescence enhancement. The decrease of the chromophore fluorescence by the aromatic compounds may result from the formation of nonfluorescent charge transfer complexes or 7~complexes (Van Durren, 1963) between the aromatic residues and the anthracycline ring. The formation of these complexes would result from the interaction between the r~-electrons of the two molecules. This interaction would be more likely to occur in the excited
296
YUEH-HSIU CHIEN and LAWRENCE LEVINE
than the ground state. The mdole group is the most etlic~ent quencher followed by the hydroxyl phenyl group: the phenyl group has the least effect. Hydroxyphenylacetic acid is a more efficient quencher than tyramine, phenylacetlc acid is a better quencher than phenylethylamlne. These differences m quenching suggest that in addition to the re-complex formation, the carboxy group may interact with the phenohc group and cause additional quenching. The degrees of quenching by lndoleacetlc acid and tryptamine are the same suggesting that the formation of nonfluorescent 7r-complexes is primarily' responsible for the observed effects with the indole compounds. This speculation is consistent with the finding that acetate quenches the flt, orescence intensity of adriamycln and adriamycmone systems. The effects of phen)lalanme and tr)ptophan could be a combination of the fluorescence enhancement caused by' their amino acid backbones and the fluorescence quenching caused by' their aromatic side chains. These combined effects can be observed more clearly m adriamycmone solutions because the adriamycinone molecule does not have the amino sugar moiety: therefore, the chromophore is fully exposed to the environment. At low amino amd concentrations, the fluorescence enhancement process dominates. As the amino acid concentration increases the re-complex quench,ng effect becomes important. Since the phenyl group is a weak ~z-complex former, the fluorescence intensity of adriamycinone is higher than its reference point e~en
at the 2 × 10-2 M level. However, phenylalanlne does show a concentratmn dependent quenching effect. We are thus suggesting that aromatm amino acid and/or carboxy groups are present at the antlbod), combining site REFERENCES
Arcamone F., Franceschl G., Orezzl P, Casslnelh G, Barbleri W and Mondelh R (19641 J Am chem. So~. 86, 5334 Bachur N R., Moore A L., Bernstem J. G and Llu A 11970} Can~er Chemother. Rep., Part I 54, 89 DiMarco A., Gaetani M. and Dongott~ t i (1963} Turnoff 49, 203. E1sen H. N. 11964p Method.~ m Mc&cal Rewarch (E&ted b) Elsen H N.}, Vol. X, p. 115 Chicago Year Book MedJcal, Chmago Lopatin D. E., and Voss E. W (1971) Blochemtstrv 10, 208 Lowry O H, Rosebrough W F, Farr A L and Randall R V (1951}J hlol Chem 193,265 Parker C. W, Yoo J. J., Johnson M C. and Godt S. M {1967) Biochemistry 6, 3408. Parker C W and Osterland (' K (19701 B~ochem~str) 9, I 0~4
Scatchard G. 11949} Ann ~.I ~' Acad. Set. 51,661). Van Duuren B. L (1963) Chem Rev. 63, 325 Van Vunakls H, Langone J. J., Rlceberg L. J and Levme L. (1974) Cancer Res. 34, 2546. Vehck S. F., Parker C. W. and Etsen H N ~19601Proc ham. Acad. Sol. U.S A. 46, 1470. Weber G. 11952) Btochem. J. 51, 155
Printed in Great Britain
FLUORESCENCE QUENCHING OF ADRIAMYCIN BY SPECIFIC ANTIBODIES* Y U E H - H S I U C H I E N t and L A W R E N C E LEVINE~ Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154, U.S.A. (Received 15 July 1974)
Abstract--The interaction of adriamycin, daunomycin, N-acetyldaunomycin, daunomycin benzoylhydrazone and adriamycinone with rabbit and goat antibodies to adriamycin results in quenching of the fluorescence intensity of these haptens. In solvents of low dielectric constant, the fluorescence of the hgands is enhanced and a peak at longer wavelength appears. With solutions of acidtc, basic and neutral amino acids, the fluorescence of the haptens is increased. With aromatic amino acids however, a concentration dependent quenching effect is observed. These results, in conjunction with data obtained when amino and carboxyl analogs of the aromatic amino acids are used, suggest that aromatic amino acid residues and possibly carboxyl groups as well are near the antibody combining sites.
INTRODUCTION Anthracycline antibiotics, such as adriamycin and daunomycin, have been shown to have antitumor activities (DiMarco et al., 1963). Van Vunakis et al. (1974) have successfully produced antibodies against adriamycin by immunizing rabbits, a goat and monkeys with adriamycin-protein conjugates. The antibodies in these antisera, as determined by radioimmunoassay, recognize the anthracycline structures of adriamycin, daunomycin and their structural analogues, adriamycinone, N-acetyldaunomyein, and daunomycin benzoylhydrazone. The anthracycline moiety of those molecules imparts unique fluorescence characteristics which have been used to determine the levels of daunomycin and its metabolites in serum and urine (Bachur et al., 1970). This haptenic anthracycline chromophore also enabled us to study the change in its fluorescence intensity when it binds to the antibody and to study the strength and properties of the antibody combining site. Huorescence dyes have provided special advantages as molecular probes to study protein small molecule interactions (Weber, 1952). Such studies have also been applied to antigen-antibody systems (Parker et al., 1967; Parker and Osterland, 1970; Velick et al., 1960; Eisen, 1964; Lopatin and Voss, Jr., 1971). We have found that the fluorescence of adriamycin and the anthracycline analogues is quenched when bound to specific antibodies. This quenching effect may represent interactions between the hapten and aromatic amino acids and carboxyl groups on residues at the antibody combining site. MATERIALS A N D M E T H O D S
Adrlamycln was obtained from the National Cancer Institute. Adriamycinone, the aglycone of adriamycin, was * Publication No. 996 from the Graduate Department of Biochemistry, Brandeis University. Supported in part by Grant IC-IOM from the American Cancer Society. t Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California. :~American Cancer Society Professor of Biochemistry (Award N. PRP-2I).
prepared by Dr. John J Langone, according to the procedure used to prepare daunomycinone from daunomycin (Arcramone et al, 1964). Daunomycin, N-acetyldaunomyctn, and daunomycin benzoylhydrazone were generous gifts from Dr. Nicholas R. Bachur of the Baltimore Cancer Research Center, NCI, Baltimore, Md. Some of the structures are shown in Table 1. The L-amino acids and tyramlne were purchased from Mann Research Laboratory while phenylacetlc acid, tryptamine and hydroxyphenylacetlc acid were purchased from Aldrich Chemical Company. Phenylethylamine was obtained from Eastman Company and 1-ethyl-3-(dlmethylammopropyl)-carbodlimlde from Ott Chemical Co. All the common salts were commercially available reagent grade chemicals used without purification. Goat and rabbit antisera were prepared according to the methods of Van Vunakls et al. (1974). Antibodies were purified by affinity chromatography. Adrlamycin was covalently linked to affinose 202 resin with water soluble 1-ethyl-3(dlmethylamlnopropyl)-carboditmide The resin conjugate (5 ml) and 5-0 ml of undiluted goat anti-adriamycin were incubated within the column overnight at 2-4°C. The resin column was then washed exhausttvely and the antibodies were eluted by allowing 1.0 ml of adrlamycin (10 mg/ml) to penetrate the resm bed, at which time the flow rate was adjusted to 1 ml per hour. The individual fractions (1 ml) were exhaustively dialyzed to remove the free adriamycin. They were assayed for protein by absorbance at 280 nm and antibody activity by radtoimmunoassay (Van Vunakls et al., 1974). Only small quantities of purified adnamycin antibodies were prepared because of the large amounts of adriamycin required for elution. No experiment was performed to test for binding of normal immunoglobulins to the adrlamycm-affinose resin. Absorption spectra were obtained on a Cary 14 spectrophotometer. A Zeiss spectrophotometer was used for absorbance measurements. Fluorescence measurements were made using a Perkin-Elmer MPF-3 fluorescence spectrophotometer. Ratios of fluorescence intensity relative to exciting light intensity were measured so as to correct for source intensity fluctuation. All experiments were carried out at room temperature. In binding expertments, the sample was excited at 467 nm, and the wavelength of emission was 555 nm. All the experiments were carried out in isotris buffer (0.01 m Tns-HC1, pH 7-4, 0.147 M NaC1 solution) unless otherwise stated. Immunoglobuhns from normal rabbit and goat sera and from immune goat and rabbit antisera were precipitated with 40% (NH4)2SO4. The precipitates were dissolved in
291
292
YUEH-HSIU C H I E N and LAWRENCE LEVINE Table 1 Average intrinsic association constants ( M Llgands
Rabbit 895C-7
Goat 7
44 x 104 5.0 x 10"* 44 x I()4
29 × It) ~' 33 ~ l0 ~ 23 , l(J"
2-4 x I()4 24 x 10"t
12 ~ 10 I 2 ,- 10 ~
Adriamycin Daunomycin N-Acetyldaunomycm Daunomycin benzoylhydrazone ~ Adriamycinone b 0 ~
OH ~L. / ~
1~ 14 /COCH3
0 ~J.
Y
OCH3 0
OH ~
13 14 /COCH20H
~
---f,-;~:.^ OH H u "
I
HO ~ NH 2
H
NH z
1)
Daunomycln
"H H
Adrlarnyctn
C-13 benzoylhydrazone h Adrlamycln aglycone As measured by equilibrium dialysis with an [~25i]_adriamycl n derivative, the /k+, of the goat antiserum was also found to be low: in the order of 10 ~ M - ~. "
A
380
420
460
500
540
580
Wavelength (nm)
a
iE
520
the equation X~ = (1, - I~I/I, Ao, where Xo is the total hgand added, I, is the fluorescence intensity of the control immunoglobulin; and I~ is the fluorescence intensity of the antiserum solution Free ligand concentration Xj was then calculated from the equation X / = X o - Xb The reciprocal values of Xb were plotted against the reciprocal values of X0, data were fitted into a straight line The reciprocal value of the intercept at l/X0 = 0 was taken as the total hgand binding sites, L, in the solution. Assuming all of the lmmunoglobulins are divalent, L/2 was taken as the antibody concentration in the solution Binding data were expressed in terms of r and c, were t is moles of hgand bound per mole of antibody and c is the free ligand concentration, r/c was plotted as a function of r (Scatchard, 1949)and the average lntrmsic association constant Ko was taken as the value of r/c at r = I
RESULTS
/
\
iI/ 550
580
610
640
Wavelength (nm)
Fig. IA. Absorption spectra of adriamycm. Adriamycm in 0'01 M Tris-HCL pH 7.4 buffer [NaC1] = 0.147 M. lB. Fluorescence spectra of adriamycin (479 x 1 0 - 6 M ) i n 0-01 M Tris-HCl, pH 7-4 ( - - ) , in the presence of 3-3 × 10-TM purified anti-adriamycIn antibody ( ), and in the presence of 3.5 mg/ml bovine serum albumin ( --). .
.
.
.
.
lsotrls buffer and dialysed before they were used for binding experiments. Protein concentration was determined by the Lowry method (Lowry, 1951). Binding experiments were carried out as follows: Three ml of the immunoglobulin solutions were titrated. Concentrated hgand solutions (0-8 mg/ml) were added from a mlcrosyringe, the contents were mixed and the fluorescence intensity was recorded after each addition. Titration of the immune lmmunoglobulins was always accompanied by a titration of nonimmune immunoglobulins (from the same species) at the same protein concentration. For each addition of the hgand solution the number of moles of ligand bound was calculated from
The absorption and emission spectra of adriamycin in isotris buffer are given in Fig. IA and B. The anthracyclic moiety exhibits a fluorescence maximum at 555 nm and a shoulder at 578 nm. Daunomycin, Nacetyldaunomycin, daunomycin benzoylhydrazone and adriamycinone all have the same absorption and emission spectra. Addition of purified antibodies to the adriamycin solution resulted in a decrease in the fluorescence yield which was not accompanied by a shift m the emission spectra (Fig. I B). This suggested that a quantitative assay for adriamycin anti-adrlamycin interaction based on the quenching of the fluorescence of adriamycin when it was bound to antibody was feasible. To test this, increments of purified antibodies were added to 4.79 × 10 -6 M adriamycin. The amount of quenching was proportional to the purified antibody added. Immunoglobulin solutions obtained from normal goat or rabbit serum did not quench the fluorescence of the antibiotic;they increased its fluorescence intensity without changing its emission spectra. Similar increases in fluorescence were observed when bovine serum albumin was added to adriamycin solutions (Fig. 1B). Since immunoglobulins from normal serum
Fluorescence Quenching of Adrtamycin by Specific Antibodies
293
5 -A 5
4 4 O
3 3
2 2
1 I
0
i
I
04
08
I
I
12
16
I
20
r
0
I 04
018
I
i 16
12
I 20
r
Fig. 2A. Binding of adnamycin by goat antiserum. Total protein concentration 5.4 mg/ml, antibody concentration 3.03 × 1 0 - 6 M. 2B. Binding of daunomycin benzoylhydrazone by goat antiserum. Total protein concentration of the serum 5.4mg/ml. Antibody concentration 3.03 x 1 0 - 6 M. Experiments were carried out in isotrls buffer. increased the fluorescence intensity of adrlamycin, although slightly, titration of the immune globulins was always accompanied by a parallel titration using nonimmune globulins at the same protein concentrations. The association constants of goat and rabbit anti-adriamycin antisera and adriamycin, daunomycin, daunomycin benzoylhydrazone, N-acetyldaunomycin and adriamycinone were determined. These data are shown in Table 1. Two typical Scatchard plots are shown in Figs. 2A and B. In order to elucidate the nature of fluorescence quenching when these anthracycline ligands are bound to specific antibodies, fluorescence emission of adriamycin was measured in dioxane-isotris buffer solutions. With increasing percentages of dioxane in the medium (and corresponding decreases in the dielectric constant) the fluorescence yield increases and a 578 nm emission peak appears (Fig. 3). Daunomycin, daunomycin benzoylhydrazone, N-acetyl daunomycin and adriamycinone all showed similar effects. The effects of different amino acids on the fluorescence spectra of adriamycin and its structural analogues were also examined. The amino acids were dissolved in isotris buffer, pH 7.4. When necessary, the pH of acidic and basic amino acid solutions were adjusted back to pH 7.4 by addition of NaOH or HC1. Tyramine was used instead of tyrosine, because of tyrosine's low solubility. Glycine, leucine, glutamlc acid and arginine increased the fluorescence intensity of the system. However, tyramine and tryptophan quenched the fluorescence intensity of all five ligands. Phenylalanine also quenched the fluorescence intensity of the adriamycin, daunomycin and daunomycin benzoylhydrazone systems. However, phenylalanine (2 × 10--" MI did not decrease the fluorescence intensity of the Nacetyldaunomycin and the adriamycinone solution to below their reference points, which is the fluorescence intensity of corresponding ligands in lsotris buffer. Nevertheless, with increasing concentrations of phenylalanine the quenching trend is similar to those of tryamine and tryptophan. The results of amino acid effects are presented in Figs. 4A and B and Figs. 5A, B, and C. Although not shown in Figs. 4 and 5, amino acid
effects at concentrations lower than 1 x 10 -3 M were also carried out but with one third of the dye concentrations used previously. In the adriamycin, daunomycin and daunomycin benzoylhydrazone systems, only tryptaphan showed an observable concentrationdependent quenching effect at concentrations between 1 x 10 -4 and 1 x 10 -3 M. Glutamic acid, arginine and leucine enhanced the fluorescence intensity of Nacetyldaunomycin but only at concentrations higher than 5 x 10-4M. Between 1 x 10 -4 and 1 x 10-3M, the fluorescence intensity of aglycone increased as the concentration of amino acids increased. In order to reveal the nature of the quenching by these aromatic amino acids, the effects of several organic compounds on the fluorescence intensity of adriamycin and adriamycinone were studied. These compounds were acetate, tryptamine and indoleacetic acid which have indole groups in common with tryptophan, hydroxyphenyl acetic acid which has the hydroxyphenyl group in common with tyrosine and tyramine, and phenylacetic acid and phenylethylamine which have the phenyl group in common with phenylalanine. These organic acids and amines were dis-
>,
ca
8
520
5~o
5~o Wavelength
61o
6~o
(nrn)
Fig. 3. Fluorescence emission spectra of adriamycm m: (1) dioxane solution; (2) dioxane-isotris buffer (80:20); (3) dioxane-lsotris buffer (60:40); (4) dioxane-isotris buffer 140:60); (5) dioxan~isotris buffer (20:80); and (6) isotris buffer
294
Y U E H - H S I U C H I E N and L A W R E N C E LEVINF 25
(B)
20
15
g o g
IO 05
13::
"'o rio
0
20I
[A mlrlO Clcld] ~
mM
llO lAmmo acldl,
2JO mM
Fig. 4A. Effect of a m i n o acids on the fluorescence emission of the adriamycm system The effects of glycme(- - • •-4,1eucine(--II--II--),glutam]cacld ( A--• ), arginme [- [] [] L phenylalanme (- A ~ ), tyramme ( - - O - Q - - J , tryptophan ( O O ) ~vere studied. The system contained 2-4 × 10-" M adriamycm in 0.01 M Tns-HC1, pH 7.4, 0 147 M NaCI solution. 4B. Adriamycmone system. The effectsofglycinel • - • ),leucme(--I--II--),glutamicacld( • - ~ ),argmme ( ~ [~ t, phenylalanme (- A z5 k tyramine ( - - ~ O -~ ), and tryptophan ( O --O--I were studied The system contained 3.5 × i0 -~ M a d n a m y c m o n e in 0-01 M T n s HC1, pH 7.4, 0-147 NaC1 soluuon I 5 -(C)
I 5f(B)
I0
~
!
g g 8
05
to
c
_s
*6
I'0 [Amino OCld],
_s
20 mM
rO [Amino
OCld],
2'0 mM
,", - - ~ - - ~ O [Amino ocld],
20 mM
Fig. 5A. Effect of amino acids on the fluorescence emission of the d a u n o m y c m system The effects of glycme ( V - - • 1, leucme ( - - I - - I I - - ) , glutam]c acid ( A - • I, arglnme ( U~ D L phenylalanlnc ( A A J, tyramme ( O - - O - - ) , and tr)optophan ( O - O ~ ) were ~tudled The system contained 2 5 × I()-~'M daunomycin m 0"01M Tris HCI, pH 74 buffer, 0-147M NaCI solution 5B Effect of amino acids on the fluorescence emxss~on of N-acetyldaunomycm s~stem The effects of glycme ( - - • • - ) , leucme ( - - I - - I I - - I , glutamlc acid ( - • •---), argimne ( ~ W ), phenylalanlne ( A ~ ), tyramme ( - - ~ - I and tryptophan ( O © ) were studied The system contained 2-3 × I 0 - 6 M N-acetyldaunomycm m 0.01 M Tris HCI, pH 7 4 buffer, 0 I47M NaC1 soluUon. 5C. Effect of amino acids on the fluorescence emission of d a u n o m y c m benzoylhydrazone system The effect ofglycine ( - - • • ), leucme (--I1--11--), glutamic acid ( - A m • I, argmme ( [] r2 I, phenylalanine ( - - A A--), tyramine ( - - ~ - O - - ) , and tryptophan ( O O -t v~ere studied. The system contained 2.1 × 10-~'M daunomycin benzoylhydrazone in 0.01 M THs-HCI pH 7,4 q 147 '~I Na('l solution
Fluorescence Quenching of Adriamycin by Specific Antlbo&es
295
Table 2. Relative fluorescenceintensity Concentranon Isotns buffer Indoleacetlc acid Tryptamme Hydroxyphenylacetic acid Tyramine Phenylacetlc acid Phenylethylamlne Acetate
1× 2× 1× 2x 1× 2× 1× 2x 1x 2× 2× 1x 2x
10-3 10 - 3 10 - 3
10 3 t0- 3 10 -' 10 - 3
10-2 10-3 10-z 10- z 10-3 10-2
Adriamycin
Adriamycinone
1.00 0.93 0'33 0.95 0.36 0.98 0-57 1"00 0'75 0.81 0-78 0.84 0-96 0.76
1.00 0-62 0.31 0.62 0.31 0.65 0.32 0.97 066 0-62 0.46 1.00 0.92 0-72
Experiments were carried out in isotris buffer. [Adrlamycin] = 2.4 x 10-6M [Adriamycinone] = 3.5 x 10 - 6 M. solved in isotris buffer and the pH of the solutions was adjusted to 7.4 by adding NaOH or HC1. The results are shown in Table 2. DISCUSSION
When adriamycin and its structural analogues, daunomycin, daunomycin benzoylhydrazone. Nacetyldaunomycin and adriamycinone are dissolved in dioxane solution, the fluorescence intensity of the system increases. Similar increases were also observed when daunomycin was dissolved in alcohol solutions (Bachur et al., 1970). These increases in solvents of low dielectric constant suggest that the small increases in fluorescence intensity when the ligands bind nonspecifically to immunoglobulinsprepared from normal goat and rabbit sera are due to factors related to the low polarity of the environment. Similar increases in fluorescence intensity were observed when bovine serum albumin which has been shown to have hydrophobic binding sites for organic dye molecules was added to the system. The excitation (467nm) and emission (555nm) maximum of these anthracyclic molecules are quite different from the excitation (280-295 rim) and emission (305-350 nm) spectra of proteins. Therefore, it is unlikely that the reduction of fluorescence of these molecules when they bind to specific antibody combining sites could be caused by energy transfer between the ligands and the protein molecules. Therefore, fluorescence quenching of the ligands could be used to determine the binding strength of the antibodies without the use of purified antibodies. Scatchard plots show that these antibodies are heterogeneous. Nevertheless, the average intrinsic association constants can give good approximations of their binding strength. Adriamycin, daunomycin, two derivatives of daunomycin, N-acetyldaunomycln and daunomycin benzoylhydrazone and adriamyclnone all have the similar binding strengths with antibodies directed toward adriamycin. This finding is consistent with the inhibition studies by Van Vunakis et al. (1974), who found that the anti-adriamycin antibodies recognized the anthracycline structure. Both by radioimmunoassay and by fluorescence quenching the relatively bulky benzoylhydrazone on the C-13 keto group has little effect on the binding strength of the I\l~l 12 4
(
system and indicates that the area of the agylcone complementary to the antibody receptor site is distant from the C-13 and C-14 group. Anthracyclic chromophores have two phenolic hydroxy groups at positions 5 and 12 and two keto groups at positions 6 and l l. Studies have shown that a phenolic hydroxy group is more aci&c in the excited state than in the ground state, and that the ionized species is nonfluorescent (Van Durren, 1963). According to Bachur et al. (1970), when daunomycin was dissolved in alcohol or 0'3 M HC1 solutions, the fluorescence yield increases and the longer wavelength peak appears. At alkaline pH values, the anthracyclic compounds no longer fluoresce. This decrease of fluorescence in alkaline solution follows closely the titration curve of the dye. The increase in fluorescence of adriamycin and its structural analogues in dioxane which not only is of lower dielectric constant but also is less of a proton acceptor than water, may be explained by the fact that the dioxane molecule has lower affinity for the phenolic proton: in addition, the less polar environment may stabilize the non-ionized phenol group more. Thus, the non-ionized fluorescent form would be favored. The effects of different amino acids on the fluorescence intensity suggest that these interactions are specific since they are observable even at 1 × 10-3 M. Acidic, basic or neutral amino acids all increase the fluorescence intensity of the system. At low concentrations of phenylalanine and tryptophan, the fluorescence intensity also increases. However, tyramine, tryptamine, indole-acetic acid, hydroxyphenylacetic acid, phenylacetlc acid and phenylethylamine fail to show similar effects, suggesting that the zwitter ion contributed by both the amino and carboxy groups on the amino acid backbone is required. However, due to the complexity of the chromophore, further studies are necessary in order to elucidate the mechanism of this fluorescence enhancement. The decrease of the chromophore fluorescence by the aromatic compounds may result from the formation of nonfluorescent charge transfer complexes or 7~complexes (Van Durren, 1963) between the aromatic residues and the anthracycline ring. The formation of these complexes would result from the interaction between the r~-electrons of the two molecules. This interaction would be more likely to occur in the excited
296
YUEH-HSIU CHIEN and LAWRENCE LEVINE
than the ground state. The mdole group is the most etlic~ent quencher followed by the hydroxyl phenyl group: the phenyl group has the least effect. Hydroxyphenylacetic acid is a more efficient quencher than tyramine, phenylacetlc acid is a better quencher than phenylethylamlne. These differences m quenching suggest that in addition to the re-complex formation, the carboxy group may interact with the phenohc group and cause additional quenching. The degrees of quenching by lndoleacetlc acid and tryptamine are the same suggesting that the formation of nonfluorescent 7r-complexes is primarily' responsible for the observed effects with the indole compounds. This speculation is consistent with the finding that acetate quenches the flt, orescence intensity of adriamycln and adriamycmone systems. The effects of phen)lalanme and tr)ptophan could be a combination of the fluorescence enhancement caused by' their amino acid backbones and the fluorescence quenching caused by' their aromatic side chains. These combined effects can be observed more clearly m adriamycmone solutions because the adriamycinone molecule does not have the amino sugar moiety: therefore, the chromophore is fully exposed to the environment. At low amino amd concentrations, the fluorescence enhancement process dominates. As the amino acid concentration increases the re-complex quench,ng effect becomes important. Since the phenyl group is a weak ~z-complex former, the fluorescence intensity of adriamycinone is higher than its reference point e~en
at the 2 × 10-2 M level. However, phenylalanlne does show a concentratmn dependent quenching effect. We are thus suggesting that aromatm amino acid and/or carboxy groups are present at the antlbod), combining site REFERENCES
Arcamone F., Franceschl G., Orezzl P, Casslnelh G, Barbleri W and Mondelh R (19641 J Am chem. So~. 86, 5334 Bachur N R., Moore A L., Bernstem J. G and Llu A 11970} Can~er Chemother. Rep., Part I 54, 89 DiMarco A., Gaetani M. and Dongott~ t i (1963} Turnoff 49, 203. E1sen H. N. 11964p Method.~ m Mc&cal Rewarch (E&ted b) Elsen H N.}, Vol. X, p. 115 Chicago Year Book MedJcal, Chmago Lopatin D. E., and Voss E. W (1971) Blochemtstrv 10, 208 Lowry O H, Rosebrough W F, Farr A L and Randall R V (1951}J hlol Chem 193,265 Parker C. W, Yoo J. J., Johnson M C. and Godt S. M {1967) Biochemistry 6, 3408. Parker C W and Osterland (' K (19701 B~ochem~str) 9, I 0~4
Scatchard G. 11949} Ann ~.I ~' Acad. Set. 51,661). Van Duuren B. L (1963) Chem Rev. 63, 325 Van Vunakls H, Langone J. J., Rlceberg L. J and Levme L. (1974) Cancer Res. 34, 2546. Vehck S. F., Parker C. W. and Etsen H N ~19601Proc ham. Acad. Sol. U.S A. 46, 1470. Weber G. 11952) Btochem. J. 51, 155
