Fluorescent enhancement in antibody-hapten interaction 1-anilinonaphthalene-8-sulfonate as a fluorescent molecular probe for anti-azonaphthalene sulfonate antibody

Immunochemist(y. Pergamon Press 1968. Vol. 5, pp. 143-153.

Printed in Great Britain

FLUORESCENT ENHANCEMENT IN ANTIBODY-HAPTEN INTERACTION 1-ANILINONAPHTHALENE-8-SULFONATE AS A FLUORESCENT MOLECULAR PROBE FOR ANTI-AZONAPHTHALENE SULFONATE ANTIBODY* t TAI-JUNE YOO~ AND CmCRLES W. PAmm~t§ Division of Immunology, Department of Internal Medicine Washington University School of Medicine, St. Louis, Missouri Received 14 September 1967; in revisedform 6 November 1967) Almtrsct--The fluorescence of 1-anilino-8-naphthalene sulfonate is profoundly influenced by its molecular environment. Rabbit antibodies capable of binding ANS have been prepared to 1-azo-8-naphthalenesulfonate and its 1-4 isomer. The quantum yield of ANS fluorescence is increased more than 100 fold in the presence of specific antibody and there is a blue s h ~ of 45 rn/~. The fluorescence of ANS is largely unaffected by normal rabbit y-globulin. There is a close correlation between the results of quantitative precipitin analysis and fluorometric titration. While changes in ANS fluorescence in various solvents have generally been attributed to differences in solvent polarity we have observed that ANS fluorescence is increased in DIO as compared with H,O suggesting that the mechanism may be more complex. This study indicates that ANS as a hydrophobic molecular probe at specific antibody site should be very useful in determination of antibody concentration, affinity and binding heterogeneity and in the study of specific antihapten antibody structure.

INTRODUCTION IT HAS been known that certain classes of organic molecules exhibit little fluorescence in an aqueous solution but become highly fluorescent upon binding with certain proteins. Several anilinoacridine and anilinonaphthalene exhibit marked enhancement of fluorescence upon binding to serum albumin [1]. l-Anilino-8-naphthalene sulfonate (1-8 ANS) was used by Weber and Young [2] to study the acid expansion of bovine serum albumin and by Stryer [3] to probe the hydrophobic haem binding regions of apomyoglobin and apohemoglobin. Gaily and Edelman [4] found that the thermal transition of Bence--Jones protein was accompanied by changes of ANS fluorescence. McClure and Edelman [5, 6] have made extensive studies of protein conformation using the fluorescent properties of 2-6 TNS (2-toludinonaphthalene-6-sulfonate). Few attempts have been made to use fluorescent enhancement in the study of h a p t e n - a n t i b o d y interactions. Berns and Singer [7] observed that antibodies to 4azobenzenearsonate produced an increase in the fluorescence of an aminobenzenearsonate substituted acridine. However, the extent of increase in the fluorescence was less than 5 per cent of the changes observed with bovine serum albumin. In a brief * Part of the work was presented at the Tenth Annual Meeting of the Biophysical Society (February 23-25, 1966), Biophysical J. VI: Al~t. 78 (1966). t Supported by N I H (U.S.), Grant AI-219 and AI-9881. **Present address: Department of Biochemistry Research, Roswell Park Memorial Instltute, Buffalo, New York. Reprint should be asked at above address. § Recipient of a research career development award. 143

144

'FAX-JuNF.Yoo and CHARI.ESW. PARKER

communication in 1962, Winkler reported that antibodies to 1-azo-4-naphthalene sulfonate considerably enhanced the lluorescence of 1-5 TNS [8]. \Ve have confirmed and extended the observations of Winkler [8] in regard to fluorescence enhancement by using well characterized hydrophobie probe, 1-8 ANS, with antibody against 1-azo-8-naphthalene sulfonate and its 1-4 isomer. We have observed that antibody binds 1-anilino-8-naphthalene sulfonate in a highly specific manner. Here we report our study on the preparation and characterization of specific ~,-globulin fraction, affinity and stoichiometry of binding and fluorescence characteristic of bound hapten. MATERIALS AND METHODS

Preparation of immunogen. Diazotization of 1-amino-8-naphthalene sulfonate (1-8 ANS) and its 1-4 isomer. One hundred mg of 1-8 ANS (Eastman Kodak Chemicals) was dissolved in water and added to 3 equivalents of HCI 1 N, 1 •35 ml, cooled in ice after which 31 mg of NaNO2 was added. The mixture was vigorously stirred and kept in an ice bucket until addition to the protein solution. Bovine ~,-globulin (B~,G) was used as the immunizing antigen and human serum albumin (HSA) was used to demonstrate precipitating antibody in the antisera. Fifty mg of B~,G and 50 mg of HSA were dissolved in ice cold 0.1 M borate buffer at p H 8 as a 1% solution. The diazonium salt solution was added dropwise with continuous stirring. The pH was maintained at 8-9 with 1 N N a O H during the coupling reaction. The pH was readjusted to 8.0 30 min alter all the diazonium salt had been added. The solution was stirred overnight and dialyzed against pH 7, 0-01 .~f P O , buffer with 0.1 ,~I NaC1 for 3 days with several changes of dialyzing solution, then finally dialyzed against 0 "01 .'~I phosphate buffer (0.008 M dipotassium phosphate and 0-002 M monosodiuin phosphate) with 0.015 3.1 NaCI at pH 7-4. Immunization preparation of 1G-immunoglobulin. Three mg of azo-B~G was injected with complete Freund's adjuvant in the footpads of random bred New Zealand white rabbits. Antiserum was collected 4-6 weeks alier the first immunization and the rabbits were then given intermittent booster injections and hled frequently. The sera were demonstrated to contain precipitating antibody by interfacial precipitin analysis with azo-HSA. ~,-Globulin fractions were prepared by (NH4).SO, precipitation at 50 per cent saturation. The ammonium sulfate ti~actions were subjected to DEAE cellulose chromatography (1 g of dry DEAE t?)r 1 ml serum). The protein and resin were equilibrated with 0.02 ~'ll phosphate butter at pH 7. The first protein peak was collected and concentrated by precipitation with (NH4) ~SO~ at 50 per cent of saturation and dialyzed against 0-01 ?~I PO4 buffer at p H 7.4. Testing hapten. The magnesium salt of l-anilino-8-naphthalene sulfonic acid (Eastman Kodak Chemicals) was recrystallized several times from water after filtration of a hot solution through Norit A and talc. The light green crystals of magnesium salt were dried at 120-130 ° for several hr. The molar absorption coefficient was 4.95 4~ 0" 1 × 10 -3 M -a at 350 mt~ [2]. The product gave a single spot on thin layer chromatography using n-propanol aqueous ammonia (2 : 1) as solvent.

145

Molecular Probe at Antibody Site

D , O was provided by Dr. S. I. Weissman, Washington University, St. Louis, Missouri and Dr. H. Box, Roswell Park Memorial Institute, Buffalo, New York. T h e latter preparation was from International Chemical and Nuclear Company, City of Industry, California. Its D t O content was 99.86 per cent and it contained tritium to the extent not more than 0.002 #c/ml.

Ultrac~arifugation. Ultracentrifugal analyses were made with a Spinco Model E Analytical ultracentrifuge operating 59780 rev/min with rotor temperature regulated at 20°C. Sedimentation coefficients were calculated by standard methods. Immunodectrophoresis. This was done following the micromethod of Scheiddigger

[9]. In the trough, anti-rabbit y-G was used (antisera prepared by Dr. Y. Yagi at Roswell Park Memorial Institute, Buffalo, New York).

Quantitative precipitia analysis. Specific precipitation was performed by mixing a constant volume of y-globulin with a variable amount of 1-8 azo-NS-HSA (1-8 azonaphthalene sulfonate coupled to h - m a n serum albumin). After incubation (60 min at 37°C followed by 2 days at 4°C) precipitates were collected and washed twice at 4°C with 0.15 M NaC1. Washed precipitates were alr-dried, dissolved in 0.25 M acetic acid and absorbancies were determined at 280 n ~ . T h e absorbancies at 280 m# were corrected for the antigen contribution as shown in Table 1. TAsLs 1.

SPECIFIC PP~CIPITATION OF ~-OLOBULIN FKACTION FROM AN rJ,msR.UM

Tube No.

Antigen* added (~g)

l 2

135 182

0.397 0.340

Absorbance t (280 m~,) (400 mp)

Alamrbance+* antibody precipitated Precipitate

O" 105 0.098

0.202 O. 150

0.211

3

16

0.303

0-043

4

60

0.454

0.063

0.327

5 6 7

lO0 0If lOO~

0.543 o.o13 o.o17

O. lOl 0.005 0.oo4

0.349§

0.367 0.310 0.273

0.424 0.513

* Ratio of absorbance 280 rap/400 m~ = 1"8 of antigen stock solution was used for the calculation of the amount of antibody; (O.D.)je0 Antibody = (O.D.)n0.a precipitate -- 1' 8 (O.D.)40,.a precipitate. t Corrected only for cuvette blank. + Corrected for control. For the control normal rabbit F-globulin was tuft. § Tube 5 has O.D. --0.349 (0.241 mg) therefore 0.4mg/ml of antibody. Fluorometric titration showed 0.43 mg/ml for this sample. [I Antibody control tube. No antibody was added to this. All other tubes had 0.6ml of y globulin fraction. ¶ Antigen control tube. No antigen was added.

146

TAI-JUNE Yoo and CHARLESW. PARKER

Equilibrium dialysis. Equilibrium dialysis experiments were carried out at 25 ° essentially by the method of Eisen [ 10]. Control experiments showed that equilibrium was attained after 24 hr. The dialysis tubing was previously washed by heating for several hours in 0"5 M acetic acid followed by soaking in distilled water [11]. One ml of ~,G-immunoglobulin was placed inside the bag and haptenic solution was placed outside. The tubes were rotated at 1 rev/min for 24 hr. The concentration of the free ANS in the outside solution was assayed by fluorescence measurement in the presence of bovine serum albumin. Fluorescence measurement. Fluorescence emission, excitation and polarization were performed on a commercial instrument (Aminco-Bowman) employing 2 monochromator at right angles to the samples. The light source was replaced by a stable d.c. operated high Xenon arc and a water jacket was built around the sample holder for temperature control. One ml ofyG-immunoglobin was transferred in 1 cm square quartz cuvette and 5 × 10 -e M hapten (ANS) was added successively until 0-2 ml had been added, then higher concentrations of ANS were added to cover all phases of binding saturation. Polarization was measured by the procedure of Chen and Bowman [12]. An automatic X-Y Recorder (Mosley) was used for the emission and excitation spectrum. The spectrum reported here (unless specified) is the direct recorder tracing which have not been corrected for the variation with wave length in the sensitivity of the detecting system. The quantum yield was calculated by following the procedure of C h e n [ 13] using 1-8 ANS as 0.004 in aqueous solution [2]. Determination of binding constants. The equilibrium constant of association K0 was calculated from fluorometrie data and equilibrium dialysis according to the logarithmic form of Sips equation [14-16]: log

n--y

--alog c +a

log K0

where ), is moles of hapten bound per moles of antibody at free ligand concentration c; n is the maximum number of moles of ligand that can be bound per mole of antibody; and a is the heterogeneity index which describes the dispersion of association constants about the average intrinsic association constant K0.

RESULTS

Immunoelectrophoresis of chromatographically isolated anti-azonaphthalene sulfonate antibodies. Antibodies against azonaphthalene sulfonate in rabbits were isolated by chromatography on DEAE cellulose as described. The preparation was tested by immunoelectrophoresis for the presence of contaminating proteins other than IgG using anti-rabbit ~,-globulin prepared in sheep, goat and horse. The results are shown in Fig. 1. By this criterion, the antibody sample contained pure IgG.

Ultracentrifugation. The picture showed one main peak $20.,~ ~ 6.8 with symmetry (Fig. 2).

"!,

! , : \ mixtm<" ol ,Voat and-rabbit y-G (lgA rich) and shecp and-rabbit y-(i J: '.1 ~i~h, xxc~<"ill the ll<>ugh in the left column. These antisera were absorbed i,, t~d>l~it l e ( ; and used in the tlough in the right colunan. Testing sample (A) ::,>, ~t:~l rabbit serum ~13~ l g M rich rabbit y-G (C) IgA rich rabbit y-(; (D). It is noted that testing sample A has pure IgG.

FIG. 2. Photograph taken with Schilieren optics during ultracentrlfugation of

Ig(;. seven mg/ml borate buffer p H 8. Rotor speed 59780 rev/min. Exposures were taken at 28 rain (left) and 68 min (right). Calculated S20,w = 6.8.

£page 146

Molecular Probe at Antibody Site

147

Binding of ANS to antibody. A striking enhancement of fluorescence accompanies the binding of 1-8 ANS to antibody. The quantum yield increases over 100 fold and emission shifts from green to blue. ANS in aqueous solution has a quantum yield o f 0.004 and an emission maximum at 515 mt~. When bound to antibody, the quantum yield is 0-5 and the emission maximum is at 470 m~ (Fig. 3). Figure 4 shows the increment of the fluorescence when hapten is added to protein. As in the case of the Fluorescence (arbitrary)

Absorption

300

350

400

450

500

550

600

m/~ FIo. 3. Fluorescence emission spectrum and absorption spectrum of 1-anilino8-naphthalene sulfonate b o u n d to anti-l-azo-4-naphthalene sulfonate antibody in 0.01 M phosphate buffer. 0" 15 M N a C 1 (pH 7.4). The emission spectrum of 1-8 ANS in buffer alone coincides with the base line.

c

Hopten

4000

~.ooo

20C

"-~

~00

/

coc, ,E_ 3

! I i

l

J ~ I

0 020 04,: 960.0~ 0-1 012 014 5 ~{0"~ M

4L

J

0.02

l

0.08 5X{O~ M

l-k~pfe'~added, ml

Fla. 4. A typical titration of I nag of IgG containing antibody at 25°C in 1.0 ml of 0.01 M phosphate buffer 0" 15 M NaCI with 1-8 ANS. The intersection of extrapolations of the first and third portions of the curve in a measure of the n u m b e r of antibody active site. Fluorescence is expressed in arbitrary units.

TAI-JuNEYoo and CnARt.ESW. PARKER

148

fluorescence quenching technique [17], the intersection of the extrapolation of the initial linear portion with that of the terminal linear portion is a measure of the total n u m b e r of antibody active sites. T h e value of antibody concentration Qbtained by the fluorometric titration is in close agreement with that obtained by precipitin analysis (precipitating antibody) (Table 1). However, the precipitation analysis is about 10-15 per cent less than the fluometric titration. This could be due to non-precipitating antibody present in the supernatant. The association constant is calculated from the non-linear portion of the titration curve. T h e relative degree of fluorescence increment (i.e., observed increment of fluorescent intensity compared with maximum increment) reflects the portion of antibody active sites bound by hapten (Fig. 4). Thus, at any given concentration of hapten (free and bound), if the titration value for total number of antibody sites is known, the fluorescence increment per unit hapten which could be obtained from the initial portion, may be used to calculate the concentration of antibody-hapten complex. T h e association constant is thus readily calculated. Figure 5 shows a Sips plot o f o n e of the samples for which K0 = 5.6 × 10 e M -1, a = 0.5.

30

0 X (0

20

, /,,

I

I

I

I ° L°~c ' T

Ko : 5 6 x IO6 o=05

IC--

1 I

2

Fio. 5. Fluorometric titration data were plotted according to the Sips equation. Antibody against 1-azo-4-naphthalene sulfonate group and 1-8 ANS were used for titration at 25°C pH 7-4. K0 was 5-6 × 106 and the heterogeneity index (a) was 0.5. The concentration of free hapten in insert is given in mt~moles/ml.

The fluorescence of ANS bound to antibody shows substantial polarization. Since the dependence of the polarization of the fluorescence upon the rotational relaxation time of macromolecules [18], it could afford a simple means of following enzymatic and chemical degradation of the antibody. The time course of fluorescence polarization of ANS-antibody during papain digestion [19] was measured and it showed a rapid fall of polarization of bound hapten [2] (Fig. 6). Polarization of ANS-antibody complex was studied with changes of temperature. The p value increased linearly

Molecular Probe at Antibody Site

149

it_.. ¢31 o

1

I

.L~

I

30

~?0

D~gestim time,

rain

Fio. 6. Time course of fluorescence polarization of antibody complex during papain digestion. Tile digestion mixture contained ! -0 mg rabbit y-globulin with 43pg anti-4, I-NS antibody: 0.02 m-moles PO4, pH 7.4; 0.003 m-moles NaEDTA: 5 × 10-T m-moles 1-8 ANS; 0.008 m-moles cysteine and 10t~g papain in I. I ml at 370C. Polarization was analyzed by the method of Chen and Bowman [12]. Noted rapid decrease ofp. Log (p x l0 s) was plotted against time. with decreasing temperature (Fig. 7). Similar results were observed by Haber et o2. [20] in antigen-antibody complex using nonspecific fluorescent label on a protein antigen.

Fluorescence 03",4NS in methanol wat# mbcture and D tO. Stryer [3] observed the emission characteristic of A N S dissolved in various alcohol varied markedly with the polarity of the solvent. As the polarity of the alcohol decreased, the quantum yield of A N S

15

g x o.

~4

x.. '\

;3

"¢\,. 12

Jo

I

EO

J

30 Temperature, *C

I

4O

FI~:. 7. The changes of polarization of 1-8 ANS antibody complex with temperature. "/'he rabbit ),..globulin 1 mg with 43 t,g ofantibody was mixed with 4 x 10-s m-molcs ANS. Total volume was 1"04ml. Polarization was plotted against temperature.

150

TAt-JuNE YOO and CXAt~Lm W. PARKER

o

._c

b_

4K)

430 450 470

490 510 53o 550 570 590 6~0 GSC) 650 WaveLength, mF

FIG. 8. Fluorescence emission spectra of 10-~ M 1-8 ANS in methanol-water mixtures. The per cent number is volume per cent methanol. Zero per cent coincides with base line. The higher the proportion of methanol, the greater quantum yield and the bluer is the emission.

emission was increased and the fluorescence maximum shifted to the blue. ANS was dissolved in various water-methanol mixture and the emission spectrum was obtained (Fig. 8). The higher the proportion the greater degree of fluorescence intensity was observed. ANS fluorescence in D 2 0 was compared with that in H20. It was found that the intensity of the fluorescence in D ~O was higher than in H ~O as shown in Fig. 9. DISCUSSION The most interesting result is that the 1-anilino-8-naphthalene sulfonate binds homologous or cross-reacting antibody specifically and stoichoimetricaUy with accompanying fluorescence enhancement. Normal 7-globulin does not bind ANS to a significant degree. This provides us a strong tool for the quantitative study of

>, ,n

L

,~lO

4"5

455

475 495

515

535

555

Wavelength,

575

595

I

615

I

635

rn/.L

Fzo. 9. Fluorescence emission spectra of 104 M 1-8 ANS in DsO-HsO mixtures. Y ~ is in arbitrary fluorescence units. The per cent number is volume per cent deuterium oxide. The fluorescence intensity was higher in DsO than in HsO.

Molecular Probe at Antibody Site

151

interaction of this specific hapten-antibody using the fluorometer. The fluorometer is basically simple, and the measurement can be made rapidly; furthermore, it is highly sensitive and specific. While fluorescence quenching and fluorescence enhancement involve basically similar techniques, the latter has some advantages over the former. Specifically, fluorescence enhancement can be done with y-globulin fractions whereas fluorescence quenching requires highly purified antibody [21]. Currently available procedures for obtaining specifically purified antibody inevitably involve losses of significant portions of the original antibody population, even if the purification is not difficult. It is of interest to note the sensitivity of fluorescence enhancement titrations. Fluorescence enhancement titration can be performed with as little as 10 pg per ml of antibody. Semiquantitative information in regard to antibody concentration and affinity can be obtained at even lower antibody concentrations. Furthermore, it is very useful to study antibody, its fragments or polypeptide chains with low binding constants which need large amounts of hapten for detectable binding [22, 23]. Antibody titers obtained by fluorometric titration have averaged 10-15 per cent higher than the titers obtained by the quantitative precipitin technique. This discrepancy could be primarily due to failure to measure all the antibody present by precipitin technique. The average intrinsic association constant, K0 and heterogeneity indices, a, were obtained by fluorometer and equilibrium dialysis. A good correlation has been obtained in an experimentally similar system using tritium labeled ¢5-dimethylnaphthalene-l-sulfonyl-lysine (DNS-lysine) with anti-DNS antibody---C. W. Parker et al. [24], radioiodine labeled l-iodonaphthalene-4 sulfonate with anti-l-azo-4naphthalene sulfonate antibody--T..J. Y o o et al. [25]. Additional experiments with antibodies of unusually high and low average affinities and with pools composed of many different sera will be required before it can be concluded that completely consistent correlations could be obtained. For the mechanism of increased ANS fluorescence, there are several lines of evidence which favor the change of ANS fluorescence being due to hydrophobic interactions. The hundred twenty-fold of increase of quantum yield and the large shift of the emission maximum to the blue (45 m#) can be attributed to the transfer of the dye from a polar aqueous environment to a highly non-polar active site on antibody. An effect of this kind was demonstrated on binding of 1-8 ANS to hydrophobic heme crevice of apomyoglobin [3]. In addition a high fluorescence quantum yield was found in solutions of proteins known to possess a hydrophic binding site [3, 5]. Stryer [3] interpreted the dependence of the emission maximum of 1-8 ANS on solvent polarity in terms of the dipole orientation mechanism of Lippert [26]. According to this theory, chromophores with higher dipole moments in the excited state than in the ground state will exhibit this particular dependence of fluorescence on solvent. The dipolar excited of the chromophore interacts with a polar solvent so as to orient the solvent dipoles, whereas the solvent shell in a nonpolar solvent is less perturbed. A lower-energy photon is emitted by the dye in a polar solvent, since some of the solvation energy of the excited state is lost on photon emission [3]. Dependence of the quantum yield on the solvent also can be explained by alterations in the probability of a non-radiative transition from the excited singlet state to the ground state in which solvent exhibits minimal interaction with excited molecule.

152

TAI-JuNE Yoo and CHARLESW. PARKER

Lewis and Calvin [27] proposed that intramolecular motion was involved in the nonradiative transition. Winkler [8] speculated that if above explanation was correct, then inhibition of intramolecular rotation would increase the probability of measurable fluorescence. Therefore, he postulated that increases in TNS fluorescence at the antibody site might be due to restriction to internal rotation of the bound dye. Even though the changes in the emission maximum and in quantum yield are not as incisive as they might be, these could depend on different aspects of the environment. In any event, the increased quantum yield of bound 1-8 ANS could be attributed to the mechanisms as listed above. However, another point to which we like to draw attention is the altered fluorescence of ANS in D~O. The fluorescence intensity of ANS in D , O was higher than in H~O. The changes of ANS fluorescence in D~O could be due to an effect on the N - H bond between aniline and naphthalene ring which would be replaced by an N - D bond in a D~O environment. The deuterated ANS would have a different vibrational energy resulting in different decay processes. Wright et al. [28] and Hutchison and Magnum [29] have demonstrated that the substitution of deuterium for hydrogen increases the observed life time of the triplet state of certain aromatic hydrocarbons. According to Robinson [30], the rate of a radiationless transition depends mainly on the magnitude of the product of vibrational overlap integrals between initial and final states. The deuterium effect arises because of the lower amplitute of the heavier atom vibrations, leading to smaller overlap products for the same electronic spacing. More recently Hirota and Hutchison [31] observed an effect of deuteration on the life time of the phosphorescence triplet state of naphthalene due to the decay process of an exponential triplet-singlet intramolecular transition. In their study of the fluorescent yield of rare earth ions, Kropp and Windsor [32] observed that there is a general and often dramatically large increase in the emission intensity when heavy water is used as the solvent. Also the DsO effect on the fluorescence of several organic molecules was observed by Stryer [33] where proton of the molecule could be exchanged in D t O environment as in the case of the N - H bond of ANS molecule [34]. In view of the effect of D~O on the fluorescence of ANS, we would raise the possibility that the antibody site might interact with the N H group of ANS altering the vibrational energy of the molecule. Such alterations might then contribute to the character of the fluorescence. In this event, the fluorescence spectrum of bound dye would be the resultant of several different effects--a hydrophobic environment, reduced molecular motion and local interactions at specific areas of the molecule, perhaps proton exchange reaction involving excited state of ANS. Those studies suggest that 1-anilino-8-naphthalene sulfonate are suitable probes of antibody site of this specific antihapten antibody. The hydrophobic region must be sterically accessible and of sufficient extent to bind the hapten. The polarity of the binding region can be evaluated by the emission maximum and the quantum yield of bound ligand. Many applications can be foreseen for the use of fluorescence enhancement in the study of antibody structure and antibody hapten interactions in these specific systems. I f present indications are borne out it should provide a sensitive and rapid means of evaluation of antibody concentration and affinity. It should be especially valuable in the study of antibody or its fragments or of separated polypeptide chains

Molecular Probe at Antibody Site

155

which fail to precipitate with antigen. By fluorescence polarization information regarding antibody molecular size and configuration can be obtained. One of the most attractive applications of fluorescence enhancement would be in the study of the antibody combining site in an attempt to obtain relatively small peptides with antibody activity. Atknowbdgonents--We wish to thank Drs S. I. Weissman and R. Berg (Washington University, Department of Chemistry) for their discussions on the theoretical aspect of the fluorescence and their gifts of deuterium oxide. We al~ extend our sincere thanks to Mrs P. Maier and Mr F. Maenza at Roawell Park Memorial Institute for their technical assistance in immunoelectrophoresis and ultracentrifuge study. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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