The precipitation reaction of bovine serum albumin coupled with p-azophenylleucine with homologous antibody was inhibited by several structurally related haptens. The isobutyl group substituent on alpha-carbon atom of the leucine residue contributed more than -5.8 Kcal/mol to the free energy of binding. This value was consistent with the free energy change expected from the transfer of n-butane from an aqueous environment to liquid n-butane. The observed contribution was explained, in terms of the hydrophobic interaction of the isobutyl group with the antigen binding site of the antibody molecule. These results were also compared with other hapten-antibody systems.</p

Hatase, Osamu

Hayashi, Hideo

Koide, Noriko

Oda, Takuzo

Tomoda, Jun

Tsutsui, Ken

English

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 31, Issue 5 1977 Article 2OCTOBER 1977Role of hydrophobic interaction inhapten-antibody bindingKen Tsutsui∗ Noriko Koide† Jun Tomoda‡Hideo Hayashi∗∗ Osamu Hatase†† Takuzo Oda‡‡∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,††Okayama University,‡‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Role of hydrophobic interaction inhapten-antibody binding∗Ken Tsutsui, Noriko Koide, Jun Tomoda, Hideo Hayashi, Osamu Hatase, andTakuzo OdaAbstractThe precipitation reaction of bovine serum albumin coupled with p-azophenylleucine withhomologous antibody was inhibited by several structurally related haptens. The isobutyl groupsubstituent on alpha-carbon atom of the leucine residue contributed more than -5.8 Kcal/mol to thefree energy of binding. This value was consistent with the free energy change expected from thetransfer of n-butane from an aqueous environment to liquid n-butane. The observed contributionwas explained, in terms of the hydrophobic interaction of the isobutyl group with the antigen bind-ing site of the antibody molecule. These results were also compared with other hapten-antibodysystems.∗PMID: 146401 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 31,289-294 (1977)ROLE OF HYDROPHOBIC INTERACTION INHAPTEN-ANTIBODY BINDINGKen TSUTSUI, Norio KOIDEl, Jun TOMODA1, Hideo HAYASHI2Osamu HATASE3 and Takuzo ODADepartment of Biochemistry, Cancer Institute, Okayama UniverJityMedical School, Okayarna 700, JapanReceived September 10, 1977Abstract. The precipitation reaction of bovine serum albumincoupled with p-azophenylleucine with homologous antibody was in-hibited by several structurally related haptens. The isobutyl groupsubstituent on a-carbon atom of the leucine residue contributed morethan -5.8 Kcaljmol to the free energy of binding. This value wasconsistent with the free energy change expected from the transfer ofn-butane from an aqueous environment to liquid n-butane. The ob-served contribution was explained, in terms of the hydrophobic inter-action of the isobutyl group with the antigen binding site of the anti-body molecule. These results were also compared with other hapten-antibody systems.The binding specificity of hapten with antihapten antibody has been exam-ined by many investigators. These investigators have related the haptenic struc-ture to the thermodynamic parameters of hapten-antibody interaction obtainedfrom the precipitation inhibition test (1, 2) and equilibrium dialysis (3), whichhave been the two major procedures used in these investigations.In this paper we report on some results of a precipitation inhibition study,and discuss the individual contributions of some chemical groups in hapten mole-cules to the hapten-antibody interaction. The haptenic group selected for thestudy was p-azobenzoylleucine, and the precipitation reaction with homologousantibody was inhibited by several structurally related haptens. The inhibitionexperiments indicate that the isobutyl group, substituent on the a-carbon atom ofthe leucine residue, contributed more than - 5.8 Kcal/mol to the free energy ofbinding. The results provide evidence that hydrophobic interaction plays animportant role in the binding of hapten and antihapten antibody.Present address:1 Department of Internal Medicine, Okayama University Medical School2 Department of Bacteriology, Okayama University Medical School3 Department of Physiology, Okayama University Medical School2891Tsutsui et al.: Role of hydrophobic interaction in hapten-antibody bindingProduced by The Berkeley Electronic Press, 1977290 K. TSUTSUI et al.MATERIALS AND METHODSPreparation of antigen. D, L-Leucine was reacted with p-nitrobenzoylchlo-ride and then, the nitro group was reduced by the method of Landsteiner andVan der Scheer (4). The purity of p-nitrobenzoylleucine (PNBL) and p-amino-benzoylleucine (PABL) was confirmed by measuring the mel ting point: PNBL,224.5-225.5°C and PABL, lBO-182°C.PABL (0.12 mmoles) was diazotized by equimolar sodium nitrite at a-5°C for60 min. The excessive sodium nitrite was removed with ammonium sulfamateuntil the solution fail to produce a blue response on starch-iodide paper. Di-azotized PABL was coupled to 0.2 g of bovine serum albumin (BSA) in 20 ml of0.01 M borate buffer, pH 9.0, by adding diazotized PABL in small portionsover a period of 2 hours, maintaining the pH at 9.0 by addition of 1 N NaOH.The solution was then kept at a-4°C overnight. After adjusting the pH to 7.0with HCl, the solution was dialyzed at a-4°C against 1,000 ml of 0.1 M NaClchanged daily with new solution for seven days.Preparation of antiserum. The dialyzed solution of antigen (BSA-conjugatedp-azobenzoylleucine; abbreviated BSAL) which contained 5 mg of protein perml solution was homogenized with an equal volume of Freund complete adju-vant. A total dose of 2 ml of this mixture was injected in divided dosages sub-cutaneously to the palmar, plantar, and abdominal skin of a young rabbit.Two weeks later, the same procedure was repeated. At the end of the sixthweek after the first injection, about 30 ml of antiserum was prepared from therabbit.Antibodies produced against antigenic determinants other than the intro-duced haptenic group were precipitated by addition of BSA at the optimumamount which produced maximum precipitation with the antiserum. The opti-mum amount was determined as described in the section on precipitation reac-tion. This reaction was completed in 60 min at 3]OC. The precipitate, whichcontained complement as well as antigen-antibody complex, was then removedby centrifugation at l5,000xg for 20 min. The supernatant was used as thespecific antiserum to the homologous hapten.The specificity of this antiserum was confirmed by the double diffusion tech-nique of Ouchterlony: the precipitine line was observed between BSAL butnot between BSA and antiserum.Assay of precipitation reaction. The antigen solution was serially dilutedwith 0.15 M saline solution buffered with 0.008 M phosphate (PBS), pH 7.0.After addition of a given amount of antiserum to the antigen solution, themixture was incubated for 60 min at 37"C, then allowed to stand for two daysat 4 Cl C. The precipitates were centrifuged, washed once with 1.0 ml of PBS andthe protein amount in the precipitate was measured by the method of Lowry etat. (5).Inhibition of precipitation by haptens. The amount of BSAL required foroptimum precipitation was determined, and the precipitation reaction at thisantigen-antibody ratio was inhibited with various haptens by the same pro-cedure described above. The reaction mixture contained: hapten 1.0 ml (PBS2Acta Medica Okayama, Vol. 31 [1977], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss5/2Role of Hydrophobic Group in Haptet;: 291in place of hapten for control), BSAL 1.0 ml (48,ug, the optimum amount) andantiserum 0.5ml (20 times diluted). The addition was in this sequence. Abbre-viations and formulae for the haptens used in this experiment are summarizedin Table 1. The nonspecific binding of haptens to serum protein is almostnegligible because of the low concentration of antiserum (6).TABLE 1. INHIBITION HAPTENS USED IN THE PRECIPITATION REACTIONBETWEEN BSAL AND HOMOLOGOUS ANTIBODYHapten Abbreviation Formu1abp-Aminobenzoylleucinep-Aminohippuric acidp-Nitrobenzoylleucinep-NitrobenzamideN-Acety1-L-1eucineD, L-Leucinep-Aminobenzoic acidPABLaPAHaPNBLaPNBANALaLeuPABAaHzN-C6H 4-CONH-CHR-COOHHzN-C6H 4-CONH-CHz-COOHOzN-C6H4-CONH-CHR-COOHOzN-C6H 4-CONHzH3C-CONH-CHR-COOHHzN-CHR-COOHHzN-C6H 4-COOHa Dissolved in 1 N NaOH, pH adjusted to 7.0 with Hel, diluted to desired concentrationswith PBS and stocked at 4°C.b -R = -CHzCH(CH3)ZRESULTS AND DISCUSSIONThe effect of haptens on the precipitation reaction of BSAL with homolo-gous antibody was tested (Fig. 1). The results were interpreted by applying thetheory of heterogeneous antibody binding sites described by Pauling et at. (1).They assumed a Gaussian distribution for the free energy of binding between anoell...-~0- 1·0u~0-0...-C::J 0·50Et1l"itic:.Q...-ut1l...0u.-7 -6 -5 -4 -3 -2 -1 0Amount of hapten (4n X10,umoles)Fig. 1. Precipitation inhibition by PABL and other structurally related haptens.The points are from experimental data and the curves are from theoretical calcula-tions (1). 0, PABL; 0, PNBL; e, NAL; 6, PAH; .., Leu; _, PNBA.3Tsutsui et al.: Role of hydrophobic interaction in hapten-antibody bindingProduced by The Berkeley Electronic Press, 1977292 K. TSUTSUI et af.haptenic group and heterogenous antibody. If the precipitation reaction betweenmultivalent hapten and antibody was interfered by a monovalent inhibitionhapten, two parameters (Ko' and a) can be calculated from the equation derivedby Pauling et at.. Ko'is the average inhibition constant of a hapten, expressed asa relative value to that for the homologous hapten (usually, Ko' for the homolo-gous hapten is taken as unity). Ko/ can be considered as a relative binding con-stant of the hapten to antibody. The heterogeneity index, a, is the standarddeviation in the error function, and is zero for a homogeneous group of bindingsites and increases with antibody heterogeneity. The experiment with the ho-mologous hapten, PABL, was carried out at nine different concentrations todetermine the degree of agreement between the experimental data and the theo-retical curve. As evident from the figure, the experimental points are in fairagreement with the theoretical curve. On other haptens, however, only a fewpoints were tested.The values of Ko' and (J for haptens employed were calculated and areshown in Table 2. Since values smaller than 10-4 for Ko' and greater than 7.0TABLE 2. CALCULATION OF THE AVERAGE HAPTEN INHIBITION CONSTANT (Ko'l,HETEROGENEITY INDEX (a) AND RELATIVE FREE ENERGY OF BINDING (b.G,e1)Hapten K o ' a b.Grel (Kcal/mol)----- ---_.__ ...._-----PABL 1.0 3.0 aPAH <10-4 8.0 >5.8PNBL 0.63 3.0 0.29PNBA <10-4 8.0 >5.8NAL 1. 5 x 10-3 7.0 4.1Leu <10-4 8.0 >5.8For abbreviations of haptens, see Fig. 1 legend.Values for K o ' and IJ were calculated from the theoretical equation of Pauling et af. (1).Values for b.Grel were expressed at 37°C.for a are not reliable, the apparent difference among these results for PAH,PNBA and Leu (Fig. I) has no significance. The relative free energy change ofbinding of a hapten to antibody (::SGrel) is also shown in Table 2. ,dGrel wascalculated from the following equation,LiGrel= -RT In K o ',where R is the gas constant and T is the absolute temperature. The difference inLlGrel between different haptens represents the differential free energy of binding.Comparisons of structurally related haptens differing in only one substitutiongroup, therefore, permit the estimation of the differential contribution of thegroups.4Acta Medica Okayama, Vol. 31 [1977], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss5/2Role of Hydrophobic Group in Hapten 293The contribution of the isobutyl group to the free energy of hapten bindingwith antihapten antibody is larger than 5.8 Kca1;mol (Table 2). The signifi-cance of nonpolar groups in the association of hapten with antibody has beenpointed out-by Nisonoff et ai. (2) and Karush (3). The contribution of a-phenylgroup in the hapten, phenyl-(benzoylamino)-acetic acid, was estimated to be 4.1Kcaljmol by Karush (3). Karush suggested that this value agrees closely withthat of the change in free energy for the transfer of 1mol of benzene from anaqueous environment to liquid benzene (- 4.1 Kcaljmol, see 7). He inferredfrom this fact that the phenyl group induces a quasi-crystalline structure of watermolecules in its immediate vicinity when the hapten is free but is in inter-molecular contact with the nonpolar side chains of the protein when the hapten isbound with the antibody. The same argument is valid in our case. The changein free energy for the transfer of 1mol of n-butane from an aqueous environmentto liquid n-butane is - 5.8 Kcaljmol (8). This value may be a little less negativefor isobutane because of the branching effect of the carbon chain. Therefore, acontribution larger than 5.8 Kcaljmol of isobutyl group strongly suggests thatthe nonpolarity of the isobutyl group is the main factor for the contribution ob-served, as the phenyl group is in the phenyl-(benzoylamino)-acetic acid system.It must be mentioned, however, that direct comparison of these two systems maynot be very proper because of the difference in the precipitation inhibition testand the equilibrium dialysis method.If the London dispersion force between the isobutyl group and its nearestneighbors in the antibody binding site is taken into account, the large contribu-tion of the isobutyl group is fully explained. To evaluate the dispersion force,however, it would be necessary to measure the temperature dependence of thecontribution, by the equilibrium dialysis technique.There is another possibility that the large LGrel observed for PAH is due tothe differential conformation around the peptide bond in PAH and PABL. IRspectra of PAH and PNBL eliminated this possibility since strong absorptionbands were observed around 1520-1530 cm- 1 (amide II) in both spectra, indi-cating transorm is the preferential conformation in both haptens.It is interesting that in some hapten-antibody systems, some nonpolar groupsseem to confer only limited contribution. In the p-azophenyltrimethylam-monium ion system (6, 9), p-(tert-butyl)-azophenyl group (steric analogue ofp-azophenyltrimethylammonium ion) contributes only 1.5 Kcaljmol more thanthe p-azophenyl group.There is only a small difference (0.29 Kcaljmol) between the contributionof the amino and the nitro group of PABL and PNBL molecules, respectively(Table 2). The position of these substituents corresponds to the diazo group inthe immunization antigen (BSAL). The contribution order of these groups is5Tsutsui et al.: Role of hydrophobic interaction in hapten-antibody bindingProduced by The Berkeley Electronic Press, 1977294 K. TSUTSUI et al.reversed in the p-azophenyl arsonate system (6, 10).Acknowledgment. We wish to thank Dr. T. Inada for measuring IR spectra of haptens.We also would like to acknowledge the continuing interest and encouragement of Prof.]. Tawara of the Department of Virology.REFERENCES1. Pauling, L., Pressman, D. and Grossberg, A. L.: The serological properties of simplesubstances. VIII. A quantitative theory of the inhibition by haptens of the precipitationof heterogeneous antisera with antigens, and comparison with experimental results forpolyhaptenic simple substances and for azoproteins. j. Am. Chem. Soc. 66, 784-792, 1944.2. Nisonoff, A., Shaw, A. R. and Pressman, D.: The nitro group as a determinant of im-munologic specificity. j. Am. Chem. Soc. 81,1418-1423, 1959.3. Karush, F.: The interaction of purified antibody with optically isomeric haptens. j.Am. Chem. Soc. 78, 5519-5526, 1956.4. Landsteiner, K. and Van der Scheer, J.: On the serological specificity of peptides. j.Exp. Med. 55, 781-796, 1932.5. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R.].: Protein measurementwith the Folin phenol reagent. j. Biol. Chem. 193, 265--270, 1951.6. Pressman, D. and Siegel, M.: The binding of simple substances to serum proteins andits effect on apparent antibody-hapten combination constants. j. Am. Chem. Soc. 75, 686-693, 1953.7. Karush, F.: Immunologic specificity and molecular structure. In Advances in Immunology.vol. 2, ed. W. H. Taliaferro and J. H. Humphrey, Academic Press, New York, p. 28,1962.8. Karush, F.: Immunologic specificity and molecular structure. In Advances in Immunology,vol. 2, ed. W. H. Taliaferro and ]. H. Humphrey, Academic Press, New York, p. 12,1962.9. Pressman, D., Grossberg, A. L., Pence, L. H. and Pauling, L.: The reactions of anti-serum homologous to the p-azophenyltrimethyl ammonium group. j. Am. Chem. Soc. 68,250-255, 1946.10. Pauling, L. and Pressman, D.: The serological properties of simple substances. IX.Hapten inhibition of precipitation of antisera homologous to the 0-, m-, and p-azophenyl-arsonic acid groups. j. Am. Chem. Soc. 67, 1003-1012, 1945.6Acta Medica Okayama, Vol. 31 [1977], Iss. 5, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss5/2

Immunologic specificity and molecular structure.

On the serological specificity of peptides.

Protein measurement with the Folin phenol reagent.

The binding of simple substances to serum proteins and its effect on apparent antibody-hapten combination constants.

The interaction of purified antibody with optically isomeric haptens.

The nitro group as a determinant of immunologic specificity.

The reactions of antiserum homologous to the p-azophenyltrimethyl ammonium group.

The serological properties of simple substances. IX. Hapten inhibition of precipitation of antisera homologous to the 0-, m-, and p-azophenylarsonic acid groups.

Role of hydrophobic interaction in hapten-antibody binding

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Role of hydrophobic interaction in hapten-antibody binding

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