Early interactions between inhibitors and antibodies to lysozyme

Immunochemistry,1973,Vol. 10,pp. 361-364. PergamonPress. Printedi~GreatBritain

EARLY INTERACTIONS BETWEEN INHIBITORS A N D ANTIBODIES TO LYSOZYME N A Z A R 1 0 R U B I O and A N T O N I O P O R T O L E S Instituto "Jaime Ferrin" de Microbiologia, Consejo Superior de Investigaciones Cientificas, Joaquin Costa, 32-Madrid. 6-Spain (Received 23 October 1972)

AbsWaet--Two lysozyme competitive inhibitors, histamine and D-acetyl-D-glucosamine, axe unable to avoid precipitation of the enzyme by its antibodies, as it was demonstrated by immunological technique& N-acetyl-D-glucosamine prevents inhibiting antibodies from neutralizing lysozyme, as kinetic experiments and persistence of enzyme-inhibitor complexes detected by nuclear magnetic resonance spectroscopy reveals. This antibody fraction seems to be non-precipitating. The altered configuration of lysnzyme in the enzyme-inhibitor complex, might be the reason of this effect. tNTROIBJCTION Different inhibition studies have been carried out on lysozyme (E.C. 3.2.1.17). The competitive inhibition of N-acetyl-D-glucosamine ( N A G ) and other small mol. wt saccharides has been previously published (Sharon, 1967). Inhibitions by histamine and imidazole derivatives were r e p o r t e d also (Shinitzky et al., 1966): they occur as the result o f binding 2 - 4 inhibitor molecules to one of enzyme and are due to the formation of a charge transfer complex with tryptophan residues. Some mechanisms were proposed to explain protection of enzyme by substrates in inhibition by antibody (Cinader, 1967). We choose N A G , the smallest tool. wt competitive inhibitor to minimize blockade of catalytic site (subsite C) and a possible steric hindrance mechanism. Moreover, reaction of the catalytic site with substrates, imposes configurational changes on the active zone o f some enzymes (Koshland, 1959, 1963; Sela et al., 1957) and this phenomenon was observed also in N A G - l y s o z y m e interaction (Blake et al., 1967). At least, two antigenic determinants were located in the lysozyme molecule (Fujio et al., 1968 a, b). The serological activity of this enzyme was partially inhibited by t r i - N A G (Fellemberg and Levine, 1967) and, as it was reported by Imanishi et al. (1969), the l y s o z y m e - a n t i l y s o z y m e complexes are dissociated using the same inhibitor, obtaining a highly inhibitory serum fraction by this method. Previous nuclear magnetic resonance studies of the interaction of acetamide sugars with lysozyme have been made by some authors (Dahlquist and Raftery, 1968; Raftery et al., 1968; Thomas, 1966) who found that, in the presence of the enzyme, the N-acetyl peak of N A G is split into two components. At the present, the in vitro effect of inhibitors were studied on the l y s o z y m e - a n t i l y s o z y m e early interaction by immunological and N M R techniques. I M M Vo~. I0, No. 6 - A

361

MATERIALS AND MI~T~ODS

Chemicals. Hen egg-white lysozyme Grade I (Lot 88B-3050) and bovine serum albumin (Lot 114B-1250) from Signm were used. D-glucosamine and N-acetyl-Dglucosamine were supplied by the Nutritional Biochemical Corporation and employed without further purification. Histamine dihydrochloride was purchased by L E F A (Spain),and 2H20 by Merck. Solutions were made up in 0-2 M phosphate buffer (pH 6-2) and the N A G one was prepared 48 hr before use to let it reach mutarotational equilibrium, For N M R experiments, a 0-I M citratebuffer(pH 5.5) was used. Enzyme assay, Enzymatic activity was tested against lyophilized Micrococcus iysodeikticus (NCTC 2665)cells by Shugar's method (Shugar, 1952) using a Beckman DBG spectrophotometer. Suspensions of 0.3 mg/ml in 0.2 M phosphate buffer (pH 7-2) were used as substrate. Before the lytic activity of the mixtures were tested, enzyme solutions were incubated with serum and/or NAG, at room temperature for 30 rain. Nuclear magnetic resonance spectroscopy. 60 MHz NMR spectra were recorded on a Model R 12 A PerkinElmer spectrometer at a probe temperature of 31°C. Chemical shifts are expressed in pans per million (ppm) using an internal acetone standard. Solutions for reactions were made in 0.5ml 0.1M citrate buffer (pH 5-5) lyophilized and then rehydrated with 0.5 ml sI-I~Oto study its NMR spectra. Antisera. The lysozyme immunization was achieved by four weekly intramuscular injections of 0.5; 1.0; 1-0 and 2-0 mgr respectively, suspended in 1 ml of sterile saline and emulsified with an equal volume of complete Freund's adjuvant (Difco). Rabbits were bled i0 days following the last injection and sera were separated by standard techniques and stored without preservative at -- 20"C.

Immunological techniques. 1% Agarose in Barbital buffer, pH 8.6 was used in double diffusion by the Ouchterlony method. The time of incubation at 37°C was 24 hr. Double diffusion tests in the presence of inhibitors were carried out by adding NAG and histamine at a final concentration of 40 mg/ml and 8 M respectively, to the agar solutions cooled to about 45°C.

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NAZARIO RUBIO and ANTONIO PORTOLES

Precipitation curves were obtained against 0-1 ml of a lysozyme solution (0.3 mg/ml) with or without inhibitors. Each tube received 0-1 ml of the different dilutions of anti-lysozyme serum and phosphate buffered saline, pH 7.4 (PBS) to 0.5 ml of final volume. The mixtures were incubated at 37*(2 for 90 rain and 4"C for 2 days. Precipitates were washed twice with PBS, disolved in 0.1 M NaOH, and their absorbances at 280 am read. RF~IJLTS

the substrate competing with N A G was produced. Apparently, antibody does not displace N A G from the El complex during the extent of the experiment but sustrate is able to do it. So, a protection of enzyme by competitive inhibitor may be found. Nuclear magnetic resonance studies. Partial N M R spectra of N A G molecule and its variations in the presence of either lysozyme or lysozymeantibody complexes are reported in Fig. 2. In this

Interactions o f N A G and anti-lysozyme serum with lysozyme action. The M. Lysodeikticus suspension lysis was tested against: a) lysozyme alone; b) lysozyme plus a N A G concentration producing about a 50 per cent inhibition; and c) the previous systems with anti-lysozyme serum as it is indicated in Fig. 1.

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Fig. I. Variations in the lytic effects of several lysozyme solutions (14/~g in 0-5ml final volume) on M. Lysodeikticus cell suspensions: (2-5 ml at 0-3 mg/ml). A) Enzyme alone; B) lysozyme preincubated with 0.25 ml antienzyme serum: C) lysozyme plus 0.4 mg of NAG; D) similar experiment as C, but treated with 0.25 ml of antiserum after the El complex had reached its equilibrium. As expected, 0.25 ml of antiserum completely inhibits the enzymatic action of lysozyme (curve B) which shows a normal apparent first order kinetic in curve A. N A G competes with the substrate (curve C) but its inhibitory action is almost abolished by the high substrate concentration. Lastly, when the complex enzyme-inhibitor (El) is incubated with antiserum (curve D), the antibody-mediated inhibition is avoided and a lysis of

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Fig. 2. Partial NMR spectra of NAG free in solution (5 x 10-2M) (a)and in presence of lysozyme (3 × 10-8 M) Co). In experiment (c) the same amounts of the enzyme are neutralized by 0.5 mi of anti-lysozyme serum and then, NAG was added to a final concentration of 5 x 10-2 M. Finally, the NMR spectrum of the same sample as B experience plus 0.5 ml of anti-lysozyme serum (d). In experiments (c) and (d), samples of lysozyme and antiserum or NAG were left standing for 30rain at 31°C before the third component was added. Acetone (0.5%) was used as an internal reference in experiment A and appears in the 7.85 ppm zone. way, two resonances were chosen for our present study (Fig. 2a) the signals centered at 4.84ppm which was assigned to the anomeric C-1 proton in his a orientation, (Thomas, 1966) and the 8.09 ppm one, originate by the methyl protons of the acetamide group (Raftery et aL, 1968). When lysozyme (3 x 10-354) is added (Fig. 2b), the double resonance of the a C-I anomeric proton of N A G (Fig. 2a) is flattened and signals shifted swiftly to a lower field.

Early Interactions between Inhibitors and Antibodies to Lysozyme

363

As proof that this effect was due to specific association between N A G and lysozyme rather that to bulk susceptibility effects in the relatively concentrated protein solution, the spectrum of nglucosamine 1 M in the presence of 5% bovine serum albumin, a similar concentration to that used with lysozyme, were also recorded. Such a spectrum is displayed in Fig. 3. it was also shown that

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Fig. 3. The aC-1 anomeric proton spectrum of n glucosamine (1 M) in the absence (bottom) and in the presence of 5% bovine serum albumin (top). Solutions were made up in 0.1 M citrate pH 5.5, lyophilized and rehydrated with 0-5 nd of'Fig). the addition of albumin produced a decrease in the height of the signals but it did not produce any chemical shift of the a C-I proton signals from their position in free N A G , nor did it produce any flattening effects. As previously reported (Raftery, 1968), two resonance (Fig. 2b) instead of one (Fig. 2a) were observed for acetamide methyl protons in presence of lysozyme and both shifted slightly to a higher field. These methyl resonances of N A G are produced by the two bound anomeric forms (Dahlquist and Raftery, 1968). The N A G molecule did not bind to the enzyme when lysozyme was neutralized by anti-lysozyme serum as was suggested by the presence of a simple resonance at 8.09ppm (Fig. 2c). Furthermore, (C-l) u proton signals appear at the same position as in free N A G solution but without recovering their primitive shape. In our reaction time, the antibodies do not displace the N A G molecules in the El complex as was revealed by the presence of split 8-09 ppm signal and the shift of the 4-84 ppm on to a lower field (Fig. 2d).

Beha viour of the enzyme-inhibitor complex in the precipitin reaction. Precipitation lines by the Ouchterlony technique can be visualized in Fig. 4. We verified that histamine failed to suppress the precipitation at low concentrations as well as at a concentration (8 M) that inh~ited enzymatic action 100 per cent. The same happens with N A G at 40 mg/ml.

Fig. 4. Double diffusion of undiluted anti-lysozyme serum (central well) against 0.3mg/ml lysozyme (well I); 0-3 mg/ml lysozyme + histamine I M (well 2): + histamine 2 M (well 3): ~-histamine 4 M (well 4); + histamine 8 M (well 5) and +40 mg/ml NAG (well 6). Mixtures of the same volumes of enzyme and inhibitor solutions were preincubated for 30rain at room temperature before putting it into the wells. To insure that precipitation do not appear as the result of a faster diffusion of inhibitors than lysozyme in the agar matrix, with possible dissociation of the El complex, the same mixtures were put into wells of inhibitor-containing agars. Identical precipitation lines were obtained in this experiment, showing that inhibitors fail to suppress precipitation. Precipitation curves were shown in Fig. 5. Both 2 I

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364

NAZARIO RUBIO and ANTONIO PORTOLES

inhibitors prevented precipitation partially when 6 and 12/~1 of the antiserum were added but this effect is overcome when higher antiserum amounts were used. A more intense precipitation in the antigen excess zone, seems to be produced with iysozymehistamine complex rather that with the lysozymeNAG complex or with lysozyme alone (Fig. 5). DISCUSSION Neither histamine nor NAG seem to avoid precipitation of lysozyme by undiluted antiserum (Figs. 4 and 5). Antibodies to the peptide related to the catalytic zone are not capable of precipitating the enzyme as reported previously (Arnon, 1968), but both El complexes are still able to bind precipitating antibodies. Nevertheless, when they are incubated with 6 and 12/~1 of antiserum both El complexes present their precipitation levels variably disminished if we compare them with that shown by lysozyme alone. This effect is abolished when higher abtibody amounts are added. Protection of the enzyme by NAG (Fig. Id) must be produced because inhibiting antibody failed to combine with the enzyme-inhibitor complex during the time of experiments. The same is suggested in Fig. 2d by the persistence of 8-09 ppm split signal; it indicates that N A G molecules are bound to lysozyme and prevent the antibody from neutralizing it as happens in Fig. 2c. Then, antibody prevents N A G from reaching subsite C and conversely, N A G prevents antibody from neutraliTJng lysozyme. The serum concentrations conditionated some variations in the precipitate levels when enzymeinhibitor complex were used (Fig. 5). This effect might be similar to that observed by Trop et al. (1972) studying the inhibitor and antibody interactions with a pronase trypsin-antitrypsin system where 28 per cent of the inhibitor was removed from the complex in the presence of an excess of antibodies. Persistence of the flattened shape of the 4-84 ppm signal (Figs. 2c and 2d) could be explained by the highly concentrated protein solution due to addition of antiserum, although the a C-1 proton

resonance signal comes back to the free NAG position in Fig. 2c and it remains at a lower field in Fig. 2d. When NAG binds to crystalline lysozyme residue 62 moves about 0.75 A, thus tending to narrow the cleft and there are also related small shifts in the area (Blake et al., 1967). As low tool wt of N A G minimize steric hindrance the altered configuration seems to prevent the fixation of the inhibiting antibody, but does not prevent the fixation of the precipitating one. Acknowledgements-The authors acknowledge the help-

ful assistance of Mr. Louis M. Bourne in the preparation of the manuscript. RBI~I~ICES Arnon R. (1968) Fur. J. Biochem. S, 583. Blake C. C. F., Johnson L. N., Mair G. A., North A.C.T., Phillips D. C. and Sarma V. R. (1967) Proc. R, Soc.

ser. B 167, 378. Cinader B. (1967) Antibodies to Biologically Active Molecules, p. 120. Pergamon Press, Oxford. Dahlquist F. W. and Raftery M. A. (1968) Biochemistry 7, 3269. Fellemberg R. yon and Levine L. (1967)lmmunochemistry 4, 363. Fujio H., lmanishi M., Nishiola K. and Amano T. (1968 a) Biken'sJ. IL 207. Fujio H., lmanishi M., Nishioka K. and Amano T. (1968 b) Biken'sJ. 11, 219. Imanishi M., Miyagawa N., ffujio H., Amano T. (1969). Biken'sJ. 12, 85. Koshland D. E., Jr. (1959) J. Cell. Comp. Physiol. 54 (Suppl. 1) 245. Koshland D. E., Jr. (1963)Ann. N. Y.Acad. Sci. 103, 630. Raftery M. A., Dahlquist F. W., Chan S. I. and Parsons S. M. (1968)J. biol. Chem. 243, 4175. Sela M., Anfinsen C. B. and Harrington W. F. (1957) Biochim. biophys. Acta 26, 502. Sharon N. (1967) Proc. R. Soc. ser. B 167, 402. Shinitzky M., Katchalski E., Grisaro V. and Sharon N. (1966) Archs Biochem. Biophys. 116, 332. Shugar D. (1952)Biochim. biophys. Acta 8, 302. Thomas E. W. (1966) Biochem. biophys. Res. Comm. 24 611. Trop M., Pinsky A. and Avtalion R. (1972) Immunology 22,531.