Bioconjugate
Chem!&v SEPTEMBER/OCTOBER 1993 Volume 4, Number 5 0 Copyright 1993 by the American Chemical Society
ARTICLES Synthesis and Characterization of a Penicillin-Poly@-Lysine) Which Recognizes Human IgE Anti-Penicillin Antibodies Enrico Caneva, Patrizia Di Gennaro, Francesca Farina, Marco Orlandi, and Bruno Rindone’ Dipartimento di Chimica Organica e Industriale, Universith di Milano, and CNR Center, Via Venezian 21, 1-20133 Milano, Italy Paolo Falagiani Lofarma Allergeni S.R.L., Viale Cassala 40,I-20143 Milano, Italy. Received August 5, 1992
Conjugatesof poly@-lysine)containing a penicillin or a penicilloylresidue were prepared and characterized by ‘H NMR and by size-exclusion HPLC. These conjugates and conjugates with human serum albumin were used in radioallergosorbent tests (RAST) for the determination of allergy toward penicillins.
INTRODUCTION
In spite of a recent claim suggesting the overestimation of allergy to penicillin (1), most research groups agree with the importance of this pathology (21, which may cause severe health problems (3). Penicillins are low molecular weight substances which are not themselves antigenic but give rise to a number of different antigenic determinants either by metabolism in vivo or by breakdown in vitro before administration (4). The major antigenic determinant is the penicilloyl group derived from the cleavage of the 8-lactam ring of penicillin (5),and many allergic reactions to penicillin are due to antibodies specific to this group (6). Other 8-ladam cleavage products may also act as haptens and are known as “minor determinants” (7). These are associated with the majority of the severe immediate reactions to penicillin G (8). Allergy to penicillin itself is not clearly documented. Skin tests are often used for the diagnosis of penicillin allergy (9-12). The materials used for these testa are essentially penicillin-protein conjugates (13). Several “in vitro” tests for the determination of allergy to penicillin derivatives are also based on penicillin-protein conjugates 1043- 1802/93/290e0309$04.0~I0
(14). These conjugates are not generally chemically wellcharacterized, and this has a consequence in the uncertainty about the structure of the determinant(& A chemical characterization of these compounds is also necessary since penicillin conjugates with tissue macromolecules (proteins, carbohydrates, lipids) are the compounds which elicit antibody formation (15,16).Moreover, knowledge of the structure of the antigenic determinants of penicillin conjugates may allow the preparation of monovalent derivatives which could be used to desensitize patients prior to administration of the drug (17).
EXPERIMENTAL PROCEDURES
Instrumentation. NMR spectra were measured in DzO using a Varian AC300 instrument. HPLC analyses were performed with a Waters 600E HPLC. The detector was a Hewlett-Packard 1040 diode-array detector. Electrophoresis was performed with a LKB 2117 Multiphor I1 instrument. The y-counter was a Packard Cobra instrument. Reagents. Poly&-lysine) (L-PLL) hydrobromide (n= 145 and 54) and human serum albumin (HSA) were 0 1993 American Chemical Society
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purchased from Sigma Chemicals;N- [3-(dimethylamino)propyl]-N'-ethylcarbodiimide (ECDI), tert-butylamine, and isobutyl chlorocarbonate were Aldrich products; penicillin G and V were Farmitalia Carlo Erba products. Membranes for dialysis were Spectra/Por products. Polystyrene beads were activated with glutaraldehyde accordingto the Italian Patent No. 1,218,348. Labeled, affinitypurified anti-human IgE from goat was a Lofarma product. Preparation of Penicillin GPoly(L-lysine)(PG-LPLL). (a) Using the Carbodiimide 3as Coupling Reagent. Penicillin G (2) (33mg, 0.09 mmol) was added to asolution of L-PLL (1) (13 mg, 0.0007 mmol) in 5 mL of 0.2 M aqueous buffer, pH 7.1 (K2HPOdKH2P04), and the resulting solution was stirred at 4 "C. After 5 min, 17 mg (0.09 mmol) of the carbodiimide 3 was added and this solution was stirred at 4 "C for 12 h. Low molecular weight reagents and byproducts were removed by dialysis for 120 h at 4 "C with a 3500 Da cutoff membrane. The product 4 (10 mg) was then obtained by lyophilization. (b) Using the Method via Mixed Anhydride. tertButylamine (12 pL, 0.115 mmol) and 15 pL of isobutyl chlorocarbonate (0.115 mmol) were added to a solution of 30 mg (0.0898mmol) of penicillin G (2) in 2 mL of pyridine and the resulting solution was stirred at 4 "C for 30 min and then poured into a solution of 15 mg of L-PLL (1) (0.00075 mmol) in 3.8 mL of a 0.2 M aqueous phosphate buffer, pH 8 (K2HPO$KH2P04), maintained at 4 "C. After 4 h a t the same temperature, the solution was dialyzed at 0 "C for 7 days with a 3500 Da cutoff membrane. The product (15 mg) was then obtained by lyophilization. Preparation of L-PLL Conjugates. L-PLL conjugates a t different pH's were prepared using the carbodiimide 3 as coupling reagent in 0.2 M aqueous phosphate buffer of the required pH. Preparation of HSA Conjugates. Penicillin G (2) (100 mg, 0.174 mmol) was added to a solution of HSA (100 mg, 0.00147 mmol) in 15 mL of 0.2 M aqueous buffer at the required pH (K2HPOdKH2P04) and the resulting solution was stirred at 4 "C. After 5 min, 67 mg (0.36 mmol) of the carbodiimide 3 was added and this solution was stirred at 4 "C for 12 h. Low molecular weight reagents and byproducts were removed by dialysis for 120 at 4 "C with a 3500 Da cutoff membrane. The product (80 mg) was then obtained by lyophilization. Conjugates at pH 11. The conjugates at pH 11 were prepared using a 0.2 M phosphate buffer alkalinized with sodium hydroxide. HPLC Analyses. SE-HPLC was performed by dissolving 2 mg of the sample in 0.03 M phosphate buffer (K2HP04/KH2P04 pH = 6.8)-0.1 M potassium chloride and injecting through a Rheodyne 100-pL loop. The instrument was equipped with a Synchropak GPC 100 (30 cm X 7.8 mm i.d.) SE column. The separation was achieved under isocratic conditions; the elution solvent was 0.03 M phosphate buffer (K2HPOdKH2P04, pH 6.8)-potassium chloride 0.1 M at a flow rate of 0.8 mL/min. Radioallergosorbent Test (RAST). A solution of the conjugate (0.1 mg/mL) was treated for 12 h at 25 "C with glutaraldehyde-activated polystyrene beads. Human serum (50 pL) were incubated with the solid phase for 3 h a t 25 "C in a horizontal shaker. After three washing cycles with 0.15 M phosphate buffer, pH 7.2, containing 0.15% BSA and 0.4 % Tween 20, each solid phase was incubated with 50 pL of anti-human goat IgE antibody labeled with 1261 (4 pCi/100 tubes) for 12 h at 25 "C in a horizontal shaker. After three further washing cycles as above, the solid phases were counted with a y-counter. The results are expressed as percent bound radioactivity.
Chart I
OAO,R'
(2) R : COCH2C,H5: R' : H ( 5 ) R : COCH2C6H5;R' : COCH,CH(CH3),
(6) R : COCH2W6H5; R ' : H
NHR
(4) R = COCH,C6H5
( 7 ) : R = COCH,C,H,
(8): R = COCH,C,H,
RESULTS AND DISCUSSION
The reaction of poly@-lysine) (L-PLL-NHz, 1, Chart I), average MW 20 kDa (181,with excess penicillin G (PG, 2), at pH 7.1, in the presence of the water-solublecarbodiimide 3 (ECDI) (19) gave, after extensive dialysis at 0 "C, the conjugate 4. This was constituted of a family of poly& lysines) derivatized at the primary amino group a t C, with a penicillin fragment. This conclusion resulted from the lH NMR spectrum in D2O. HI and H, appeared at 6 5.45 and 5.53 with a J = 4 Hz. Hence, they were in the same position and had the same multiplicity as the corresponding protons in PG. In particular, the coupling constant showed also that the chirality at c6 and C7 had been retained. The aromatic hydrogens of the penicillin residue appeared a t 6 7.75 as a multiplet. The hydrogens Ha, Hg, H,, Ha, and H, of the poly(L-lysine) residue resonated as multiplets a t 6 4.29,1.77,1.45,1.77, and 3.00, respectively. These attributions were confirmed by a 'H-COSY experiment (Figure 1). The integration of the aromatic protons and Ha suggested that the family of derivatized poly@-lysine) molecules constituting the conjugate contained, on average, 10% penicillin residues. This value indicates that an average amount of 14.1 molecules of PG were attached to each poly(L-lysine) molecule. Hence, the conjugate consisted of an average distribution of one residue of PG for 11.0 L-lysines in L-PLL. This evaluation of percent haptenization of a polypeptide or a protein turned out to be by far more precise that the spectrophotometric determination which was per-
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Penicillin-PoIy(L4ysine) Conjugates
I
I I
Figure 2. Electrophoretic analysis of PG-L-PLL (prepared at pH 7.1). T h e plate was incubated with B. subtilis. Growth was inhibited in the positions where PG-PLL had migrated. '
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'
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Figure 1. *H-COSY spectrum of PG-L-PLL (prepared at pH 7.1) in
DzO.
formed by monitoring the absorbance a t 260 nm. A t this wavelength the penicillin residue has its maximum, and L-PLL does not absorb. For the quantitative evaluation of percent haptenization the molar extinction coefficient (e) of PG (2) was used, and this introduced an approximation to the measure since the value of for PG is expected to be different from that of PG-L-PLL. In fact, a 5.5% haptenization resulted from this method. The high percent haptenization of L-PLL obtainedwith this synthetic method is of value. I t has been pointed out that a too low percent haptenization of the protein carrier lowers the effectiveness of the conjugate in eliciting wheal and flare in penicillin-sensitized human subjects (20). Using penicilloylated BSA as the immunizing antigen in mice, it was found that an epitope number higher than 11 penicilloyl residues per protein molecule induces significant antibody formation after a single injection, whereas 0.6 penicilloyl residues per BSA molecule do not induce penicilloyl-specific antibodies (21). A further indication of the presence of the intact penicillin residue in the conjugate was obtained by putting a gel containing a culture of Buciltus subtilis (22)over the plate which had been used in an electrophoretic analysis of 4. The growth of the microorganism was inhibited in the area to which 4 had migrated, showing that 4 had retained the antibacterial property typical of intact penicillin G23(Figure 2). Size-exclusion HPLC (SE-HPLC) analysis of the conjugate was performed monitoring the eluate at 220 nm (the absorption maximum of PLL) and a t 260 nm (the absorption maximum of PG). A peak absorbing a t 220 nm appeared a t the elution time of L-PLL, and in its tail there appeared the absorption a t 260 nm due to where the penicillin residue was concentrated (Figure 3a). Other authors have prepared penicillin-poly(L-lysine) conjugates. An alkaline pH was generally used. In particular, a recent synthetic method (24) forms a benzylpenicilloyl-poly(L-lysine) (BPO-LPLL), and this conjugate has been used in the determination of IgE antibodies to the BPO determinant (25). We repeated this preparation at pH 9.5 in the scope of giving a structure to this conjugate by lH NMR spectroscopy. A blank experiment showed that penicillin G is transformed a t pH 9.5 into a 2:l mixture of two diastereoisomers of penicilloic acid (7) as shown by the lH NMR spectrum (Figure 4). In fact, the two methyl groupsof the major stereoisomer resonated
a t 6 1.27 and 1.55 as singlets. Those of the minor diastereoisomer resonated a t 6 1.09 and 1.63. H1 and H, resonated at 6 4.26 and 5.10 as multiplets. Hence, a downfield shift occurred with both protons, indicating the cleavage of the /3-lactam ring. The presence of two diastereoisomers could be attributed to the racemization of the chiral center at CS in alkaline conditions. Hence, the conjugate of PG and L-PLL prepared a t alkaline pH was likely to consist in two diastereoisomeric families of compounds. In fact performing the reaction a t pH 11 gave a conjugate whose 'H-NMR spectrum showed a 16% haptenization. The signals of H1 and Hm in the 'H-COSY spectrum (Figure 5) suggested a benzylpenicilloyl-poly(L-lysine) structure (8) for this conjugate. In fact, a multiplet a t 6 5.10 attributable to the diastereoisomeric Hm was observed in this spectrum. H1 resonated in the same region of Ha of L-PLL and could not be observed. The SE-HPLC analysis (Figure 3b) confirmed that this conjugate was different from that prepared a t pH 7.1. An alternative procedure for the preparation of PGL-PLL consisted in the formation of the mixed anhydride 5 by reaction of 2 with isobutyl chlorocarbonate in pyridine. The reaction of 5 with L-PLL a t pH 8.5 gave a conjugate which showed a 16% haptenization of L-PLL. The SEHPLC analysis is shown in Figure 3c. Three peaks containing the aromatic chromophore were present. The SE-HPLC retention time and the 'H-NMR analysis of the mixture showed that one of them was PG-L-PLL. Hence, this procedure was not suitable for the preparation of chemically defined conjugates. The use of L-PLL of lower molecular weight (5-10 kDa) gave results very similar to those reported above. The fact that different penicillin derivatives were present in the conjugates independent of the conjugation procedure stimulated us to prepare conjugates of human serum albumin (HSA) and PG or PV. These are in fact used is solid-phase assays (26) and BPO-HSA conjugates were found in sera of penicillin-treated patients (27). A reaction performed a t pH 7, where a prevalence of unchanged penicillin molecules should be attached to HSA, gave a conjugate which was analyzed by SE-HPLC (Figure 3d). PG-HSA had the main peak a t 8.8 min and two further peaks a t 11.1 and 11.7 min. A blank experiments showed that HSA is eluted a t 7.8 min in the same conditions. Hence, haptenization of HSA resulted in a longer elution time. This chromatographic behavior suggests that PGHSA fits the pores of the SE-HPLC material more efficiently than HSA. This occurs generally either when the molecular size is decreased (e.g. by a fragmentation reaction) or when folding of the polypeptide chain has
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c
- LC
0
5
220.4
A
of PG-PLL
15
10
20
Time (min)
80
2
m
M
0
Figure 4. 'H-NMR spectrum of BPO (prepared at pH 9.5) in
E
0
5
10
15
20
25
Time (min)
___
0
5
LC LC
225.4 241.2
10
'
of PG-PLL of PG-PLL
15
20
Time (min)
Figure 5. 'H-COSY spectrum of PG-L-PLL (prepared at pH 11) in D2O.
0
4
a
12
16
20
Time (min) Figure 3. SE-HPLC analysis (eluent, 0.1 M phosphate buffer, pH 6.8; column, Synchropack GPC 100at a flow of 0.8 mL/min) of crude PG-L-PLL (a) prepared at pH 7.1 with ECDI, (b) prepared at pH 11 with ECDI, and (c) prepared via mixed anhydride, (d) SE-HPLC analysis (eluent, 0.1 M phosphate buffer pH 6.8; column, Synchropack GPC 100 at a flow of 0.8 mL/min) of crude PG-HSA prepared at pH 7.1 with ECDI.
occurred. Folding of the HSA structure upon haptenization may be the reason of this behavior. These data indicated that a conformational change had occurred on
HSA upon haptenization. Interestingly, this change was not noticed in PG-L-PLL. All these conjugates and the analogues prepared using penicillin V (6, PV) were then used ina radioallergosorbent test (RAST) (28). The conjugates were immobilized in polystyrene beads and used to test the presence of human IgE antibodies in the sera of some patients known to be allergic to penicillin. They were workers professionally exposed to penicillins and having a clinical anamnesis of allergy to penicillins. The results are shown in Table I. First of all, RAST positivity was obtained using conjugates prepared by reaction of PG or PV with HSA or PLL a t pH 11without any conjugation agent. This suggests that the penicilloyl-poly(L-lysine) determinant thus formed was recognized by I g E s of patients. A blank experiment performed in the same conditions at pH 7 did not give any RAST positivity. The second point is the difference in RAST positivity independent of the pH of the conjugation reaction. This difference was noted in patient MC but not in patient AG. The RAST positivity a t neutral pH 7, where the penicillin
Penicillin-Poly(L-lysine) Conjugates
Table I. RAST Activity Observed after Incubating Sera of Allergic Patients with PG and PV Conjugates of HSA and PLL Prepared under Different Conditions pH of conj % RAST reagent conj patient protein hapten4 activity PG 11 12.5 AG HSA PV 11 10.3 HSA PG 7 ECDI 7.1 HSA PG 8 ECDI 5.9 HSA 9 PG 5.7 HSA ECDI 10 ECDI PG 8.9 HSA ECDI PV 6.0 7 HSA ECDI PV 8.4 8 HSA ECDI PV 9.5 10 HSA PLL PG 7 ECDI 9.6 PV 7 ECDI 7.8 PLL 17.8 MC HSA PG 11 16.7 HSA PV 11 15.7 PLL PG 11 PLL PV 11 14.2 HSA PG 7 ECDI 1.2 HSA PG 8 ECDI HSA PG 9 ECDI 21.0 HSA PG 10 ECDI 2.6 HSA PV 7 ECDI 5.9 HSA PV 8 ECDI 15.1 HSA PV 9 ECDI HSA PV 11 ECDI 2.8 1.8 PLL PV 7 ECDI 14.8 PG 11 F HSA 15.0 HSA PV 11 PG 11 L HSA 1.1 HSA PV 11 0.9 PV 11 2.1 S HSA 1.7 PG 11 HSA Background bound radioactivity = 0.2%.
nucleus is present in the conjugate, suggests that antipenicillin IgE antibodies are present in the serum of the patient. In conclusion, lH NMR and SE-HPLC analysis of penicillin-protein conjugates allowed the percent haptenization and structure of the conjugate to be obtained. This is especially useful when the hapten may undergo modifications during the haptenization procedure. ACKNOWLEDGMENT
This work was supported by a CNR grant (Piano Finalizzato Chimica Fine). We thank our students Ezio Bolzacchini, Anna Brocca, and Monica Odiardo for their collaboration in the experimental work. LITERATURE CITED (1) Surtees, S.J., Stockton, M. G., and Gietzen, T. W. (1991) Allergy to penicillin, fable or fact? Br. Med. J. 302,105-106. (2) Pearson, C. R. (1991)Allergy to penicillin. Br. Med. J. 302, 1462. Walley, T., and Coleman, J. (1991)Br. Med. J . 302, 1462-1463. McSharry, C., Lewis, C., and Kirkwood, E. (1991) Br. Med. J . 302,1463.Surtees, S.J., Stockton, M. G., Gietzen. T. W. (1991)Br. Med. J. 302,1463. (3) Sogon, D. D. (1984)Penicillin Allergy. J. Allergy Clin. Zmmunol. 74,589-593. (4) De Weck, A. L. (1974)Low Molecular Weight Antigens, in Sela. The Antigens, Vol. 2,pp 141-248,Academic Press, New York. (5) Beeley, L. (1984)Allergy to Penicillin. Br. Med. J.288,511512. (6) Erffmeyer, J. E. (1981)Adverse reactions to penicillin. Ann. Allergy 47,288-300. (7) Levine, B. B., and Redmond, A. P. (1969)Minor Haptenic Determinant-Specific Reagents of Penicillin Hypersensitivity in Man. Znt. Arch. Allergy Appl. Zmmunol. 35,445-455.
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(8)Levine, B. B., Redmond, A. P., and Fellner, A. J. (1966) Penicillin Allergy and the Heterogeneous Immune Response of Man to Benzylpenicillin. J.Clinical Invest. 45,1895-1906. (9)Ressler, C., and Mendelson, L. M. (1987)Skin Test for Diagnosisof Penicillin Allergy. Current Status. Ann. Allergy 59, 167-170. (10)Van Dellen, R. D. (1981)Skin Testing for Penicillin Allergy. J. Allergy Clin Zmmunol. 68,169-180. (11)Van Arsdel, P. P., Jr. (1978)Adverse Drug Reactions. In Allergy,Principles andPractice (E. Middleton, Jr., C. E. Reed, and F. F. Ellis, Eds.) pp 1153-1154. (12) Sullivan, T. J., Wedner, H. J., Shatz, G. S., Yecies, L. D., and Parker, C. W. (1981)Skin testing to detect penicillin allergy. J. Allergy Clin. Zmmunol. 68, 171-180. (13) Parker, C. W. (1967)Conjugation of penicillin and its derivatives. In Methods in Immunology and Zmmunochemistry (C. A. Williams, and M. W. Chase, Eds.) Vol. 1, pp 133143. (14) Wide, L., Bennich, H., and Johansson, S. G. 0. (1967) Diagnosis of allergy by an in vitro test for allergen antibodies. Lancet 2,1105-1107. (15) Kristoffersson, A., Ahlstedt, S., Petterson, E., and Svird, P. 0. (1977)A radioimmunoassay for Detection of Penicilloylated Protein Contaminants in Penicillin Preparations. Znt. Arch. Allergy Appl. Zmmunol. 55, 13-22. (16) Landsteiner, K. (1962)In The Specificity of Serological Reactions, Dover, New York. Eisen, H. N. (1959)In Cellular and Humoral Aspects of the Hypersensitive State (H. S. Lawrence, Ed) Haeber, New York. (17) Schwartz, M. A. (1969) Chemical Aspects of Penicillin Allergy. J. Pharm. Sci. 58,643-661. (18) Wakana,H., Shigaki,T., andsaito, N. (1982)Intramolecular alpha helix-beta structure-random coil Transitions in Polypeptides. Biochem. Chem. 16,275-285. (19)Khorana, H. G. (1953)The Chemistry of Carbodiimides. Chem. Rev. 53,145-166. (20)Levine, B., and Redmond, A. P. (1968)The Nature of the Antigen-Antibody Complexes Initiating the Specific Whealand-Flare Reaction in Sensitized Man. J . Clin. Invest. 47, 556-567. (21)Kristofferson, A., Ahlsted, S., and Svird, P. 0. (1977) Antigens in Penicillin Allergy. Znt. Arch. Allergy Applied Zmmunol. 55,23-28. (22) Jackson, G. E. D., and Strominger, J. L. (1984)Synthesis of Peptidoglycan by High Molecular Weight Penicillin Binding of Bacillus Subtilis and Bacillus Stearothermophilus. J . Biol. Chem. 259,1483-1490. (23)Stanier, R. Y., Ingraham, J. L., Wheels M. L., and Painter, P. R. (1988)The Microbial World,Prentice Ha& Englewood Cliffs, NJ, pp 158-166. (24) Edwards, R. G., Speck”, D. A., and Dewdney,J. M. (1982) Development and use of three radioallergosorbent tests in the diagnosis of penicillin allergy. Znt. Arch. Allergy Appl. Zmmunol. 68, 352-357. (25) Blanca, M., Mayorga, C., Perez, M., Suau, R., Juarez, C., Vega, J. M., Carmona, M. J., Perez Estrada, M., and Garcia, J. (1992) Determination of IgE antibodies to the benzyl penicilloyldeterminant. A comparison between poly-L-lysine and human serum albumin as carriers. J.Zmmunol. Methods 153,99-105. (26) Lafaye, P., and Lapresle, C. (1988)Fixation of penicilloyl groupsto albumin and appearance of anti-penicilloylantibodies in penicillin-treated patients. J. Clin. Invest. 82,7-12. (27)Lapresle, C., and Wal, J.-M. (1979)The binding of penicillin to albumin molecules in bisalbuminemia induced by penicillin therapy. Biochim. Biophys. Acta 586, 106-111. (28) Ceska, M., Eriksson, R., and Varga, J. M. (1972)Radioimmunosorbent assay of allergens. J.Allergy Clin.Zmmunol. 49, 1-9.
