Anal. Chem. 1992, 64, 977-980
977
Mechanistic Studies of Electrostatic Potentials on Antigen-Antibody Complexes for Bioanalyses Ping Yu Huang and Cheng S. Lee* Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County Campus, Baltimore, Maryland 21228 A concept lnvolvlng the use of antlbodles conjugated with reporter molecules for the direct sensing on the subtle changes In the local electrostatic envlronment of antigenantibody complexes due to the antigen binding was proposed and demonstrated. The studies lncludlng the effect of solution conditions on the extent and magnitude of the electrostatic potentlals around the antlgen-antibody complex and the quantitathre dependence of the distance between the reporter molecule and the antigen-antibody blnding site on the effectiveness of the reporter molecule for senslng the antlgenantibody blndlng event are presented and discussed. Basic understanding of Induced changes in the electrostatic potentials on the antlgen-antlbody complex is essential for Impiementlng the proposed dlrect sensing mechanlsm.
INTRODUCTION The speed and specificity of immunochemical complex formation has encouraged the research and development of numerous antibody-based analytical procedures and immunosensors. Independence from the need to mix reagents with the sample for transducing the antigen-antibody reaction represents a real conceptual difficulty and is crucial for any "probes" using immunotechnology.' A concept involving the use of antibodies conjugated with reporter molecules for directly sensing the antigen-antibody reaction was therefore proposed and studied.2 The properties of reporter molecules were sensitive to subtle changes in the electrostatic environment around the binding site due to the antigen-antibody binding. The resulting change in the properties of reporter molecules could then be specifically monitored by the transducing element without the need of other reagents. This proposed direct sensing mechanism was demonstrated with the use of protein A-IgG complex as the model system.2 The binding of IgG to protein A contributed to an additional electric field around protein A. The presence of this additional electric field changed the local electrostatic environment and the local pH around the protein A-IgG complex. This change in the local pH was measured by monitoring the fluorescence property of fluorescein molecules as reporter molecules conjugated with protein A before and after the introduction of IgG. The previous theoretical analyses and experimental resulb with the use of protein A-IgG complex also indicate that the fundamental knowledge of induced electrostatic potentials around the antigen-antibody complex is critically needed for *To whom all correspondence should be addressed.
unraveling the proposed direct sensing mechanism. Therefore, the objective of this study is to advance our basic understanding in the induced electrostatic potentials around the complex. More specifically, (i) the fundamental relationship between the macroenvironment (pH and ionic strength in the bulk solution) and the microenvironment (electrostatic potential and pH around the complex) and (ii) the quantitative dependence of the distance between the reporter molecule and the binding site on the effectiveness of the reporter molecule for sensing the binding event are investigated. To achieve the above objectives, the electrostatic potential contour map3-9as an excellent tool for studying the electrostatic potentials at each amino acid residue on the protein surface is used. The contour map is generated by combining the structure data of protein measured by X-ray crystallography with the three-dimensional Poisson-Boltzmann equation. The structural data obtained from the Protein Data Bank at the Brookhaven National Laboratory'O provide the spatial coordinates of amino acid residues within protein. The finite difference Poisson-Boltzmann computation method developed by Honig et al.3-9using protein data format as the input describes the extent and distribution of electrostatic potentials arouhd the protein surface at specific solution conditions. Protein A contains four highly homologous domains with equal affinity for the Fc region of IgG. To simplify the analysis, only the crystal structure of fragment B of protein A is used for this study. The localization of the binding sites for protein A and IgG are around the tyrosine residue of protein A and at the CH2CH3domain of Fc part of IgG. The electrostatic potentials contributed by the Fab part of IgG is probably insignificant because of its large separation distance from the binding site. This conclusion was supported by the experiments carried out in our laboratory. No significant difference in the increase in fluorescence intensity of protein A, fluorescein conjugate due to the binding of IgG whole molecule or only Fc fragment of IgG was observed. We therefore generate the contour maps more readily by using the Fc fragment of IgG. The electrostatic potential contour maps of fragment B of protein A and the complex of fragment B with Fc part of IgG are generated at different solution conditions such as pH and ionic strength by using the 1FC2 data file from the Protein Data Bank. Even though the structures of fragment B, Fc part of IgG, and the complex might change with solution conditions, the same structures obtained from X-ray crystallography are still used for the generation of contour maps. These conformational changes are most likely influence the short-range interactions such as
0003-2700/92/0364-0977$03.00/00 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
hydrogen binding and steric interactions, but should not affect significantly the long-range forces such as electrostatic interactions.
EXPERIMENTAL SECTION Fragment B of protein A conjugated with fluorescein (conjugation ratio: 1.0 fluorescein per fragment B molecule) was purchased from Molecular Probes (Eugene, OR) and further purified in our laboratory in a DEAE column (Waters Chromatography, Milford, MA) with a linear elution gradient of ammonium hydroxide/ammonium chloride from 0.1 to 0.5 M.11J2 Monopotassium phosphate and dipotassium phosphate were obtained from Sigma (St. Louis, MO). Buffer solutions were prepared with water of HPLC grade. All buffer solutions were adjusted to the appropriate pH with 1M NaOH or 1M HC1. Fc part of human IgG was purchased from Accurate Chemical (New York, NY). Fluorescence emission spectra of fragment B of protein A conjugated with fluorescein were measured by a Perkin-Elmer MRF-66 fluorescence spectrophotometer. Once the maximum fluorescenceemission wavelength of fragment B conjugated with fluorescein had been determined, the fluorescence intensity measurements at the maximum emission wavelength were then carried out by a simple fluorometer such as a Pandex Fluorescence Concentration Analyzer (PFCA). DelPhi, the computer program based on the finite difference Poisson-Boltzmann equation, was obtained from Professor Honig in Columbia DelPhi was run in a GTX 210 Silicon Graphic Workstation with the use of 1FC2 structure data fiie from the Protein Data Bank. The 1FC2 file describing the spatial coordinatesof amino acid residues within the complex of fragment B and Fc part of human IgG was deposited by Deisenhofer based on his X-ray crystallographic studies.13 The calculated results of the electrostatic potentials around the macromolecular surface were displayed in three dimensions with the use of a computer program, Quanta. Tryptic digestions of fragment B and fragment B, fluorescein conjugate were performed following the same procedure elsewhere.14 Trypsin was obtained from Sigma (St. Louis, MO). The digestion reactions were allowed to proceed for 16 h and stopped by heating the solutions at 100 "C for 2 min. The solution pH was then adjusted to pH 10 with 1 M NaOH. The original phosphate buffer concentration, 50 mM, was also diluted t o 10 mM for the following tryptic mappings. Tryptic mappings of fragment B and fragment B, fluorescein conjugate,were examined by using capillary zone electrophoresis. The instrumentationfor Carrying out capillaryzone electrophoresis A fused silica capillary with a 50-pm i.d. was given e1~ewhere.l~ and 150-pm 0.d. from Polymicro Technologies Inc. (Phoenix, AZ) was used for the separations of peptide fragments. The highvoltage power supply was obtained from Spellman High-Voltage Electronics (Plainview, NY). On-column detection of peptide fragments was carried out with a Linear Instruments variablewavelength detector (Reno,NV). The cathode end of electric field was always at the detector end. Peptide fragments were introduced into the capillary by using the electromigration injection method. RESULTS AND DISCUSSION Contours of electrostatic potentials on the fragment B of protein A and the complex surface of fragment B with Fc part of IgG at 1mM phosphate buffer and pH 6.7 are shown in Figure 1. Fragment B of protein A at pH 6.7 carries a net negative potential which is shown as blue in Figure lb. This is because the isoelectric point (PI)of protein A is 5.116J7lower than pH 6.7. However, the binding of Fc part of IgG to fragment B as shown in Figure l b clearly contributes to an additional positive electric potential around the complex. The CH2CH3domain of Fc part of IgG which is the localization of the binding site especially possesses a strong positive potential (the intense red color) as shown in Figure lb. In fact, there are seven lysine residues between the amino acid sequence 310 and 340 in the CH2 domain of Fc part of IgG.13 This preliminary use of electrostatic potential contour map not only further supports the proposed direct-sensing mechanism but also demonstrates its ability as a fundamental tool
t t L
Flgure 1. (a, top) The space-filling models of fragment B of protein A and the complex of fragment B with Fc part of IgG. Fragment B is red, Fc part of IgG is yellow. (b, bottom) The electrostatic potential contour maps of fragment B and the complex of fragment B with Fc
part of IgG. The polarity of potential is represented by the color gradient from red to blue. Positive potential is red, neutral potential is green, and negative potential is blue. The solution conditions are 1 mM phosphate buffer and pH 6.7.
for studying the electrostatic potentials on and around the antigen-antibody complex. In the experimental measurements, the changes in the fluorescence intensities of fragment B of protein A, fluorescein conjugate, before and after the introduction of Fc part of IgG were measured at the different solution conditions. The effect of solution conditions on the changes in the fluorescence intensities of fragment B, fluorescein conjugate, is quite complicated. This is because the pKa of fluorescein is influenced by the ionic strength of the s o l ~ t i o n . ~The ~ J ~equilibrium binding constant of fragment B with Fc part of IgG, K , is also dependent on the solution pH and ionic strength. Even though these two parameters have no effect on the changes of electrostatic potentials due to Fc binding, they still influence the changes in the fluorescence intensities, which can alter the sensing efficiency of the measurement. For example, the percentage of change in fluorescence intensity can increase when the equilibrium binding constant, K , increases. This is because a larger fraction of the fragment B can be bound by Fc part of IgG with the larger K. In order to investigate the effect of solution conditions on induced electrostatic potentials around the fragment B-Fc complex, the quantitative assessments for the K , pK,, and electrostatic potentials must be studied separately. The fluorescence intensities of fragment B, fluorescein conjugate
ANALYTICAL CHEMISTRY, VOL. 04, NO. 9, MAY 1, 1992
in the absence of Fc, I,, at the different pH's but the same ionic strength were obtained. These 1;s were used to construct a baseline which described the relationship between fluorescence intensity and pH at the specific ionic strength. Different baselines were generated for different ionic strengths. The fluorescence intensity of the mixture of fragment B and Fc at complete binding saturation, I,, was measured at specific pH and ionic strength. The I, in comparison with the baseline at the same specific ionic strength was used to estimate the change in local pH due to the binding of Fc part and IgG. This local pH change measured indirectly was then free from complications of K and pK, changes and ready for comparison with predictions obtained from electrostatic potential contour maps. The analyses of contour maps of fragment B and the complex of fragment B with Fc part of IgG predicted the polarity and magnitude of electrostatic potentials around each amino acid residue of fragment B before and after the binding of Fc. The local pH around each residue of fragment B before and after the binding of Fc could then be estimated as
Table I. Comparison between the Local pH Changes Measured Experimentally and the Predictions Obtained from the Electrostatic Potential Contour Maps predicted pH changes around measured pH changes lysine 154 lysine 168
phosphate buffer concentration and pH 1 m M at pH 6.7 10 m M at pH 6.7 100 m M at pH 6.7 1 m M at pH 6.4 10 m M at pH 6.4 100 m M at pH 6.4
0.86 0.52 0.27 0.95 0.61 0.28
1.28 0.91 0.58 1.42 1.03 0.60 6
0.77 0.43 0.13 0.86 0.50 0.15
&5
or
where the subscripts 0 and bulk represent the concentration of ions in the immediate vicinity of each residue and in the bulk solution, respectively. The cp, is the electrostatic potential at each residue of fragment B. The binding of Fc to the fragment B changes the cpo and then changes the local pH around each residue of fragment B. The k and T are the Boltzmann constant and the absolute temperature, respectively. This is the so-called Boltzmann distribution of ions in the electric field.20 Depending on the location of amino acid residue which fluorescein was conjugated, the change in the local pH around the fluorescein molecule could then be predicted. However, several lysine residues of fragment B such as lysine 123, 126, 154, 168, 169, and 177 were available for fluorescein conjugation. To simplify the analysis, only the fragment B, fluorescein conjugate, with average molecular fluorescein/ protein (F/P) ratio of 1from Molecular Probes, Inc. was used for the experimental measurements. The local pH change measured experimentally at complete binding saturation with Fc part of IgG was then used to compare with the predicted local pH change. Through this comparison, we could possibly determine the location of lysine residue which was preferentially conjugated with fluorescein at fragment B. Based on the structure of fragment B displayed in three dimensions, lysine 126 and lysine 177 are sterically hindered from the conjugation of fluorescein. In addition, the conjugation of fluorescein at lysine 123 is right in the binding site of fragment B. The conjugation of fluorescein a t lysine 123 would severely interfere the binding activity of fragment B with Fc part of IgG. The binding constant of fragment B, fluorescein conjugate measured from this study equal to 8 X lo7 M-' is in close agreement with those reported in the literature which ranged from 4 X lo7to 2 X lo8 M-l.16J7 Thus, it is unlikely that lysine 123,126, and 177 of fragment B were the preferential locations for fluorescein conjugation. As shown in Table I, the predicted local pH changes at the remaining lysine locations, lysine 154 and lysine 168 (or 169), were then compared with the local pH changes measured experimentally at various solution conditions. The local pH changes including the experimental measurements and the predictions from the electrostatic potential contour maps all decreased with the increase of ionic strength. This was because more counterions were available at the higher ionic strength to balance the electrostatic potentials induced by the
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Time (min) Figure 2. Zone electrophoretic separation of peptide fragments obtained from tryptic digestion of fragment B of protein A: buffer, phosphate 10 mM at pH 10; capillary, 50-pm 1.d. and 150-pm o.d., length to detector 16 cm; voltage 1 kV and 10 s for injection, 3 kV for electrophoresis; absorbance at 200 nm. The peak number is the same as the peptide fragment number given in Table 11.
binding of Fc. In addition, the local pH changes all increased with the decrease of solution pH. Fc part of IgG carried more positive charges when the solution was more acidic. The comparison between the experimental results and the predictions indicated that lysine 168 (or lysine 169) was the possible preferential location for fluorescein conjugation at fragment B. In addition, the predicted local pH changes around lysine 154 were much greater than around lysine 168 (or lysine 169) especially at 10 and 100 mM phosphate buffer. Clearly, the efficiency of our direct sensing mechanism is critically dependent upon the location of fluorescein conjugation. To confirm the location of fluorescein conjugation at fragment B of protein A, tryptic mappings of fragment B and fragment B, fluorescein conjugate, were examined by using capillary zone electrophoresis. A total of eight peptide fragmenta was obtained from the tryptic digestion of fragment B and summarized in Table 11. As shown in Figure 2, these eight peptide fragments were then separated in capillary zone electrophoresis. The identification of each peptide fragment was carried out by correlating the migration velocity of peptide fragment with the parameter, charge/(molecular eight)^/^, used by Richard et al.21The theoretical charges on our peptide fragments at pH 10 were calculated by using Skoog and Wichman's As shown in Figure 3, an excellent correlation of migration velocity of our peptide fragments with their charge/(molecular eight)^/^ was also observed in this study. For tryptic mapping of fragment B with fluorescein conjugation, the separation of peptide fragments was monitored by the absorbances at 200 nm for peptides (not shown) and at 495 nm for fluorescein (shown in Figure 4). For the de-
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
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Table 11. Peptide Fragments Obtained from Tryptic Digestion of Fragment B of Protein A peptide fragment number
amino acid sequence in the peptide fragment
1 2 3
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Glu-Gln-Gln-Asn-Ala-Phe-Try-Glu-Ile-Leu-His-LeuPro-Asn-Leu-Asn-Glu-Glu-Gln-Arg Ala-Lys(l68) Ala-Lys(168)-Lys(169)
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Time (min) Flgure 4. Zone electrophoretlc separation of peptide fragments obtained from tryptic digestion of fragment 6,fluorescein conjugate: buffer, phosphate 10 mM at pH 10; capillary, 50-pm 1.d. and 150-pm o.d., length to detector 16 cm; voltage 1 kV and 10s for injection, 3 kV for electrophoresis; absorbance at 495 nm. The peaks correSpondlng to the peptide fragments 6 and 8 wlth fluorescein conjugation are indicated wlth numbers 6 and 8, respectively.
Charge/(Molecular Figure 3. The correlation between the migration velocity of peptide The peptide fragment and the value of its charge/(molecular fragments obtalned from the tryptic digestion of fragment 6 are shown as the open circles. The number shown In the figure is the same as the peptkle fragment number given in Table 11. The peptide fragments 6 and 8 conjugated wlth fluorescein are shown as the open triangles.
tection of peptide fragments, the peak height of peaks representing peptide fragments 6 and 8 decreased significantly in comparison with the peak height shown in Figure 2. Furthermore, two additional peptide peaks from tryptic mapping of fragment B with fluorescein conjugation were observed around the migration time of 7 min. These two additional peptide fragments were the two peaks shown in Figure 4 with the strongest absorbance at 495 nm. As shown in Figure 3, the migration velocities of these two additional peptide fragments were correlated very well with the charge/(molecular eight)^/^ of peptide fragments 6 and 8 conjugated with fluorescein. The conjugation of fluorescein increased the electrophoretic velocities of peptide fragments 6 and 8 toward the anode end. However, all the solutes in the capillary tubing were pumped by a strong electroosmotic flow at pH 10 toward the cathode end, the detector end. The overall migration velocities of peptide fragments 6 and 8 therefore decreased with the conjugation of fluorescein. Lysine 168 and lysine 169 were the two available locations for fluorescein conjugation at the peptide fragments 6 and 8, respectively. Tryptic mappings of fragment B and fragment B, fluorescein conjugate, supported the previous conclusion that lysine 168 and lysine 169 were the preferential locations for fluorescein conjugation. To implement our proposed direct sensing mechanism, it is essential to advance our understanding in the distribution and the change in the electrostatic potentials on the antigen-antibody complex due to antigen binding. This preliminary application of the electrostatic potential contour map demonstrates its ability as a tool in the molecular scale for
studying the electrostatic potentials on the antigen-antibody complex. It is expected that the results obtained from this project in the near future will provide the fundamental insight and possible rules for relating the microenvironment around the binding site with the macroenvironment in the bulk solution. In addition, rules for determining the location of amino acid residue for conjugation and for choosing the appropriate reporter molecule can be obtained. ACKNOWLEDGMENT
Support for this work by the Designated Research Initiative Funds of the University of Maryland at Baltimore County is gratefully acknowledged. We thank Chin-Tiao Wu for performing the separations of peptide fragments in capillary zone electrophoresis. REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)
North, J. R. Trends Biotechnol. 1985, 3, 180. Lee, C. S.; Huang, P. Y.; Ayres, D. M. Anal. Chem. 1991, 63, 464. Gilson, M. K.; Honig, B. H. Nature (London) 1987, 330, 84. Sharp, K.; Fine, R.; Honig, B. H. Science 1987, 236, 1460. Gilson, M. K.; Honig, B. H. Proteins: Struct. Funct. Genet. 1988, 3, 32. Gilson, M. K.; Honig, B. H. Proteins: Shuct. Funct. Genet. 1988, 4, 7. Soman, K.; Yang, A. S.; Honig, 6. H.; Fletterick, R. BlochemisttyI989, 2 8 , 9918. Sharp, K. A.; Honing, B. H. Chem. Scr. 1989, 29A, 71. Sharp, K. A.; Honig, 6. H.; Harvey, S. C. Biochemlstty 1990, 29, 340. Bernsteln, F. C.; Koetzle, T. J.; Wllllams, G. J. B.; Meyer, F. F.; Brice, M. D.; Rodgers, J. R.; Kennard, 0.; Shlmanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112, 535. The, T. H.; Feltkamp, T. E. W. Immunology 1970, 18, 865. The, T. H.; Feltkamp, T. E. W. Immunobgy 1970, 18, 875. Deisenhofer, J. Biochemistry 1981, 20, 2370. SJodahl, J. Eur. J. Biochem. 1977, 73, 343. Lee, C. S.; Wu, C. T.; Lopes, T.; Patel, 6. J. Chromatogr. 1991, 559, 133. Surolia, A.; Pain, D.; Khan, M. I. Trends Blochem. Sci. 1982, 7, 74. Langone, J. J. Adv. Immunol. 1982, 32, 157. Saarl, L. A.; Seitz, W. R. Anal. Chem. 1982, 54, 821. Haugland, R. P. Handbook of Fluorescent Probes and Research Chemlcais; Molecular Probes, Inc.: Eugene, 1989; p 30. Israelachvlli, J. N. Intermolecular and Surface Forces ; Academic Press: New York, 1985; Chapter 12. Richard, E.; Strohl, M.; Nlelsen, R.; Farb, P. Third International Symposium on High Performance Capillary Electrophoresis 1991, Poster Paper PT-19. Skoog. B.; Wichman, A. Trends Anal. Chem. 1986, 5, 82.
RECEIVED for review October 24,1991. Accepted January 27, 1992.
