Anal. Chem. 1997, 69, 4994-5000
Synthesis and Characterization of Insulin-Fluorescein Derivatives for Bioanalytical Applications Nathaniel G. Hentz,† John M. Richardson,† J. Richard Sportsman,‡ Janet Daijo,‡ and G. Sitta Sittampalam*,†
Research Technologies and Proteins, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46252, and Terrapin Technologies, 750 Gateway Boulevard, South San Francisco, California 94080
Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had ∼10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active. Fluorescent probes are widely used in bioanalytical applications because fluorescence-based detection offers a number of advantages over radioisotopic detection. Fluorescent probes are generally more stable and thus have longer shelf lives, more versatile, and safe (i.e., no special handling precautions and easy, inexpensive disposal), and the sensitivity can approach or equal that of radioisotopic methods. As the development of high-throughput screens (HTS) increases throughout the pharmaceutical industry,1 so does the generation of waste. Because of the high sensitivity attainable, a common methodology involves the use of radioimmunoassays. In addition, since the radiolabel is usually small compared to the protein/peptide, its effects toward the biological activity are usually considered negligible. In HTS, scintillationproximity assay is a popular assay method because it can be †
Eli Lilly and Co. Terrapin Technologies. (1) Broach, J. R.; Thorner, J. Nature 1996, 384, 14-16. ‡
4994 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
performed without the requirement of a separation step.2 However, fluorescence technologies are gaining widespread use as an alternative to radiolabeled methods because of the cost of licensing, tougher, more costly disposal regulations, special handling procedures/training, etc. As a result, fluorescence-based instruments for HTS are now commercially available.3 In fact, fluorescence polarization has seen an explosive growth in the number of applications over the past decade. Binding interactions such as protein-ligand, protein-protein, protein-DNA, etc., are readily observable by fluorescence polarization in a homogeneous format.4-6 Fluorescence polarization is particularly amenable to the clinical laboratories and pharmaceutical industries because of its homogeneous nature, requiring no separation steps.3 Because of its popularity, fluorescein is the predominant fluorophore of choice used in many bioanalytical applications (i.e., protein-protein interactions, assays, DNA tags, gene expression, analytical detection, etc.).7 This is due in part to its relatively high molar absorptivity and quantum yield, its solubility in water, and its visible excitation and emission wavelengths at 495 and 520 nm, respectively. Fluorescein is also used in techniques such as fluorescence microscopy, and flow cytometry because an argon ion laser (spectral line at 488 nm) can be used. In this paper, insulin was chosen as a model protein for fluorophore labeling studies in part because of its pharmacological importance, relatively small size (51 amino acids divided into an A-chain and a B-chain), several reactive groups, and widely studied and characterized structure and function.8,9 In addition, insulinfluorescein is commercially available and is used in a number of applications. However, except for one report,10 very little is found in the literature about the full characterization and biological activity of such insulin species (or any protein species) labeled with a fluorophore. A number of different insulin conjugates have been synthesized and structurally characterized.8 Specifically, moieties such as fluorophores,10-20 chromophores,21 biotin,22 sugars,23-25 saporin,26 and iodine27-29 have been chemically conjugated to insulin through (2) Bosworth, N.; Towers, P. Nature 1989, 341, 167-168. (3) Rogers, M. V. Drug Discovery Today 1997, 2, 156-160. (4) Checovich, W. J.; Bolger, R. E.; Burke, T. Nature 1996, 375, 254-256. (5) Lunblad, J. R.; Laurance, M.; Goodman, R. H. Mol. Endocrinol. 1996, 10, 607-612. (6) Phizicky, E. M.; Fields, S. Microbiol. Rev. 1995, 59, 94-123. (7) Haughland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes: Eugene, OR, 1997. (8) Blundell, T.; Dodson,G.; Hodgkin, D.; Mercola, D. Adv. Protein Chem. 1972, 26, 279-402. (9) Pitts, J. E.; Bajaj, M. Immunol. Clin. Exp. Diabetes 1984, 3-49. S0003-2700(97)00726-9 CCC: $14.00
© 1997 American Chemical Society
the primary amine-,9-26 tyrosine-,27-29 and cysteine-reactive groups.11 The synthesis and purification of both monoiodonated27 and diiodinated insulin28 species have been reported in which the effects of location and extent of iodination were studied. The biological activity with respect to insulin receptor binding27,28 and insulin-specific endosomal protease29 was also determined for the purified iodoinsulin species. It should also be noted that all four possible monoiodinated insulin isomers were purified by HPLC.29 As mentioned earlier, insulin has been previously labeled with fluorescein isothiocyanate (FITC) and characterized for a number of applications.10,13,17-20 The biological activity of the insulin-FITC derivatives has been reported to differ from that of native insulin.10,13 Bromer et al.10 provided the first biological activity information on different insulin-FITC species by performing a mouse convulsion test and immune hemolysis inhibition assay. The activity of a mixture of two monosubstituted insulins was reported to be 40% of native insulin while the di- and trisubstituted species had virtually no biological activity. To date only three insulin-FITC species have been isolated at any one time: a mixture of mono-, di-, and trisubstituted by anion exchange10 or two monosubstituted and one disubstituted by reversed-phase HPLC.30 Finally, since it has been reported that the order of reactivity of the three primary amines is B1(Phe) > A1(Gly) >> B29(Lys) and derivatives of insulin differing in only the position of modification could be separated,10,20,23 it was hypothesized that different insulin-FITC conjugates could be synthesized and purified, and therefore, the binding characteristics of the different conjugates could be studied systematically. Fluorescence polarization assays for insulin have been previously described, but to our knowledge only mixed species of insulin-FITC conjugates were used.31,32 (10) Bromer, W. W.; Knapp-Sheehan, S.; Berns, A. W.; Arquilla, E. R. Biochemistry 1967, 6, 2378-2388. (11) Myhre, D. V.; McCracken, M. S.; Keough, T.; Hill, J. C.; Macfarlane, R. D. Biomed. Environ. Mass Spectrom. 1988, 17, 113-119. (12) Maisano, F.; Gozzini, L.; de Hae¨n, C. Bioconjugate Chem. 1992, 3, 212217. (13) Tietze, F.; Mortimore, G. E.; Lomax, N. R. Biochim. Biophys. Acta 1962, 59, 336-346. (14) Jobba´gy, A.; Jobba´gy, G. M. J. Immunol. Methods 1973, 3, 399-410. (15) Nanami, M.; Zaitsu, K.; Ohkura, Y. Biol. Pharm. Bull. 1993, 16, 99-102. (16) Pinto, D. M.; Arriaga, E. A.; Sia, S.; Li, Z.; Dovichi, N. J. Electrophoresis 1995, 16, 534-540. (17) Jobba´gy, A.; Jobba´gy, G. M. J. Immunol. Methods 1973, 2, 371-382. (18) Bailey, I. A.; Garrat, C. J.; Penzer, G. R.; Smith, D. S. FEBS Lett. 1980, 121, 246-248. (19) Shimoyama, R. J. Clin. Endocrinol. Metab. 1981, 53, 502-506. (20) Maeda, H.; Ishida, N.; Kawaucji, H.; Tuzimura, K. J. Biochem. 1969, 65, 777-783. (21) Bredehorst, R.; Wemhoff, G. A.; Kusterbeck, A. W.; Charles, P. T.; Thompson, R. B.; Ligler, F. S.; Vogel, C. W. Anal. Biochem. 1991, 193, 272-279. (22) Fabry, M.; Brandenburg, D. Biol. Chem. Hoppe-Seyler 1992, 373, 143150. (23) Gorman, J. J.; Corino, G. L.; Mitchell, S. J. Eur. J. Biochem. 1987, 168, 169-179. (24) Baudys, M.; Uchio, T.; Mix, D.; Wilson, D.; Kim, S. W. J. Pharm. Sci. 1995, 84, 28-33. (25) Mehvar, R. Drug Dev. Ind. Pharm. 1994,20, 395-404. (26) Ippoliti, R.; Lendaro, E.; Bellelli, A.; Fiani, M. L.; Benedetti, P. A.; Evangelista, V.; Brunori, M. Nat. Toxins 1996, 4, 156-162. (27) Frank, B. H.; Peavy, D. E.; Hooker, C. S.; Duckworth, W. C. Diabetes 1983, 32, 705-711. (28) Maceda, B. P.; Linde, S.; Sonne, O.; Gliemann, J. Diabetes 1982, 31, 634640. (29) Seabright, P. J.; Smith, G. D. Biochem. J. 1996, 320, 947-956. (30) Schultz, N. M.; Huang, L.; Kennedy, R. T. Anal. Chem. 1995, 67, 924-929. (31) Nithipatikom, K.; McGown, L. B. Talanta 1989, 36, 305-309. (32) Yamguchi, Y.; Hayashi, C.; Miyai, K. Anal. Lett. 1982, 15, 731-737.
This paper describes for the first time the full characterization of all four insulin-FITC conjugates by the following: (1) labeling human insulin with fluorescein isothiocyanate through the primary amines as a function of time, pH, and FITC/insulin ratio; (2) purification of fluorescein-labeled insulins; (3) facile structural characterization of the fluorescein/conjugate by proteolytic peptide mapping via HPLC-electrospray mass spectrometry; (4) demonstrating binding of the different insulin-FITC species to antiinsulin antibody in the development of a fluorescence polarization assay; (5) determining biological activity by an insulin receptor autophosphorylation assay. EXPERIMENTAL SECTION Chemicals. Deionized water was obtained from a Milli-Q UF Plus water purification system (Millipore Corp., Bedford, MA). All working solutions were diluted in 100 mM potassium phosphate, pH 7.4, containing 100 µg/mL acetylated bovine γ-globulin (PanVera Corp., Madison, WI). Zinc-free human insulin was obtained from Lilly Research Laboratories (Indianapolis, IN). Fluorescein isothiocyanate (isomer I) and Staphylococcus aureus strain V8 Type XVII protease and monoclonal mouse anti-human insulin were purchased from Sigma (St. Louis, MO). HPLC-grade acetone and acetonitrile were provided by Mallinckrodt (Paris, KY). Polyclonal guinea pig anti-porcine insulin was obtained from ICN Biochemicals (Costa Mesa, MO). Instrumental Procedures. All HPLC separations were performed with Vydac C18 (5-µm dp) columns: 250 × 4.6 mm and 250 × 10 mm for analytical and preparative runs, respectively. Solvent delivery was accomplished by a dual-pump gradient system (Thermo Separation Products ConstaMetric 3500), where flow rates of 1.00 and 2.50 mL/min were maintained for the 4.6and 10-mm-i.d. columns, respectively. The following gradient was used in all HPLC determinations: 0-15 min (85% to 65% A), 1525 min (65% to 35% A), and 25-32 min (35% A), where A was 0.1% trifluoroacetic acid (TFA) in deionized water and B was 90% acetonitrile/10% deionized water/0.1% TFA. The samples were injected by a Model AS-1007 HPLC automatic sampling system (BioRad, Hercules, CA). For the preparative runs, a 1000-µL injection loop was used, and for analytical runs, a 20-µL injection loop was used. Peaks were monitored by UV absorbance at 220 nm (Applied Biosystems 757 absorbance detector) in tandem with fluorescence detection (Hewlett-Packard 1046A programmable fluorescence detector), where the excitation and emission wavelengths were set at 495 and 520 nm, respectively. The fluorescence spectra of insulin-FITC were determined on a PC1 photon counting spectrofluorometer (ISS, Champaign, IL). The fluorescence polarization experiments were carried out on a Beacon fluorescence polarization system (PanVera Corp.) equipped with fluorescein excitation and emission filters. The sample volume used throughout the assay development was 1000 µL. Electrospray mass spectra were obtained using a Perkin-ElmerSciex API III triple-quadrupole mass spectrometer (Thornhill, ON, Canada) equipped with a pneumatically assisted electrospray source. Samples were infused into the source of the mass spectrometer at a flow rate of 10 µL/min using a syringe pump. Analyses were performed in the positive ion detection mode. Scans were acquired over a range of 900-2100 u in 0.1 u intervals for a dwell time of 1 ms/interval. A total of 5-10 scans were accumulated. Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Preparation of Insulin-Fluorescein Conjugates. Reaction conditions were a modification of Bromer et al.10 and Shimoyama.19 FITC was dissolved in acetone (1 mg in 200 µL) and added dropwise to a 2.0-mL solution containing the appropriate amount of zinc-free human insulin dissolved in either a 100 mM carbonate buffer (pH 8.5) or 100 mM phosphate buffer (pH 7.0). Each buffer contained 200 µM EDTA to prevent aggregation of insulin. Upon addition of FITC, the reaction vials were protected from light and allowed to mix at room temperature via an end-over-end rotator. The reactions were monitored by HPLC with respect to time. To stop the reaction, the mixtures were placed on a gel filtration column (20 × 1.5 cm) with a bed volume of 25 mL containing BioRad P-2 polyacrylamide gel pre-equilibrated with phosphatebuffered saline (PBS), pH 7.1. After the appropriate fractions were collected, the samples were analyzed by HPLC. The appropriate insulin-FITC peaks were then purified by reversed-phase HPLC, lyophilized, and reconstituted with PBS. The insulin-FITC samples were then divided into 20-µL portions and kept frozen until use. Autophosphorylation Assay of Insulin Receptor. Insulin receptors from IM9 cells were purified by wheat germ agglutinin affinity chromatography. The insulin receptors were further immunopurified in situ in an adaptation of an ELISA described by Hagino et al.33 All steps required washing with 20 mM Tris buffer, pH 7.4, containing 0.05% Tween 20 (TBST) 5 times except prior to blocking, where only 3 washes were used. Assays were performed in a total of 100 µL. Briefly, 96-well Nunc Maxisorp plates were coated 16 h at 4 °C with 0.5 µg of anti-HIR (MAB3, Katatech, Inc.) in 100 µL of 50 mM NaHCO3, pH 9.0. Nonspecific binding was blocked with 200 µL of 1% BSA/TBST for 30 min at 56 °C. Then 0.85 ng of WGA-enriched insulin receptor in 100 µL of WGA buffer (50 mM HEPES, pH 7.6 containing 0.1% Triton X-100 and 1 mg/mL Bacitracin) was captured overnight at 4 °C. Unbound material was washed away, and insulin or fluoresceinlabeled insulin was added to the wells followed by a 1-h incubation. Dilutions of insulins were in 50 mM HEPES-buffered saline, pH 7.6, containing 0.1% Triton X-100, 0.05% BSA, 2 mM MnCl2, 10 mM MgCl2, and 10 µM ATP, and the reaction ran 1.5 h at room temperature. After washing, 1 µg/mL horseradish peroxidaseconjugated PY20 (Zymed) in 50 mM HEPES-buffered saline containing 0.05% Tween 20, 1% BSA, 2 mM orthovanadate, and 1 mg/mL Bacitracin was added. After a 1-h incubation at room temperature, the plate was washed again, and 100 µL of TMB substrate (KBL) was added. The plate was read kinetically at 650 nM. Staphylococcus aureus Digestion of Insulin-Fluorescein Conjugates. The different insulin-fluorescein conjugates were separated by HPLC and analyzed by electrospray mass spectrometry to determine the degree of substitution. To locate the positions where the fluoresceins were attached, an S. aureus V-8 Type XVII protease digestion was employed. Each purified conjugate was dissolved in 50 µL of 0.01 M HCl to a concentration of 2 µg/µL. Following the addition of 200 µL of 0.1 M HEPES buffer, pH 7.5, and 40 µL of enzyme (1 mg/mL), the reaction was allowed to incubate for 6 h at room temperature. To quench the digestion, one volume (290 µL) of 1.25 M ammonium sulfate, pH 2.0, was added. The digests were then subjected to LC-MS for (33) Hagino, H.; Shii, K.; Yokono, K.; Matsuba, K.; Yoshida, H.; Hosomi, M.; Okada, Y.; Kishimoto, Y.; Hozumi, M.; Ishida, T. Diabetes 1994, 43, 274280.
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Figure 1. UV chromatogram of a commercially available insulinFITC preparation. The fluorescent peaks (determined by fluorescence detection) are denoted by asterisks.
peptide analysis to determine the change in the fragmentation pattern with respect to unlabeled insulin and identify the position of the fluorescein. Purification of Anti-Insulin Antibodies. Monoclonal antiinsulin (mAb) and polyclonal anti-insulin (pAb) were purified from the respective sera by use of a Protein A affinity purification kit (BioRad). Once the appropriate fractions were collected and pooled in PBS, pH 7.1, the antibody sample was divided into 100µL portions and stored frozen until use. The total IgG protein concentrations for mAb (0.33 mg/mL) and pAb (1.2 mg/mL) were determined by BCA protein analysis (Pierce, Rockford, IL), using mouse IgG as a standard. RESULTS AND DISCUSSION Fluorescein-Labeled Insulin. It has been shown previously that certain residues or regions of residues are important for the binding of insulin to insulin receptors.34,35 Also, it has been observed that commercially available insulin-FITC contains a variety of insulin species (Figure 1). Therefore, to gain a better understanding of the binding between fluorescein-labeled insulin and anti-insulin antibody, only a single species of labeled insulin was sought whether it was mono-, di-, or trisubstituted. To identify the reaction conditions that would selectively label insulin at the A1 (Gly), B1 (Phe), or B29 (Lys) residues (Figure 2), four different reaction conditions were employed: (A) 1:1 at pH 7.0, (B) 3:1 at pH 7.0, (C) 1:1 at pH 8.5, and (D) 3:1 at pH 8.5, where the molar ratios denote FITC/insulin. Since the pKa’s of the two N-terminal R-amino groups are reported as 8.4 and 7.1 for A1 and B1, respectively, and >9.8 for the -amino group (B29),36,37 it was hypothesized that insulin could be selectively labeled at the different positions. The insulin-FITC conjugation reactions were monitored at different time intervals by HPLC over a 20-h period. It was found that in the case of pH 7.0, 3:1 (FITC/insulin) the predominant species of insulin-FITC after 30 min was peak 2 (Figure 3A). Within 4 h, equal amounts of peaks 2 and 3 appeared. Small amounts of other insulin-FITC species began to appear as well (peaks 4 and 5, Figure 3A). For the pH 7.0, 1:1 (FITC/insulin) (34) Scha¨ffer, L. Eur. J. Biochem. 1994, 221, 1127-1132. (35) Pullen, R. A.; Lindsay, D. G.; Wood, S. P.; Tickle, I. J.; Blundell, T. L.; Wollmer, A.; Krail, G.; Brandenburg, D.; Zahn, H.; Gliemann, J.; Gammeltoft, S. Nature 1976, 259, 369-373. (36) Gao, J.; Mrksich, M.; Gomez, F. A.; Whitesides, G. M. Anal. Chem. 1995, 67, 3093-3100. (37) Hefford, M. A.; Oda, G.; Kaplan, H. Biochem. J. 1986, 237, 663-668.
Figure 2. Primary structure of human insulin. Arrows indicate reactive primary amine sites for FITC conjugation. Regions I, II, III, and IV are the different fragments obtained after digestion by S. aureus V-8 protease. The solid lines represent the disulfide linkage between the appropriate cysteine residues.
reaction the predominant insulin-FITC species after 20 h appeared as peak 3; virtually no other insulin-FITC species were identified (Figure 3B). The pH 8.5 reaction conditions yielded a similar pattern except that the reactions occurred much faster and there were larger amounts of peaks 4 and 5 formed by the end of 20 h (Figure 3C). Since the purpose of this report was to examine all four insulin-FITC species, the 3:1 reaction carried out at pH 8.5 for 20 h was chosen for further characterization (i.e., it had the greatest number of insulin-FITC species). The fluorescein substitution for peaks 1-5 (Figure 3C) was determined by electrospray mass spectrometry. It was confirmed that peak 1 was indeed unlabeled insulin, while peaks 2 and 3 were monosubstituted, peak 4 was disubstituted, and peak 5 was trisubstituted (Table 1). Each of peaks 2-5 were then subjected to S. aureus V-8 proteolysis to determine the positional substitution of fluorescein in the insulin molecule (Figure 2). It was expected that any proteolytic fragment with a fluorescein attached to it should result in a shift in retention time compared to the respective unlabeled fragment. Although the peaks could probably have been identified by comparison of retention times (Table 2), unambiguous identification was needed due to the different chromatographic conditions from that reported in literature.38 By HPLC-MS peptide mapping, it was confirmed that peak 2 (Figure 3C) was labeled at A1, peak 3 was labeled at B1, peak 4 was labeled at A1 and B1, and peak 5 was labeled at A1, B1, and B29 (Table 3). After characterization of the insulin-fluorescein conjugates, the rest of the pH 8.5 reaction mixture was purified by semipreparative HPLC. The fractions were collected, pooled, lyophilized, and reconstituted with PBS, pH 7.1. BCA protein analysis was carried out to determine the concentrations of the respective insulin-FITC conjugates. Binding Studies. To determine the incubation time needed for binding equilibrium to take place between antibody and insulin-FITC, concentrations of 1 nM mAb and 250 pM insulinFITC (B1 conjugate) were chosen to pursue the kinetics study. The binding was monitored over time for each of the four different insulin-FITC conjugates until the fluorescence polarization signal did not change. As shown in Figure 4, 15-20 min is required for (38) Darrington, R. T.; Anderson, B. D. J. Pharm. Sci. 1995, 84, 275-282.
Figure 3. UV chromatograms of (A) pH 7.0, 1:1 (FITC/insulin) after 30 min, 4 h, and 20 h reaction times; (B) pH 7.0, 3:1 (FITC/insulin) after 20 h. (C) UV chromatogram of the insulin-FITC reaction at pH 8.5, 3:1 (FITC/insulin). The fluorescent peaks (determined by fluorescence detection) are denoted by asterisks. The numbered peaks in (C) were collected and subjected to electrospray mass spectrometry followed by proteolytic peptide mapping. Table 1. Molecular Mass of Insulin-Fluorescein Species As Determined by Electrospray Mass Spectrometry after Separation by Reversed-Phase HPLC no. of fluoresceins
calcd mass (amu)
0 1 2 3
5807.6 6197.4 6586.4 6975.8
1
2
peak no. 3
6196.8
6196.9
4
5
5807.6 6586.6 6976.1
maximum binding with both mAb and pAb. Therefore, in all further experiments, an incubation time of 15 min was chosen. Using all four insulin-FITC conjugates a binding-saturation experiment was performed to determine the amount of mAb to use throughout the competition experiments. With the concentrations of each conjugate (denoted A1, B1, A1B1, and A1B1B29) held constant at 250 pM (the lowest concentration that yielded Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Table 2. HPLC Retention Times (min) for the Insulin and Insulin-FITC Fragments after S. aureus V-8 Protease Digestion of the HPLC-Purified Peaks from Figure 3C
proteolytic fragment A(1-4) B(22-30) A(18-21)-B(14-21) A(5-17)-B(1-13) A(1-4)b B(22-30)b A(5-17)-B(1-13)b
insulin standard 5.9 13.6 14.1 18.7
peak no.a 3 4
2
5.9 13.7 14.2
13.7 14.1 18.8 21.7
5
13.6 14.1 21.7
23.2
23.2
14.2 21.7 21.5 23.3
a Peak 2 is monosubstituted; peak 3 is monosubstituted; peak 4 is disubstituted; peak 5 is trisubstituted. b Fluorescein-labeled fragments.
Table 3. Molecular Mass of Insulin and Insulin-FITC Fragments after S. aureus V-8 Protease Digestion As Determined by LC-MS (Electrospray)
proteolytic fragment A(1-4) B(22-30) A(18-21)-B(14-21) A(5-17)-B(1-13) A(1-4)a B(22-30)a A(5-17)-B(1-13)a a
calcd mass insulin (amu) standard 416.5 1116.3 1377.5 2969.4 805.9 1505.7 3358.8
417.3 1116.2 1377.4 2969.3
peak nos. 2
3
4
5
417.2 1116.0 1116.3 1115.9 1377.3 1377.2 1377.6 1377.1 2969.2 806.3 806.4 806.4 1505.4 3359.1 3359.3 3358.9
Fluorescein-labeled fragments. Figure 5. Binding saturation plot to determine the optimum amount of (A) monoclonal and (B) polyclonal anti-insulin antibody to use for the competition experiments. The concentration of each insulin-FITC conjugate was 250 pM.
Figure 4. Incubation time for insulin-FITC (250 pM B1 insulinFITC conjugate) with mAb (1 nM) and pAb (84 nM).
reproducible fluorescence polarization signals), different amounts of mAb were added to the cuvettes containing labeled insulin. As shown in Figure 5A, the binding saturation occurs at ∼2 nM IgG for mAb. Therefore, an amount of mAb corresponding to 1 nM was used for further competition experiments. This value translates to the minimum amount of mAb required that yields the greatest change in fluorescence polarization. Similarly, an amount corresponding to 84 nM IgG for pAb was chosen as the optimum Ab concentration to pursue the competition studies (Figure 5B). Human insulin was then used to compete with the fluoresceinlabeled human insulin conjugates toward both mAb and pAb. A range of insulin concentrations (10 pM to 1 µM) was examined. 4998 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
The insulin and insulin-FITC were mixed in the cuvette prior to the addition of antibody (mAb or pAb). In both cases, the concentration of labeled insulin was held constant at 250 pM. Figure 6A shows the results of the competition experiments for all four conjugates using mAb. A similar trend can be observed for the pAb competition experiment (Figure 6B). The order in sensitivities between the different insulin-FITC conjugates for both mAb and pAb are as follows: B1 > A1 > A1B1 > A1B1B29. The IC50’s, calculated by a four-parameter nonlinear regression program written in-house (RIASYS, Eli Lilly, Indianapolis, IN), are shown in Table 4. Note that the IC50’s obtained with the pAb are almost 1 order of magnitude lower than those for the mAb. This can be attributed to the fact that the polyclonal antibody preparation contains antibodies with a range of specificities toward insulin, whereas the monoclonal antibody binds to a single epitope on the insulin molecule. Since the pAb preparation contains a greater range of specificities, it was expected to yield different binding affinities toward the insulin-FITC conjugates. Note that the IC50 calculated for the trilabeled insulin is not very reliable since the change in fluorescence polarization (bound vs unbound) is very small (Figure 6). These data can also provide some additional information about the insulin-FITC structural conformation as well. The fluorescence polarization for the unbound monosubstituted insulin-FITC (e.g., at high insulin concentrations, Figure 6) is ∼170 mP, while that for the multisubstituted species is ∼110 mP. Theoretical
Table 5. Biological Activity (Expressed as EC50) of the Four Insulin-FITC and Unlabeled Insulin Species As Determined by an Autophosphorylation Assay Using WGA-Purified Human Insulin Receptor
Figure 6. Binding competition between insulin and each of four fluorescein-labeled conjugates using (A) mAb and (B) pAb as the binder. In each case, 250 pM insulin-FITC was mixed with unlabeled human insulin before a 15-min incubation with the appropriate antibody. Table 4. Comparison of Analytical Characteristics Determined from the Insulin-FITC Conjugates and pAb and mAb Antibodies detectable amt, nM
polyclonal Ab A1 B1 A1B1 A1B1B29 monoclonal Ab A1 B1 A1B1 A1B1B29
IC50, nM
min
max
4.41 5.86 5.79 2.2
0.92 0.64 1.21 1.22
21.8 52.6 26.8 4.2
1.14 2.41 3.62 7.01
309.5 306.2 649.3 30.4
21.4 31.3 58.5 15.7
predictions indicate that the molecules should all have the same fluorescence polarization if they are identical (e.g., the same rotational correlation and molecular volume).39 If the conformation of the multisubstituted insulin-FITC molecules changed such that the FITC moieties were further from the insulin molecule, then the FITC moiety would be able to rotate more freely than in the monosubstituted case. This would, in turn, reduce the FP signal. The difference in FP signal may also be attributed to the (39) Lackowitz, J. R. In Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983; pp 112-151.
insulin species
EC50, nM
A1 B1 A1B1 A1B1B29 human insulin pork insulin
13.7 1.11 29.9 558 1.43 1.42
differences in the microenvironment around the FITC, thus altering fluorescence lifetime, rotational correlation time, or quenching. This paper also describes the determination of the biological activity of the insulin-FITC conjugates. The four insulin-FITC species, and unlabeled human insulin from the same lot, were all subjected to an autophosphorylation assay using WGA-purified human insulin receptor that was further purified by antibody capture on an ELISA plate. As indicated in Table 5, the activity of the insulin-FITC species varies with respect to the position and degree of substitution. The B1 conjugate was found to be the most biologically active species, approximately equivalent to the unlabeled human and pork insulin standards. This indicates that the B1 position is not significantly involved in the binding to the receptor. The A1 and A1B1 insulin-FITC molecules show a ∼10-fold decrease in biological activity suggesting that the A1 site plays a role in binding toward the insulin receptor. In addition, the trisubstituted insulin-FITC species, A1B1B29, shows almost a 100-fold decrease in biological activity. This may be a result of the B29 position playing an important role in receptor binding, or the conformation of the trisubstituted insulin may be altered such that the receptor binding region on the insulin molecule is no longer recognizable. These data agree with previous work by Schaffer34 and Pullen et al.,35 where a range of insulin residues were either chemically modified or altered by point mutation. In both cases, the A1 position was found to play an important role in the binding of insulin to its receptor.34,35 The FITC label at residue B29 also seems to contribute to the demise of the binding toward the receptor, which is consistent with data reported by Pullen et al.35 In this case, the B29 residue is near the secondary binding site of insulin toward the insulin receptor. CONCLUSION Four different insulin-FITC conjugates were prepared, separated, and fully characterized, both structurally and biologically. Reaction conditions were developed such that the insulin could be modified with FITC at the A1, B1, or A1B1 positions almost selectively by altering the pH, time, and molar ratio of FITC to insulin. All four species were separated from each other by reversed-phase HPLC. In characterizing the insulin-FITC conjugates, it was shown that the degree and position of substitution can dramatically effect the structural conformation and thus the biological activity of a fluorophore-labeled protein. We have shown that insulin labeled with FITC at the A1 or B1 position exhibits similar binding toward anti-insulin antibodies. However, the sensitivity of such the assay described in this paper was found to be highly dependent on the type of antibody. On the other hand, the A1 and B1 insulin-FITC conjugates were found to dramatically effect the biological activity. Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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For the first time, the biological activity of all four insulinFITC derivatives have been measured by a tyrosine kinase phosphorylation assay. The B1 derivative was shown to be fully active as expected from the residues involved in receptor binding. This demonstrates that the B1 derivative can be as effective in biological measurements as radiolabeled insulin provided the fluoresceinated insulin is appropriately purified and characterized. Based on these studies, work is currently underway in our laboratory to synthesize and characterize other fluorescently labeled proteins and peptides which retain full biological activity. Work is also currently underway in our laboratory toward the development of a fluorescence polarization assay for insulin using insulin receptors as well as the development of a fluorescence polarization-based HTS using insulin-FITC.
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ACKNOWLEDGMENT We thank David Sharknas (Eli Lilly and Co.) for helpful suggestions regarding the S. aureus V-8 protease digestion for the insulin-FITC peptide mapping, and Steven Kahl (Eli Lilly and Co.) for helpful discussions regarding the preparation of the manuscript.
Received for review July 7, 1997. Accepted October 7, 1997.X AC970726M
X
Abstract published in Advance ACS Abstracts, November 15, 1997.
