Anal. Chem. 1999, 71, 384-387
Characterization of Allosteric Insulin Hexamers by Electrospray Ionization Mass Spectrometry D. Fabris* and C. Fenselau†
Structural Biochemistry Center, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250
Electrospray mass spectrometry is used at pH 8.0 in combination with accurate mass measurements to confirm the multiplicity of insulin in stable noncovalent complexes with Zn(II) ions. Determination of the nature and number of ligands involved in Zn(II) chelation is in agreement with crystal and solution structures. Counting the number of ligands participating in each center allows deduction of the geometric configuration of the ligand field and gives indirect information about the conformational state of the insulin monomers in solution. Since its discovery in 1921, insulin has been the object of intense studies aimed at elucidating its synthesis, storage, transport, and receptor interactions. The biologically active form of insulin is the monomer, but hexameric insulin binding two Zn(II) ions is produced and stored in the β-cells of the pancreas before release into the bloodstream. X-ray crystallography1-4 and other spectroscopic techniques5-8 have described the conformational properties of these noncovalent complexes in crystals and in solution, showing that insulin hexamer is an allosteric protein complex capable of adopting three distinct conformations designated as T6, T3R3, and R6 (Figure 1). In each subunit, the N-terminal residues (B1-B9) of the B-chain may assume an extended (T state) or a helical conformation (R state). This conformational change is forced by the binding of phenolic ligands to six hydrophobic pockets. In the T state, Zn(II) ions assume an octahedral geometry requiring three His (B10) residues and three water molecules in each coordination center. In the R state, the conformational change forces Zn(II) ions to adopt a tetrahedral coordination requiring three His (B10) and a single anion or smallmolecule ligand. Hexamers are assembled from three dimers, † Present address: Department of Chemistry and Biochemistry, University of Maryland College Park, College Park, MD 20742. (1) Adams, M. J.; Baker, E. N.; Blundell, T. L.; Harding, M. M.; Dodson, E. J.; Hodgkin, D. C.; Dodson, G. G.; Rimmer, B.; Vijayan, M.; Sheat, S. Nature 1969, 224, 491-495. (2) Bentley, G. A.; Dodson, E. J.; Dodson, G. G.; Hodgkin, D.; Mercola, D. Nature 1976, 261, 166-168. (3) Derewenda, U.; Derewenda, Z.; Dodson, E. J.; Dodson, G. G.; Reynolds, C. D.; Smith, G. D.; Sparks, C.; Swenson, D. Nature 1989, 338, 594-596. (4) Smith, G. D.; Swenson, D. C.; Dodson, E. J.; Dodson, G. G.; Reynolds, C. D. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 7093-7097. (5) Kaarsholm, N. C.; Ko, H.-C.; Dunn, M. F. Biochemistry 1989, 28, 44274435. (6) Brader, M. L.; Dunn, M. F. Trends Biochem. Sci. 1991, 16, 341-345. (7) Brader, M. L.; Kaarsholm, N. C.; Lee, R. W.-K.; Dunn, M. F. Biochemistry 1991, 30, 6636-6645. (8) Choi, W. E.; Borchardt, D.; Kaarsholm, N. C.; Brzovic, P. S.; Dunn, M. F. Proteins 1996, 26, 377-390.
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Figure 1. (a) Schematic depiction of Zn(II) ion chelation by insulin in the T state and in the R state and (b) allosteric equilibria between the different hexameric forms, T6, T3R3, and R6 (adapted from Brader et al.6).
which can be symmetric (dimeric T or dimeric R) or asymmetric (mixed T and R dimers), and are related by an exact 3-fold axis of symmetry, with the two Zn(II) ions located on the 3-fold axis. The factors affecting the conformational change are of paramount importance for the design of pharmaceutical preparations for diabetes. For example, common preparations for subcutaneous injection are produced with addition of phenolic preservatives9 and contain R6 zinc/insulin crystals.3 The “4-zinc” insulin,4 also described clinically as “slow-acting” because it is slow to dissolve and enter the blood stream, has been identified with the T3R3 hexamer.5 Little is known about the relationship between the conformation of the hexameric complex and the conformation of released monomeric insulin. Insulin monomer has played an important role in the development of ionization techniques for mass spectrometry of large molecules, providing a benchmark for the development of plasma desorption (PD),10-12 fast atom bombardment (FAB),13-15 and electrospray ionization (ESI).16-18 Electrospray at near neutral pH (9) Brange, J. Galenic of Insulin; Springer-Verlag: Berlin, 1987. (10) Sundqvist, B. U. R.; Macfarlane, R. Mass Spectrom. Rev. 1985, 5, 421. (11) Demirev, P.; Fenselau, C.; Cotter, R. J. Int. J. Mass Spectrom. Ion Processes 1987, 78, 251-258. (12) Unger, S. E.; Brange, J.; Lauritano, A.; Demirev, P.; Wang, R.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1988, 2, 109-112. (13) Barber, M.; Bordoli, R. S.; Elliott, G. J.; Tyler, A. N.; Bill, J. C.; Green B. N. Biomed. Mass Spectrom. 1984, 4, 182-186. (14) Fenselau, C.; Yergey, J.; Heller, D. Int. J. Mass Spectrom. Ion Phys. 1983, 53, 5-20. (15) Matsuo, T. Mass Spectrom. Rev. 1989, 8, 203-236. (16) Meng, C. K.; Mann, M.; Fenn, J. B. Z. Phys. D 1988, 10, 361-368. (17) Loo, J. A.; Udseth, H. R.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1986, 17, 411-414. 10.1021/ac980753s CCC: $18.00
© 1999 American Chemical Society Published on Web 12/10/1998
offers an exceptionally gentle desorption technique, which enables direct observation of intact noncovalent complexes after desorption into the gas phase.19 In this work, the capability of electrospray mass spectrometry is tested for characterization of different insulin hexamers produced in solution by incubating insulin monomer with Zn(II) ions and various nonprotein ligands. Accurate mass measurements are shown to provide complete identification of the ligands and characterize the stoichiometry of the allosteric complexes, thereby providing indirect information on the coordination geometry of zinc and the conformational state of the insulin monomers in the complexes. EXPERIMENTAL SECTION Chemicals and Sample Preparation. Bovine insulin was purchased as the sodium salt (Boehringer Mannheim Co., Indianapolis, IN), dissolved in 10 mM ammonium acetate (pH 8.0), and desalted by ultrafiltration on an Ultrafree-0.5 centrifugal filter (Millipore Co., Bedford, MA) with a 5000 Da molecular weight cutoff. Concentration was measured by UV spectrometry (6010 molar absorbtivity at 276 nm). A solution of ZnCl2 (atomic adsorption grade, Sigma Chemical Co., St. Louis, MO) was also prepared in 10 mM ammonium acetate (pH 8.0). Aliquots were added to desalted insulin in a 1:2 molar ratio, which provides a slight excess of Zn(II) ions to insulin monomer over the 1:3 ratio desired in the final hexameric species. After overnight incubation at room temperature, the solution was desalted 3 times by ultrafiltration to remove the slight excess of Zn(II) ions. This stock solution was stored at -20 °C. Prior to ESI-MS analysis, the solution was diluted with ammonium acetate buffer, pH 8.0, to obtain a final concentration of 120 µM insulin monomer (or about 20 µM insulin hexamer). Different conformational states of the allosteric complex were obtained by treating the stock solution of insulin and Zn(II) with phenol (Sigma Chemical Co., St. Louis, MO) in a 1:50 or a 1:200 molar ratio (insulin monomer to phenol) and then adding appropriate amounts of ammonium chloride, ammonium iodide, or other ligands, as suggested by Dunn.6-8 Solutions were incubated at least 15 min at room temperature. Storage overnight at 4 °C did not produce noticeable spectral variations. After incubation, solutions were diluted with ammonium acetate buffer, pH 8.0, to give a final concentration of 120 µM (insulin monomer) for ESI-MS analysis. The final pH was adjusted to 8.0 by adding small volumes of 0.1 M acetic acid or 2% ammonium hydroxide. Standards for accurate mass measurements, R-chymotrypsinogen from bovine pancreas (Worthington Biochemical Co., Freehold, NJ) and carbonic anhydrase II from bovine erythrocytes (Sigma Chemical Co., St. Louis, MO), were of the highest available grade and were dissolved directly, without further purification, in 10 mM ammonium acetate (pH 8.0) to final concentrations of 50 µM. Mass Spectrometry. All experiments were performed on a JEOL (Tokyo, Japan) HX110/HX110 four-sector mass spectrometer equipped with an Analytica of Branford (Branford, CT) thermally assisted electrospray source. Each spectrum was the averaged profile of 20-50 linear scans, with a duty cycle of 15(18) Smith, R. D.; Loo, J. A.; Ogorzalek-Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (19) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23.
Figure 2. Electrospray mass spectrum of desalted bovine insulin without addition of ZnCl2.
20 s each. Resolution was set to 500 (10% valley) by adjusting the slit widths. For high mass accuracy, the external standard method was employed in which analyte and standards are introduced into the source by consecutive injections using a loop injection system without mixing and without stopping the data acquisition. For this study, different charge states of R-chymotrypsinogen (25 666.1 Da) were used to bracket the analyte peaks and mass assignments were obtained by direct manual interpolation. The threshold was raised above the asymmetric baseline before each peak of interest was centroided. Control experiments with holocarbonic anhydrase II20,21 gave an observed mass of 29109.8 ( 3.7 Da, which is in good agreement with 29 107.8 Da, the average molecular weight calculated from the protein sequence chelating one Zn(II) ion and one molecule of water. Apocarbonic anhydrase was also used as a control. The accuracy of this measurement was 0.01%. To ensure optimal spray stability, solutions of analyte at pH 8.0 and methanol sheath solvent were delivered by separate syringe pumps at a 1 µL/min flow rate. Aliquots of 10 µL were introduced into the source through a loop injection system. RESULTS AND DISCUSSION The electrospray ionization mass spectrum of a 120 µM solution of desalted insulin, pH 8.0, is shown in Figure 2. The analysis was performed near neutral (native) conditions to preserve the noncovalent associations, as described in the Experimental Section. To evaluate the tendency of insulin monomer to aggregate in solution, desalted insulin was analyzed without any addition of zinc. At least three species were detected in addition to the monomer, with observed molecular masses corresponding to the tetramer, hexamer, and decamer of insulin with no trace of metals or other ligands. The wide scan range used for this experiment provided detection of several charge states for each species, allowing for unambiguous identification of the different aggregates. Figure 3 presents the electrospray mass spectrum of the solution obtained after incubation of insulin monomer with ZnCl2. Only one major species was observed with significant intensity and a range of charge states. The accurate mass measurement produced a mass corresponding to a hexamer containing six insulin monomer units, two Zn(II) ions, and six molecules of water, i.e., Ins6Zn2(H2O)6. Table 1 provides a summary of accurate mass (20) Fenselau, C.; He, T.; Antoine, M.; Hathout, Y.; Fabris, D. A chemical mechanism for inactivation of anticancer drugs by Metallothionein. Presented at the 4th International Metallothionein Meeting, Kansas City, MO, September 17-20, 1997. (21) Zaia, J.; Fabris, D.; Wei, D.; Karpel, R.; Fenselau, C. Protein Sci. 1998, 7, 2398-2404.
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Figure 3. Electrospray mass spectrum of desalted bovine insulin after addition of ZnCl2 and overnight incubation.
Figure 5. Electrospray mass spectrum of the stock solution of bovine insulin and Zn(II) after treatment with phenol in a 1:200 molar ratio.
Table 1. Accurate Mass Measurements of Insulin Hexamers mass (Da) composition
proposed conformation
calcda
Ins6Zn2(H2O)6 Ins6Zn2Ph(H2O)3 Ins6Zn2Ph2 Ins6Zn2PhCl Ins6Zn2I2
T6 T3R3 R6 R6 R6
34 640.4 34 680.4 34 720.4 34 661.9 34 786.2
obsdb 34 641.0 ( 6.5 34 680.1 ( 1.1 34 722.2 ( 22.9 34 663.5 ( 17.2 34 775.8 ( 3.4
a Based on an average molecular mass of 5733.6 Da for bovine insulin. b R-Chymotrypsinogen (25 666.1 Da) was used as external standard. Standard deviations are calculated from mass assignments obtained from three different charge states.
Figure 4. Electrospray mass spectrum of an aliquot of the stock solution of bovine insulin and Zn(II) after treatment with phenol in a 1:50 molar ratio.
measurements for the different allosteric complexes compared to the average molecular weights calculated from the protein sequence and the masses of zinc and different ligands. It is important to note how the presence of Zn(II) ions forces insulin to coordinate with a highly specific stoichiometry, as compared to the more diverse aggregation stoichiometries observed when zinc is totally absent (Figure 2). The comparison between these spectra, acquired under the same instrumental conditions, appears to remove the possibility of artifactual effects from the ionization technique on the coordination stoichiometry, which appears to be determined by the composition of the solution following the chelation rules inferred from other techniques.6-8 In this case, the identity of the ligands and the stoichiometry of the hexameric complex observed in Figure 3 is in good agreement with an octahedric coordination geometry, with a conformational state corresponding to T6. The spectrum obtained from the stock solution of insulin and Zn(II) after treatment with phenol in a 1:50 molar ratio (insulin monomer to phenol) is presented in Figure 4. As for the previous sample, only one major species was detected in the presence of zinc. The accurate mass measurement matches that for a complex including six insulin monomers, two zinc ions, one phenol, and 386 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
three molecules of water, i.e., Ins6Zn2Ph(H2O)3. This stoichiometry would correspond to a hexamer containing one octahedric and one tetrahedric center, described as T3R3. The possibility of a 1:1 mixture in solution between Ins6Zn2(H2O)6 and Ins6Zn2Ph2 is excluded by the accurate mass assignment (Table 1) and by the symmetric and narrow shape of the peaks. Figure 5 shows the electrospray ionization mass spectrum of the stock solution of insulin and Zn(II) treated with phenol in a 1:200 molar ratio (insulin monomer to phenol). The accurate mass corresponds to a hexamer containing six insulin monomers, two zinc ions, and two phenol molecules, i.e., Ins6Zn2Ph2, suggesting that both metal centers have assumed a tetrahedric coordination geometry, defined as R6. Accurate mass measurements for insulin hexamers formed with different anionic ligands are included in Table 1. They provide stoichiometry and ligand composition and predict conformations consistent with the previously defined principles.6-8 Control injections of holocarbonic anhydrase II20,21 performed just before each insulin experiment consistently produced an accuracy of (3.7 Da (0.01%) or better, as described in the Experimental Section. The wider standard deviations reported for some of the insulin measurements reflect very closely the intrinsic difficulty of spraying solutions of very high ionic strength, i.e., spray instability and signal suppression. From these measurements, it was not possible to determine whether phenol or phenoxy ion is involved in zinc chelation. The pKa of phenol is 10.0; however, this may be lowered in the presence of metal ions by stabilization of the conjugate anion, allowing phenoxy ions to be involved in the ligand field, in analogy with other anionic ligands like I-, Cl-, and SCN-.5-8 The transition between the T and the R states is thought to be mediated by binding of phenol to six hydrophobic pockets, which induces a conformational change in the N-terminal portion of the insulin B-chain.5-8 Hydrophobic interactions acquire stabilization energy when water is removed from the region were the interaction occurs. The survival of hydrophobic interactions in a completely anhydrous environment and their role in stabilizing protein conformations in the gas phase are under intense study.22,24 In this work, binding of phenol to the six hydrophobic pockets was not detected in the gas phase, as indicated by the observed molecular masses. The preservation in the gas phase of the correct stoichiometry for the different allosteric forms of insulin hexamers (22) Wolynes, P. G. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2426-2427. (23) Kaltashov, I.; Fenselau, C. Proteins 1997, I27, 165-170. (24) McLafferty, F. W.; Guan, Z.; Haupts, U.; Wood, T. D.; Kelleher, N. L. J. Am. Chem. Soc. 1998, 120, 4732-4740.
suggests that the conformational change causing the T-R transition (extended versus helical) produces stable conformers, which is in agreement with the observation of stable elements of secondary structure in the gas phase.23,24 CONCLUSIONS On the basis of earlier X-ray crystallographic and spectroscopic work, the nature of zinc-insulin chelates reveals the allosteric conformation of insulin hexamers in solution. Thus, electrospray ionization mass spectrometry provides a rapid and relatively sensitive method to characterize the presence, the ligand com-
position, and even the allosteric conformation of hexamers formed in solutions of insulin. ACKNOWLEDGMENT The authors thank Dr. J. Zaia for helpful discussions. This work was supported by NIH Grant GM 21248.
Received for review July 10, 1998. Accepted October 29, 1998. AC980753S
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