Bioconlugete Chem. 1992, 3, 291-294
?gl
Synthesis and Characterization of Singly Modified (Carboxyferroceny1)cytochrome c Derivatives Antonius C. F. Gorren,+Man Ling Chan,Brian R. Crouse, and Robert A. Scott’ Departments of Chemistry and Biochemistry and the Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602. Received February 5, 1992 Carbodiimide-activated coupling chemistry has been used to covalently attach 1,l’-dicarboxyferrocene (dcFc) to the e-amine of surface lysine residues of horse heart cytochrome c. Conditions have been found that optimize the production of singly modified (dcFc)cytochrome c derivatives and the presence of one free carboxylate per modification site allows separation and purification of about 10 of these derivatives by cation-exchange chromatography. Reversed-phase HPLC tryptic peptide mapping techniques have been used to identify the attachment sites of eight pure (dcFc)cytochrome c derivatives (at lysines 7,8,13,25,60,72,73, and 100). Through-space distances from these lysines to the nearest heme edge span the 6-16 A range and these derivatives should prove useful in exploring the distance dependence of long-range intramolecular electron transfer in cytochrome c.
INTRODUCTION
In recent years much research has been devoted to the fundamental mechanism of long-range biological electron transfer and to the dependence of the electron transfer rate on parameters like driving force or distance of separation of acceptor and donor sites (1-5). One method for varying the donor-acceptor distance in proteins is to attach a second redox center to amino acid side chains a t different locations on the surface of a single-site redox protein. This approach was originally taken by Gray and Isied and their co-workers, who coupled Ru(NH&, to His33 of horse heart cytochrome c (6-8). Similar derivatives have since been obtained for a number of other proteins as well (9-18). Alternatively, other metal complexes can be attached to different surface groups of cytochrome c. For example, recent efforts in this laboratory have generated cytochrome c derivatives with the cobalt cage complex Co(diAMsar)l coupled to the carboxylates of several aspartic and glutamic acid residues (19,20). The presence of 19 lysine residues on horse heart cytochrome c suggests that covalent attachment strategies directed a t lysine e-amines may result in numerous derivatives with a range of probeheme distances. Millett and co-workersgenerated a number of such derivatives with [Ru(bpy)~(dcbpy)l (bpy = 2,2’-bipyridine; Hzdcbpy = 4,4‘-dicarboxy-2,2‘-bipyridine)as the lysine-attached moiety (21,22). We have recently attached [Ru(NH& in]Z+ (inH = 4-carboxypyridine, isonicotinic acid) to cytochrome c lysines (20). As part of our continuing effort to generate series of probe-labeled cytochrome c derivatives with which to study distance effects of biological electron transfer, we have
* Address all correspondence to Robert A. Scott, Department of Chemistry, University of Georgia, Cedar Drive, Athens, GA 30602-2556. + Present address: Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, the Netherlands. Abbreviationsused bpy, 2,2’-bipyridine;Co(diAMsar), [1,8diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosanyl]cobalt dcbpy2-,4,4’-dicarboxy2,2’-bipyridine; dcFc, 1,l’-dicarboxyferrocene; DPV, differential pulse voltammetry; EDC, l-ethyl-3[3-(dimethylamino)propyl]carbodiimide;ICP/AES, inductively coupled plasma/atomic emission spectrometry; in-, isonicotinate (4-carboxylatopyridine);NHE, normal hydrogen electrode.
used a ferrocene derivative as a lysine-modifying reagent. Heller and co-workers have demonstrated that l-ethyl3-[3-(dimethylamino)propyllcarbodiimide(EDC) will activate the carboxyl group of carboxyferrocene toward nucleophilic attack by surface lysine e-amines of glucose oxidase, generating derivatives with about 12 ferrocenes per protein molecule (23). We have chosen to use 1,l’dicarboxyferrocene (dcFc) in a similar reaction to generate singly labeled cytochrome c derivatives. The presence of the negative charge from the second carboxylate allows the separation of a number of these (dcFc)cytochrome c derivatives from native cytochrome c and from one another. EXPERIMENTAL PROCEDURES
Cytochrome c (Sigma type VI) was purified according to a literature method (24) and 1,l’-dicarboxyferrocene was synthesized according to literature methods (25-27). Anal. Calcd for ClpHloO4Fe: C, 52.59; H, 3.68. Found: C, 52.58; H, 3.71. Coupling Reaction. 1,l’-Dicarboxyferrocene (dcFc) (3 mmol) was suspended in 9 mL water and the pH increased with concentrated NaOH to a value between 7.5 and 8.0, a t which stage the dcFc dissolved. A cytochrome c solution (15 pmol in 6 mL of 100 mM potassium phosphate (KPi) buffer, pH 7.0) was then added and the pH adjusted to 7.5. All subsequent steps were carried out in the dark. The reaction was started by the addition of solid EDC (0.3 mmol) and the mixture was stirred for 1 h at room temperature, followed by exhaustive ultrafiltration (Amicon YM 5 membrane). Separationand Purification of (dcFc)cytochromes c. A crude separation was achieved on a 2.5 X 70-cm column of S-SepharoseFast Flow resin (PharmacidLKB), using a pH 8.0 elution buffer containing 4 mM NaPi and 1mM ascorbic acid and a linear gradient running from 0 to 200 mM NaCl in 1 L. Further purification of the derivatives was accomplished by FPLC on a HR 5/5 Mono S column with the same elution buffer and gradients in the range between 0 and 100 mM NaC1. Methods. Absorbance spectra were measured on a Cary 219 UV/visiblespectrophotometer. Heme c concentrations of cytochrome c solutions were measured using e410 = 1.05 X lo5M-’ cm-l(28). Electrochemical measurements were performed by differential pulse voltammetry (DPV) using an EG&G Princeton Applied Research Model 264A polarographic analyzer/stripping voltammeter (connected to 0 1992 American Chemical Society
292
Bloconjugate Chem., Vol. 3, No. 4, 1992
Scheme I
Gwen et al. I
I
I
I
I
60
8
m 40 T dcFc
-
/
20 0 80
a MFE 815 M Plotamatic XY-recorder) with a Au working electrode, a Ag/AgCl reference electrode (both from BioAnalytical Systems),and a Pt wire as the counter electrode. All electrochemical measurements were carried out in the presence of 100 mM NaC1,50 mM NaPj (pH 8.0), and 10 mM 4,4'-bipyridine. ICP/AES metal analyses were performed by the Institute of Ecology, University of Georgia. Tryptic digests and reversed-phase HPLC peptide maps were obtained as previously described (29)using a Waters 600E HPLC with a Delta-Pak CIScolumn (300 A, 5 pm; 3.9 mm X 15.0 cm). Tryptic peptide sequencing was performed by the Molecular Genetics Instrumentation Facility, University of Georgia. Through-space distances from modification sites (lysine e-amine nitrogens) to the nearest porphyrin ring carbon of the heme (and their standard deviations) were estimated by time-averaging such distances from structures calculated at each ps of a 100-psmolecular dynamics simulation of solvated horse heart cytochrome c (30)using the Amber 3 software package (31). These computations will be described in detail elsewhere (Conrad, D. W., Zhang, H., Stewart, D., Scott, R. A,, manuscript in preparation).
30
40
50
.e-
u L--L--L---'o 20 30 40 30 40 50
-10
20
30
-0 20 30 40 50 20 Elution Volume (mL)
30
40
50
50
RESULTS
Figure 1. Cation-exchangeseparation of the (dcFc)cytochrome c derivatives. (a) Separation of the crude reaction mixture on a Pharmacia LKB FPLC HR 5/5 Mono-S column. In all cases, buffer A is 4 mM NaPi, pH 8.0, with 1 mM ascorbic acid, and buffer B is buffer A plus 100 mM NaCl. All flow rates were 1.0 mL min-1. Rechromatographic purifications of subfractions under the same conditions are shown for fractions 2a (b), 2b (c), 3 (d), 4 (e), 5b (0, and 5c (g).
Coupling Reaction and Product Separation. Reaction of cytochrome c with dcFc in the presence of EDC (Scheme I) resulted in a highly reproducible mixture of protein products that are separable by cation-exchange chromatography. Figure l a shows the FPLC Mono-S elution pattern for a typical product mixture. Rechromatography on FPLC Mono-S (Figure lb-g) allowed separation of a number of subfractions, suggesting the formation of at least 13 distinct (dcFc)cytochrome c derivatives. ICP/ AES determination of total iron and spectrophotometric determination of heme c concentration demonstrated that fractions 2-6 each had a total iron to heme ratio of about 2, indicative of the presence of one dcFc moiety per cytochrome c molecule. Fraction 7 (Fe:heme, 1:l)was the unmodified protein, whereas fraction 1 (not shown; Fe: heme, 12:l) eluted before the start of the gradient and consisted of a mixture of free dcFc and cytochrome c that had incorporated two or more ferrocenes. The ratio of modified to unmodified cytochrome c varied with reactant concentrations. When EDC or dcFc was omitted from the reaction mixture, no modification was observed. The reaction conditions employed in Figure 1 (cyt c:EDC:dcFc, 1:20:200; 1 mM cyt c ) were chosen to minimize the formation of multiply modified derivatives. Increasing the EDC and dcFc concentrations (cyt c:EDC: dcFc, 1:30:300) resulted in formation of more singly modified protein but also produced significant amounts of doubly modified protein. The reaction pH (7.5) was chosen at about the higher of the pK, values of the dcFc
carboxylate groups (6.5 and 7.6 (32)). Variation of the reaction pH between 7.3 (at lower values the solubility of dcFc in water decreases rapidly) and 8.0 did not appreciably change the overall yield but did change the relative proportions of fractions 2-6. Chromatographic separation was performed at pH 8.0 to ensure that the free carboxylate group of the bound dcFc was deprotonated. This improved the separation of the modified derivatives, presumably owing to the larger change in charge distribution. During the incubation with dcFc, the cytochrome c heme became partly reduced, significantlycomplicatingthe separation and identification of modified derivatives. For this reason, ascorbic acid was added to the elution buffer to keep the derivatives fully reduced. It is remarkable that, although the extent to which cytochrome c was reduced during the coupling reaction varied from one experiment to another, the relative degree of reduction of the different fractions followed a regular pattern, always systematically decreasing with increasing fraction number. It is essential that the (dcFc)cytochrome c derivatives be kept in the dark. When two samples of product mixture were stored overnight at 4 "C, one in the dark and one in room light, FPLC of the former resulted in the usual elution pattern, whereas the elution profile of the latter was completely different, indicating that exposure to light caused the derivatives to decompose. Differential pulse voltammetry of the (dcFc)cytochrome c derivatives showed oxidation waves for both the cy-
(dcFc)cytochromes c
Bioconjugate Chem., Vol. 3, No. 4, 1992 293
Table I. Assignment of Attachment Site for Purified (dcFc)cytochrome c Fractions of Figure 1 FPLC corresponding estimated elution estimated % sequence of modified modification fraction no. heme-Lys N, fraction purity fragments missing from tryptic mapa tryptic fragment site from refs 21,22 distance ( h b 2al 190 T3, T4 IFVQX: T3 Lys 13 2B 6.2 i 0.6 T11, Tll’, T12’, T10/11, T10/11’ Lys 72 3A 10.2 f 0.9 3 100 4a 100 T3 Lys 8 3B 14.8 f 0.7 4b 80 T12, T12’, Tll’, T10/11’ Lys 73 14.7 f 0.6 5bi 85 GXK: T2’ Lys 7 4’ 12.9 f 0.5 5c 100 GGXHL T5/6 Lys 25 4A 14.0 f 0.7 6a 100 XATNE T18’ Lys 100 6AB 14.8 f 0.6 6bi 100 T10, T11, Tll’, T10/11, T10/11’ Lys 60 5B 15.5 f 0.5 Horse heart cytochrome c tryptic fragments are named as follows: T1, (1-5); T2, (6-7); T2’, (6-8); T3, (9-13); T4, (14-22); T5, (23-25), T6, (26-27); T7, (2638); T7’, (28-39); T8, (40-53); T9, (54-55); T10, (5640); T11, (61-72), Tll’, (61-73); T12, (74-79); T12’, (73-79); T13, (80-86);T14, (87-88); T15, (89-91); T16, (92-97); T17, (98-99); T18, (101-104); T W , (100-104). * Estimated by molecular dynamics simulations as described in Experimental Procedures. ~~~
60
site as Lys-13. In derivatives for which modified lysines connected two fragments neither of which appeared in the native map, identification was accomplished by collecting and sequencing the new double fragment. The results for eight of the purest fractions are summarized in Table I.
40
DISCUSSION
8 20 w
Millett and co-workers recently obtained a number of singly modified cytochrome c derivatives that have [Ru(bpy)z(dcbpy)l attached to single lysine residues (21,22). Perhaps not surprisingly, the lysines that were modified (by dcbpy initially) in those experiments and the order of cation-exchange chromatographic elution of the resulting derivatives closely parallel the results of this study. The elution order seems to reflect the positions of the modified lysines projected along an axis that coincides with the calculated molecular dipole axis of cytochrome c (33).Since the positive end of the dipole is located near the exposed heme edge, modification nearest the heme causes the largest change in the molecular dipole, altering the cationexchange mobility the most, predicting that the dcFcheme distances should increase with increasing fraction number. As shown in Table I, this trend is approximately observed. The incorporation of a second redox-active center at different positions in a single-site-redoxprotein is the most straightforward method to investigate the way in which distance affects long-range electron transfer in proteins. A complete understanding of the effects of donor-acceptor distance and intervening medium on electron transfer rates will require a large body of rate data on different proteins with different donorlacceptor combinations. The necessity of this is evident from the fact that thus far a clear but unexplained discrepancy exists between the rates found with cytochrome c and myoglobin on one hand (12), and the blue copper proteins on the other (14,16, 17). Results obtained recently in this laboratory with cytochrome c , modified a t carboxylate groups with Co(diAMsar), showed an insignificant distance dependence (20). Determination of the intramolecular electron transfer rates from the heme to the carboxyferrocenyl group in the derivatives reported here, which have estimated dcFcheme distances that range from about 6 to 16 A (Table I), may shed more light on the relationship between electrontransfer rate and distance.
I
a
h
0 ;
W
40 20 I
YO
I
I
I
I
30 40 50 60 70 Elution Volume (mL)
0
Figure 2. Reversed-phase HPLC tryptic maps of (a) native cytochrome c and (b) subfraction 2al (Figure Ib). Note the absence of tryptic fragments T3 and T4 in b and the appearance of the major new peak at an elution volume of about 68 mL, which was identified by sequencing as the double-modified fragment T3(dcFc)T4. (See footnote a of Table I for tryptic fragment assignments.)
tochrome c heme at 265 mV (vs NHE) and of the dcFc moiety at 760 mV. The latter value, which is 100 mV higher than that observed for free dcFc, was found at both pH 6.4 and 8.0. The optical absorbance spectra of the modified fractions were very similar to that of native cytochrome c , as might be expected considering the relatively low absorbance of free dcFc [A, (E): 446 nm (238 M-l cm-’1, 306 nm (1.04 X lo3 M-l cm-’)I. Identification of Attachment Sites. To determine the attachment site of dcFc the purified derivatives were subjected to trypsin digestion followed by reversed-phase HPLC. Since dcFc-modified lysines are no longer recognized as cleavage sites by trypsin, modification generally results in the disappearance from the peptide map of the two fragments that are connected by the modified lysine, with the concomitant appearance of a new peak representing the modified double fragment. Since the HPLC elution positions of the tryptic fragments of native cytochrome c are known (20) (Figure 2a), the modification site could in most cases be determined from the tryptic map alone. An example is shown in Figure 2b, in which the reduction in intensity of peaks assigned to T3 and T4 and sequencing of the major new peak eluting after all native fragments allowsthe assignment of the modification
ACKNOWLEDGMENT
Dr.Greg Frauenhoff is acknowledged for the synthesis of 1,l’-dicarboxyferrocene. Hui Zhang and Dr. David Stewart are gratefully acknowledged for help with the molecular dynamics simulations. The University of Georgia
204
Bloconlugate Chem., Vol. 3, No. 4, 1992
Computing and Networking Services provided Cyber 205 time for these computations. Gary Brayer is acknowledged for providing the atomic coordinates for horse heart cytochrome c. B.R.C. acknowledges summer support from the NSF Research Training Group Award to the Center for Metalloenzyme Studies (DIR 90-14281). LITERATURE CITED (1) Marcus, R. A,, and Sutin, N. (1985) Electron Transfers in Chemistry and Biology. Biochim. Biophys. Acta 811, 265322. (2) Scott, R. A., Mauk,A. G., and Gray, H. B. (1985) Experimental Approaches to Studying Biological Electron Transfer. J . Chem. Educ. 62,932-938. (3) McLendon, G. (1988) Long-Distance Electron Transfer in Proteins and Model Systems. Acc. Chem. Res. 21, 160-167. (4) Williams,R. J. P. (1989)Electron Transfer in Biology. Biochem. Int. 18,475-499. (5) Gray, H. B., and Malmstrom, B. G. (1989) Long-Range Electron Transfer in Multisite Metalloproteins. Biochemistry 28, 7499-7505. (6) Yocom, K. M., Shelton, J. B., Shelton, J. R., Schroeder, W. A., Worosila, G., Isied, S. S., Bordignon, E., and Gray, H. B. (1982)Preparation and Characterizationof a Pentaammineruthenium(II1) Derivative of Horse Heart Ferricytochrome c. Proc. Natl. Acad. Sci. U.S.A. 79, 7052-7055. (7) Winkler, J. R., Nocera, D. G., Yocom, K. M., Bordignon, E., and Gray, H. B. (1982) Electron-Transfer Kinetics of Pentaammineruthenium(III)(histidine-33)-Ferricytochrome c . Measurement of the Rate of Intramolecular Electron Transfer between Redox Centers Separated by 15 8, in a Protein. J . Am. Chem. SOC.104,5798-5800. (8) Isied, S. S., Kuehn, C., and Worosila, G. (1984) RutheniumModified Cytochromec: Temperature Dependence of the Rate of Intramolecular Electron Transfer. J. Am. Chem. SOC.106, 1722-1726. (9) Therien, M. J., Selman, M., Gray, H. B., Chang, I. J., and Winkler, J. R. (1990) Long-Range Electron Transfer in Ruthenium-ModifiedCytochromec: Evaluation of PorphyrinRuthenium Electronic Couplings in the Candida krusei and Horse Heart Proteins. J . Am. Chem. SOC.112, 2420-2422. (10) Osvath, P., Salmon, G. A., and Sykes, A. G. (1988) Preparation, Characterization, and Intramolecular Rate Constant for Ru(I1) Fe(II1) Electron Transfer in the Pentaammineruthenium Histidine Modified Cytochrome c551 from Pseudomonas stutzeri. J . Am. Chem. SOC.110, 7114-7118. (11) Crutchley, R. J., Ellis, W. R., and Gray, H. B. (1986) Long Range Electron Transfer in Pentaammineruthenium(His-48)Myoglobin. Frontiers in Bioinorganic Chemistry (A. V. Xavier, Ed.) pp 679-693, VCH, Weinheim, Germany. (12) Axup, A. W., Albin, M., Mayo, S. L., Crutchley, R. J., and Gray, H. B. (1988) Distance Dependence of Photoinduced Long-Range Electron Transfer in Zinc/Ruthenium-Modified Myoglobins. J . Am. Chem. SOC.110, 435-439. (13) Margalit, R., Kostic,N. M.,Che, C.-M.;Blair,D. F., Chiang, H.-J., Pecht, I., Shelton, J. B., Shelton, J. R., Schroeder, W. A,, and Gray, H. B. (1984) Preparation and Characterization of Pentaammineruthenium(histidine-83)azurin: Thermodynamics of Intramolecular Electron Transfer from Ruthenium to Copper. Proc. Natl. Acad. Sci. U.S.A. 81, 6554-6558. (14) Kostic, N. M., Margalit, R., Che, C.-M., and Gray, H. B. (1983) Kinetics of Long-DistanceRuthenium-to-Copper Electron Transfer in [Pentaamminerutheniumhistidine-83Jazurin. J . Am. Chem. SOC.105, 7765-7767. (15) Farver, O., and Pecht, I. (1989) Preparation and Characterization of a Ruthenium Labeled Rhus Stellacyanin. FEBS Lett. 244, 376-378.
-
Gorren et el.
(16) Farver, O., andPecht, I. (1989) Long-Range Intramolecular Electron Transfer in Rhus uernicifera Stellacyanin: A Pulse Radiolysis Study. FEBS Lett. 244, 379-382. (17) Jackman, M. P., McGinnis, J., Powls, R., Salmon, G. A., and Sykes, A. G. (1988) Preparation and Characterization of Two His-59 Ruthenium-Modified Algal Plastocyanins and an Unusually Small Rate Constant for Ruthenium(I1) -Copper(11) Intramolecular Electron Transfer over -12 A. J . Am. Chem. SOC.110,5880-5887. (18) Jackman, M. P., Lim, M.-C., Salmon, G. A., and Sykes, A. G. (1988) Preparation of a Penta-a”ineruthenium(II1) Derivative of Chromatium uinosum HiPIP and the Kinetics of Intramolecular Electron Transfer. J. Chem. SOC.,Chem. Commun. 179-180. (19) Conrad, D. W., and Scott, R. A. (1989)Long-RangeElectron Transfer in a Cytochromec Derivative Containing a Covalently Attached Cobalt-CageComplex. J. Am. Chem. SOC.11I,34613463. (20) Scott, R. A., Conrad, D. W., Eidsness, M. K., Gorren, A. C., and Wallin, S. A. (1990) Long-Range Electron Transfer in Cytochrome c Derivatives with Covalently Attached Probe Sigel, and Complexes. Metal Ions in Biological Systems (H. A. Sigel, Eds.) Vol. 27, pp 199-222, Marcel Dekker, New York. (21) Pan, L. P., Durham, B., Wolinska, J., and Millett, F. (1988) Preparation and Characterization of Singly Labeled Ruthenium Polypyridine Cytochrome c Derivatives. Biochemistry 27, 7180-7184. (22) Durham, B., Pan, L. P., Long, J. E., and Millett, F. (1989) Photoinduced Electron-Transfer Kinetics of Singly Labeled Ruthenium Bis(bipyridine) Dicarboxybipyridine Cytochrome c Derivatives. Biochemistry 28, 8659-8665. (23) Degani, Y., and Heller, A. (1987) Direct Electrical Communication between ChemicallyModified Enzymes and Metal Electrodes. 1. Electron Transfer from Glucose Oxidase to Metal Electrodes via Electron Relays, Bound Covalently to the Enzyme. J . Phys. Chem. 91, 1285-1289. (24) Brautigan, D. L., Ferguson-Miller, S., and Margoliash, E. (1978)Mitochondrial Cytochrome c: Preparation and Activity of Native and Chemically Modified Cytochromes c. Methods Enzymol. 53, 128-164. (25) Benkeser, R. A., Goggin, D., and Schroll, G. (1954) A Route to Monosubstituted Ferrocene Compounds. J . Am. Chem. SOC.76, 4025-4026. (26) Mayo, D. W., Shaw, P. D., and Rausch, M. (1957)Effect of Tetrahydrofuran on the Lithiation of Ferrocene. Chem.Ind. 1388-1389. (27) Rausch, M., Vogel, M., andRosenberg, H. (1957) Derivatives of Ferrocene. I. The Metalation of Ferrocene. J . Org. Chem. 22,900-903. (28) Margoliash, E., and Frohwirt, N. (1959) Spectrum of HorseHeart Cytochrome c. Biochem. J . 71,570-572. (29) Conrad, D. W. (1990)Long-Range Intramolecular Electron Transfer in Cobalt-Labeled Cytochrome c. Ph.D. Thesis, University of Illinois at Urbana-Champaign. (30) Bushnell, G. W., Louie, G. V., and Brayer, G. D. (1990) High-ResolutionThree-Dimensional Structure of Horse Heart Cytochrome c. J . Mol. Biol. 214, 585-595. (31) Weiner, S. J., Kollman, P. A., Nguyen, D. T., and Case, D. A. (1986)An All Atom Force Field for Simulations of Proteins and Nucleic Acids. J . Comput. Chem. 7, 230-252. (32) Woodward,R. B., Rosenblum, M.,and Whiting, M. C. (1952) A New Aromatic System. J . Am. Chem. SOC.74,3458-3459. (33) Koppeno1,W. H.,andMargoliash,E. (1982)TheAsy”etric Distribution of Charges on the Surface of Horse Cytochrome c. Functional Implications. J . Biol. Chem. 257, 4426-4437. Registry No. dcFc, 1293-87-4;cytochromec, 9007-43-6;lysine,
56-87-1.
