Interaction of a Novel Fluorescent Analog of Interferon-γ with

retained over half of its original biological activity. Binding of py-IFN-γ to human amnion WISH cells showed appreciable enhancement in fluorescence...
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Bioconjugate Chem. 1997, 8, 459−465

459

Interaction of a Novel Fluorescent Analog of Interferon-γ with Transformed Cells Ayala Falach,† Ilana Nathan,‡ Stavanit Baram,† Nurith Porat,† Alexander Dvilansky,‡ and Abraham H. Parola*,† Department of Chemistry, Faculty of Natural Sciences, and Unit of Hematology, Faculty of Medical Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Received September 30, 1996X

A fluorescent analog of human recombinant interferon-γ (IFN-γ) was prepared for the first time. The recovered pyrene-labeled IFN-γ (py-IFN-γ), with an estimated seven pyrene molecules per IFN-γ, retained over half of its original biological activity. Binding of py-IFN-γ to human amnion WISH cells showed appreciable enhancement in fluorescence polarization from 0.055 to 0.215 and in fluorescence lifetime from 56 to 80 ns. The ratio of the vibronic peaks did not change, indicating that the pyrene molecules remained in water environment even after binding. Py-IFN-γ provides a novel tool for unraveling the mechanism of the initial interaction between this antiproliferative lymphokine and its target, cancer cell membrane receptors. Its fluorescence could provide the means to follow receptor recycling when it occurs.

INTRODUCTION 1

IFN-γ is a glycoprotein, produced by T lymphocytes after stimulation with mitogens or antigens, which exerts pleiotropic biological effects. In addition to its antiviral properties, it has antiproliferative, differentiating, and immunoregulatory activities (De Maeyer and De MaeyerGuignard, 1988; Kalvakolanu and Borden, 1996). Of great interest is the antiproliferative activity of IFN-γ, which has been shown to be more potent than that of other interferons in blocking the growth of several tumor cells in vivo and in vitro (Rubin and Gupta, 1980). Two IFN-γ polypeptides self-associate to form noncovalent antiparallel homodimers with an apparent molecular mass of 34 kDa (Ealick, 1991). To elicit cell responses, the initial interaction between IFN-γ and the target cell occurs at specific membranal receptors (Farrar and Schreiber, 1993). The functionally active IFN-γ receptor is composed of at least two speciesspecific components, a ligand binding subunit, R chain, and an additional subunit, β chain, that were cloned (Hemmi et al., 1994; Soh et al., 1994; Lundell and Narula, 1994). Ligand binding provokes increased phosphorylation on serines, threonine (Mao et al., 1990), and tyrosine of the receptor chains and regulates the association of the R and β receptor subunits with the tyrosine kinases JAK-1 and JAK-2 (Igarashi et al., 1994; Darnell et al., 1994; Kaplan et al., 1996) and the transcription factor p91 (Greenlund et al., 1994). The role of the JAK-STAT pathway in the signaling mechanism of IFN-γ was described (Heim et al., 1995). Although progress was made in the study of the signal transduction cascade, operating after ligand binding, the mechanism underlying the initial step of interaction * Author to whom correspondence should be addressed [telephone (972) 7 6472454; fax (972) 7 6472943; e-mail aparola@ bgumail.bgu.ac.il]. † Department of Chemistry. ‡ Unit of Hematology. X Abstract published in Advance ACS Abstracts, June 1, 1997. 1 Abbreviations: IFN-γ, interferon-γ; py-IFN-γ, pyrenesulfonyl chloride labeled IFN-γ; VSV, vesicular stomatitis virus; BSA, bovine serum albumin; PBS, phosphate-buffered saline (154 mM NaCl/10 mM sodium phosphate buffer, pH 7.4); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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between IFN-γ and target cell membranes is not yet clear. Thus, association of the two receptor subunits upon exposure to the ligand was recently described, but the detailed initial cell surface events that initiate signaling are still unresolved (Marsters et al., 1995). IFN-γ labeled with radioactive iodine (125I) was used to study binding to its receptor in intact human cells (Rubinstein et al., 1987; Branca and Baglioni, 1981; Finbloom et al., 1985). The number of binding sites per WISH cell was shown to be 50 000-70 000 with a Kd value of 7.3 × 10-9 M at 4 °C (Sarkar and Gupta, 1984). We thought that fluorescent-labeled IFN-γ would be a promising tool for studying these initial steps. It, together with other lipid specific membranal probes, would report on dynamical responses of various membrane components and provide an opportunity to study lipid-lipid and lipid-protein interactions in the IFN-γ activated membrane (Parola, 1993). In particular, IFNγ, which is labeled by a microenvironmentally sensitive fluorescent probe, should be able to report both on its immediate surroundings and on dynamical changes in the receptor after binding IFN-γ (e.g. receptor recycling through the membrane). Such information cannot be obtained by radioactive reporters. To this end we have prepared a fluorescent-labeled IFN-γ and characterized its specific interaction with membranal receptors, using both steady-state and dynamic fluorescence. The label was a pyrene derivative, having a relatively long lifetime and vibronic peaks sensitive to its environment. MATERIALS AND METHODS

Preparation of Pyrene-Labeled IFN-γ. Recombinant IFN-γ, 108 units/mg (Mory et al., 1986; Novick et al., 1982), was graciously donated by D. Novick, The Weizmann Institute of Science, Rehovot, Israel. In a typical labeling experiment, 2.4 × 105 units of IFN-γ in 0.1 M bicarbonate buffer, pH 9.2, was mixed with pyrenesulfonyl chloride (3.12 × 10-4 M) in ethylene glycol at a 3:1 ratio. This mixture was incubated for 3 h with stirring at room temperature. The reaction was terminated by incubation with 0.2 M lysine for 30 min at room temperature. Excess free label was removed on a Sephadex G-25 column, with PBS containing 0.1% gelatin as an elution buffer. The fluorescent-labeled fractions of IFN-γ (py-IFN-γ) were collected. For preparation of py© 1997 American Chemical Society

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IFN-γ for amino acid analysis the labeling procedure had the following modifications in the final stage of purification: (1) Excess free lysine and pyrenesulfonyl chloride were removed by ultrafiltration, using a Centricon filter with 10 000 molecular mass cutoff. (2) Alternatively, to avoid the presence of residual lysine, ethanolamine was used for termination of the pyrenesulfonyl chloride labeling of IFN-γ. Excess ethanolamine, free and bound to the fluorescent label, was removed either by repetitive extractions or by precipitation with TCA. Aqueous solutions of labeled and control IFN-γ were lyophilized and redissolved in 70 µL of sodium citrate buffer, pH 2.2, at room temperature. Fifty microliters was injected to the amino acid analyzer. Amino Acid Analysis. Amino acid analyses of unlabeled and pyrenesulfonyl chloride labeled IFN-γ were carried out on Dionex Bio LC at the Weizmann Institute of Science, Rehovot, Israel. Protein Assay. Protein was determined according to the Bio-Rad method; absorption at 595 nm was read on a Gilford spectrophotometer (Sedmak and Grossberg, 1977). IFN-γ Assay. Antiviral activity was determined in duplicates with a microtiter inhibition-of-cytopathic-effect assay against VSV on monolayers of WISH cells. Titers are reported as antiviral units, based on the National Institute of Health Human IFN-γ reference reagent Go23-901-530 (Eppstein et al., 1985). SDS-PAGE. Special steps were undertaken in the preparation procedure of py-IFN-γ to be able to detect its band(s) on the SDS-PAGE. Higher initial concentration of protein (Reprogene, Rehovot), 0.5 µg/µL, was used in the labeling step. To avoid the dilution encountered by gel filtration, py-IFN-γ was either loaded on 2 mL Sephadex G-25 columns, and the void volume collected following centrifugation (1400g, 2 min) or directly analyzed by 15% SDS-PAGE, following boiling in Laemmli buffer. Fluorescence Measurements. Fluorescence spectra were taken on a Perkin-Elmer MPF-44 spectrofluorometer. Steady-state fluorescence polarization was measured on MPF-44, SLM 4800, and Gregg-MM spectrofluorometers (Parola, 1993). Unless otherwise indicated, all measurements were carried out at 4 °C. Fluorescence lifetime was measured on the SLM 4800 (Lakowicz et al., 1979) or the Gregg-MM (an ISS upgrade of the SLM 4800). Data obtained on the SLM 4800 were analyzed in terms of two lifetimes; both lifetime and differential phase studies were carried out on the Gregg-MM system as described in detail before (Porat et al., 1988; Parola et al., 1994). The data were analyzed using the ISS187 decay analysis software. Membrane Purification. WISH cell cultures (human amnion cell line), grown in MEM medium containing 10% fetal calf serum, were collected by scrapping with a rubber policeman on ice and centrifuged twice at 800g for 7 min at room temperature, and the pellet was washed twice with Hank’s buffer (Ip and Cooper, 1980). The cells were resuspended in HEPES (2 mM) buffer, pH 7.2, containing 0.25 M sucrose (106 cells/mL). The cells were subjected to homogenization (7 s, setting of 5-6, 10 times) by Polytron (Kinematica GndH), and Na2EDTA (0.5 mM) was added. The suspension was centrifuged twice at 5500g for 15 min at 4 °C. The supernatants were subsequently pelleted at 100000g for 60 min at 4 °C. This membrane-enriched fraction was resuspended in 0.3 mL of HEPES buffer, pH 7.2, containing 25% sucrose and kept frozen at -70 °C. Binding of Py-IFN-γ to Membranes. WISH cell membranes (10 mg/mL) were washed with PBS, sus-

Falach et al.

pended in 0.5 mL of PBS, and then incubated for 1 h with py-IFN-γ (6000 units) in the presence of BSA, 3 mg/mL. Control membranes incubated without interferon and membranes labeled in the presence of large excess (500 times) of unlabeled IFN-γ were similarly set in parallel. The latter sample served to determine nonspecific labeling of membranes by py-IFN-γ. Following incubation, the samples were centrifuged at 100000g for 30 min and the supernatants collected. The membranes were washed with PBS and finally suspended in 0.5 mL of PBS. Binding of Py-IFN-γ to WISH Cells. WISH cells, harvested at confluency, were suspended in 0.3 mL of buffer, 1 × 107 cells/mL, and incubated for 1 h at 4 °C with py-IFN-γ (5000 units) in the presence of BSA, 3 mg/ mL. The extent of nonspecific binding was determined by incubating the cells in the presence of an excess (500 times) of unlabeled IFN-γ . At the end of the incubation, cells were centrifuged at 1000g for 5 min. Cells were washed twice with PBS and resuspended in 1 mL of PBS containing 1% NP-40. RESULTS

Labeled IFN-γ. a. Preparation and Biological Activity. The reaction of pyrenesulfonyl chloride with IFN-γ requires basic pH. At these conditions IFN-γ retains most of its biological activity. The reaction started at pH 9.2 and dropped to 8.5 at the end. Optimal incubation temperature and time were employed. Excess label was treated with lysine to prevent reaction with gelatin, which was used to prevent the adsorption of IFN-γ to the Sephadex G-25 column. The activity yields were checked during the labeling procedure. Optimal recovery results indicated 33% loss in activity at the end of the labeling, after the addition of lysine. Another 13% loss was noticed after the separation of labeled IFN-γ on the Sephadex G-25 column, resulting in a total activity loss of 46%. A similar loss in activity was encountered by IFN-γ subjected to the same labeling conditions in the absence of label molecules. Since there is no change in the amount of protein during the labeling step, the only protein loss could occur at the separation step, due to the trapping of IFN-γ molecules in the column. If this is true, the loss in activity resulting from IFN-γ inactivation is only 33%. Figure 1 shows good correlation between the fluorescence emission and the biological activity of the eluted fractions of pyrene-labeled IFN-γ. The insert in Figure 1 shows good separation of the labeled IFN-γ from free pyrenesulfonyl chloride bound to lysine. Gel electrophoresis analysis of the labeled as compared with the unlabeled IFN-γ on SDS-PAGE resulted in a single band of both labeled and unlabeled IFN-γ (Figure 2). A single band was obtained when excess py-IFN-γ was removed either by Sephadex G-25 columns or by the 15% SDSpolyacrylamide gel. This result indicates a rather homogeneous population of labeled protein. It excludes the presence of two kinds of protein molecules, labeled and unlabeled, which should render two separate bands; in the case of hetetogeneous labeling, band broadening shoud have resulted. b. Fluorescence Characteristics. The emission spectra of diluted pyrene solutions depend on the nature of the solvent. Comparison between polar and nonpolar solvents shows a relative rise in the emission intensity of the symmetry forbidden vibronic transitions, which results in remarkably altered fluorescence emission profiles of the pyrene moiety (Lianos and Georghiou, 1979a). To verify this phenomenon in pyrenesulfonyl chloride, we studied its spectra in various solvents. Marked

Pyrenesulfonyl Analog of Interferon-γ

Bioconjugate Chem., Vol. 8, No. 4, 1997 461

Figure 1. Elution profile of pyrene-labeled IFN-γ on Sephadex G-25 column (0.8 × 14 cm). Elution buffer consisted of 0.1% gelatin in PBS. Fractions containing 0.5 mL were collected at a flow rate of 6 mL/h. Fluorescence was measured at λmax(ex) ) 346 nm; λmax(em) ) 376 nm. IFN-γ activity was measured as described under Materials and Methods. Insert shows separation of free excess label from py-IFN-γ.

Figure 2. SDS-PAGE analysis of py-IFN-γ. Recombinant IFN-γ was labeled with pyrenesulfonyl chloride as described under Materials and Methods. For comparison, IFN-γ was treated similarly without label molecules. IFN-γ was separated on 15% SDS-polyacrylamide gel and stained with Coomassie blue. Lane a, Mr markers; lanes b-d, unlabeled IFN-γ; lanes e-g, py-IFN-γ: lanes b and e, 6 µg; lanes c and f, 2 µg; lanes d and g, 1 µg of protein.

differences in the vibronic profile of the fluorescence emission of pyrenesulfonyl chloride are noted (Figure 3). Table 1 shows a summary of all the solvents studied, indicating both spectral shifts and changes in the two major emission peaks ratio. While spectral maxima slightly shifted, the ratio among the three major vibronic peaks varied markedly in the solvents. In THF, the major vibronic band appears at 420 nm; in ethylene glycol, ethanol, and water, the major bands are at 377 and 395 nm. The ratio among these peaks generally increases in more polar solvents, yet other factors, e.g., solvent basicity (hydroxyl nature), may contribute to this ratio too. In American white oil, which is used to estimate the viscosity of biomembranes, the ratio between the first two vibronic modes of pyrene is 1.25, although the dielectric constant is very low. The range between the ratio of 1.25 in American white oil and 1.98 in water is wide enough to report on environmental changes upon binding to membrane receptors. Py-IFN-γ

Figure 3. Fluorescence emission spectra of py-SO2Cl (1 × 10-6 M) in various solvents: A, water (- - -), ethylene glycol (s), tetrahydrofuran (- ‚ -); B, water (- - -), ethanol/water 1:1 ratio (s), ethanol (- ‚ -); insert, American white oil. All uncorrected spectra were taken on the same MPF-44 spectrofluorometer.

in PBS containing 0.1% gelatin shows a fluorescence spectrum similar to that of pyrenesulfonyl chloride dissolved in water, with a peak ratio of 1.77 (Figure 4), which is remarkably different from that obtained for pyrenesulfonyl chloride in ethylene glycol (see Table 1). Excimer emission at 480 nm, with an intensity of 8.5% of the major monomer emission at 376 nm, is noted (not shown). Steady-state fluorescence polarization (Table 2)

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Table 1. Vibronic Fluorescence Structure of Py-SO2Cl in Various Solvents: Comparison with Free and Membrane-Bound Py-IFN-γ

solvent American white oilc THF ethylene glycol ethanol ethanol/water 1:1 water py-IFN-γ in buffer py-IFN-γ bound to membranes py-SS-ADA

ratio of major dielectric emission peaks of λmax1/λmax2 b constant pyrene-SO2-Xa (nm) ∼2-2.40

81.00 ND ND

1.25 1.21d 0.16 0.93 0.76 0.85 1.04 1.98 1.77 1.75

377/398 377/398 380/430 380/395 386/402 377/395 377/395 377/392 376/395 376/395

ND

1.70

377/395

2.95 32.00 24.30

aX

is Cl for free label in various solvents and IFN-γ. b Usually (and as shown in Figure 2), pyrene exhibits three vibronic peaks: (1) at ∼380, (2) at ∼395, and (3) at ∼420; (1) and (2) denote the first ones. c Range of values for various alkanes with C8-C12. d Obtained on the Gregg-MM; all other values were recorded on the MPF-44 spectrofluorometer.

and unlabeled IFN-γ, revealed that pyrenesulfonyl chloride labeling resulted in the elimination of 7 of the 21 lysine residues. No other amino acid residue was affected. Binding of Py-IFN-γ to Purified WISH Cell Membranes and Intact WISH Cells. The fluorescence of py-IFN-γ after binding to purified WISH cell membranes is shown in Figure 5. Binding specificity is evident from binding studies of py-IFN-γ in the presence of excess unlabeled IFN-γ. The specificity of binding averaged at 77.1 ( 5.5%. Figure 6 shows the same experiment with whole cells. Because of increased scattering, the fluorescence spectra had to be taken after extraction with NP-40. Again, binding specificity was proved. Table 2 shows fluorescence properties of py-IFN-γ after binding to the membranes. Fluorescence polarization increased 4-fold relative to free py-IFN-γ, indicating immobilization. Figure 7 is a representative phase and modulation lifetime analysis for py-IFN-γ in buffer, assuming a three-lifetime model. The long lifetime component, 56.3 ns, is the major contributor (R ) 0.56) and should be considered the most relevant in data analysis, since unlabeled cells have autofluorescence with two-lifetime components which coincide with the short and middle lifetime values of pyrene, i.e. 10.8 ns (R ) 0.55) and 1.5 ns (R ) 0.45). The increased pyrene fluorescence lifetime, in accord with Perrin’s formula, supports our assignment of the rise in fluorescence polarization to the immobilization of py-IFN-γ. Rotational correlation times show the same trend (Table 2): Binding of py-IFN-γ to its receptor lengthens rotational correlation times. DISCUSSION

Figure 4. Emission spectra of py-IFN-γ (s) in PBS containing 0.1% gelatin, py-SO2Cl in ethylene glycol (- - -), and unlabeled IFN-γ (- ‚ -) in PBS. Fluorescence intensity was measured at λmax(ex) ) 346 nm. Excitation and emission slits were 2 and 4 nm, respectively.

revealed increased polarization values upon binding to IFN-γ. Amino Acid Analysis. Py-IFN-γ for amino acid analysis was carefully purified from excess pyrenesulfonyl chloride and, in addition, ethanolamine was substituted for lysine in the termination step of the reaction, as described under Materials and Methods. Amino acid hydrolysis profiles obtained by HPLC, for both unlabeled and pyrenesulfonyl chloride labeled IFN-γ, revealed the content of each amino acid in nanomoles. The nanomoles of each residue analyzed was normalized to that of either alanine or glycine, and the ratios between labeled and unlabeled samples were determined. The comparison among three repeats of such data, obtained for labeled

The present work was aimed at obtaining a biologically active fluorescent lymphokine, e.g. IFN-γ, which would be used to study the dynamical consequences of binding to its receptor on target cells. Fluorescently tagged IFN-γ would shed light both on the initiation of the signaling process at the membrane level and on the phenomenon of receptor recycling, when it occurs (Uzgiris et al., 1982). Others reported on internalization and translocation of IFN-γ, even into the nucleus (Bader and Wietzerbin, 1994). Fluorescently tagged IFN-γ would reveal such phenomena by reporting the change in the receptor’s rotational correlation time. A prerequisite is the preparation of a fluorescently modified but still selectively binding and biologically active IFN-γ. To the best of our knowledge, this was not hitherto reported in the literature. Pyrenesulfonyl chloride was chosen as a suitable probe for the following reasons: (1) It has a relatively long fluorescence lifetime (>20 ns). (2) The excitation maximum of pyrene (346 nm) is beyond the range of tryptophan excitation (280-290 nm). (3) Pyrene has a relatively high quantum yield, 0.7-0.8 (Turro, 1978), and a relatively large absorptivity coefficient (40 000 M-1 cm-1) (Knopp and Weber, 1967), allowing its detection even at very low concentrations (10-8 M). (4) It has a large Stokes shift, which is particularly important for scattering biological systems. (5) The pyrene molecule has an appreciable environmental sensitivity, which is expressed in the variability of fluorescence lifetime and in the variable ratio of the vibrational emission bands (Lianos and Georghiou, 1979a,b; Lianos et al., 1980). (6) The pyrene moiety is known for its excimer emission, which enables determination of adjacent pyrene labeling sites on proteins. Thus, while in py-SS-ADA with 1:1 molar ratio of pyrene to protein no excimer emission was observed (Porat et al., 1988), in py-IFN-γ with seven

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Pyrenesulfonyl Analog of Interferon-γ Table 2. Fluorescence Parameters of Free and Membrane-Bound Py-IFN-γa sample

polarization

py-SO2Cl in ethylene glycolb

0.000

py-SO2Cl in American white oil

0.025 ( 0.001

py-IFN-γ in buffer

0.055 ( 0.010

py-IFN-γ bound to membranes

0.215 ( 0.110

lifetimes (ns) τ1 ) 18.50 τ2 ) 5.00 τ1 ) 30.89 ( 1.49 τ2 ) 5.17 ( 1.20 τ3 ) 1.14 ( 0.26 τ1 ) 56.35 ( 1.07 τ2 ) 13.88 ( 0.30 τ3 ) 1.95 ( 0.06 τ1 ) 80.40 ( 5.22 τ2 ) 16.16 ( 1.07 τ3 ) 1.94 ( 0.10

R1 ) 0.95 R2 ) 0.05 R1 ) 0.891 ( 0.006 R2 ) 0.061 ( 0.007 R3 ) 0.048 ( 0.006 R1 ) 0.561 ( 0.002 R2 ) 0.323 ( 0.002 R3 ) 0.116 ( 0.002 R1 ) 0.327 ( 0.009 R2 ) 0.308 ( 0.011 R3 ) 0.365 ( 0.009

χ2

rotational correlation time (ns)

χ2

NDc

ND

ND

5.4

5.00 ( 0.33c

1.7

0.6

8.24 ( 0.35

1.5

2.3

44.97 ( 13.25c,d

2.2

a All measurements, except b, were done on the upgraded Gregg-MM multifrequency phase modulation spectrofluorometer, using HeCd laser [λmax(ex) ) 325 nm], L shape, and two Pyrex filters in the emission; the polarization results are the mean of at least four independent measurements; the results of lifetime and rotational correlation time are of at least eight frequencies, except d, which represents the analysis of only four frequencies; analysis was based on σphase ) 0.400 and σmod ) 0.008, except c, for which σphase ) 0.600; lifetime was measured using the magic angle against POPOP as a reference; rotational correlation time was determined by analysis of phase only, using the long lifetime and a linear combination of the two shorter lifetimes. Standard deviation is shown. b Polarization was measured on the MPF-44 Perkin-Elmer spectrofluorometer; lifetime was measured on the SLM 4800 phase modulation spectrofluorometer using a combination of two filters: bandpass and Pyrex, at the excitation and emission maxima. c ND, not determined.

Figure 5. Binding of pyrene-labeled IFN-γ to WISH cell membranes: total binding of py-IFN-γ (s); nonspecific binding done in the presence of excess IFN-γ (- - -); unlabeled membranes (- ‚ -). Fluorescence intensity was measured at λmax(ex) ) 346 nm.

pyrene moieties bound to one protein (see below) the excimer emission is seen. The reaction between pyrenesulfonyl chloride and an amine moiety (e.g. lysine side chain) yields a sulfonamide bond, which is stable in both acidic and basic conditions (Porat et al., 1988). The amino acid sequence of IFN-γ (Gray et al., 1982; Rinderknecht et al., 1984) shows numerous NH2 moieties, potentially capable of binding pyrenesulfonyl chloride. Amino acid analysis indicated that seven lysine residues of IFN-γ were covalently bound to the labels. We attempted to verify the number of pyrenesulfonyl chloride molecules bound to IFN-γ by spectroscopic measurements. The difficulty in estimating this number arises from the low quantity of IFN-γ available, even at high level of activity. We could not detect either the protein or the attached pyrene in pyIFN-γ in the UV absorbance spectrum of the finally

Figure 6. Binding of pyrene-labeled IFN-γ to WISH cells: total binding of py-IFN-γ (s); nonspecific binding done in the presence of excess IFN-γ (- - -); unlabeled cells (- ‚ -). Fluorescence intensity was measured at λmax(ex) ) 346 nm.

labeled product, which was further diluted in the last step of column separation. Accordingly, the number of pyrene moieties was estimated by comparing the relative pyrene fluorescence of the labeled IFN-γ with that of pyrenelabeled adenosine deaminase of a similar, 45 000, molecular mass. The calculation is based on the assumption of similar fluorescence quantum yields of pyrene, when attached to either one of these proteins (in both proteins, pyrene fluorescence emission had similar lifetimes and emission profiles). On the basis of the molar extinction coefficient of the pyrene-labeled adenosine deaminase,  ) 40 000 M-1 cm-1 (Knopp and Weber, 1967) at λex(max) ) 346 nm, and the protein assay, the number of pyrene molecules attached to adenosine deaminase was experimentally determined. From the relative fluorescence of the two pyrene-labeled proteins and the ratio of protein concentration (calculated for IFN-γ from its specific activity after labeling), we estimated that about eight

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Figure 7. Fluorescence lifetime of py-IFN-γ bound to WISH cell membranes. Measurements were done on the upgraded Gregg-MM multifrequency phase modulation spectrofluorometer, using He-Cd laser [λmax(ex) ) 325 nm], L shape, and two Pyrex filters. Lifetime was measured using the magic angle against POPOP as a reference.

pyrene moieties were attached to each IFN-γ molecule, in agreement with the amino acid analysis. Obviously, this number is based on gross assumptions. The binding of seven to eight pyrene moieties to each molecule of IFN-γ accounts for our ability to detect the fluorescence of IFN-γ even at the very low protein level. In spite of the large molar ratio of pyrene per IFN-γ, over 50% of its activity is retained. A similar loss in activity was encountered by IFN-γ subjected to the same labeling conditions in the absence of label molecules, indicating that the labeling per se did not cause the loss in activity. Statistically, it is inconceivable that half of the protein was labeled by 14 pyrene moieties while the other half, which remained unlabeled, is the one responsible for the observed 54% remaining activity. This was further verified by the SDS-PAGE studies exhibiting only a single band for both the labeled and the unlabeled IFNγ. Excessive labeling was avoided because it resulted in reduced IFN-γ activity; the activity of py-IFN-γ with a 3-fold increase in fluorescence intensity was about 25% of the activity of py-IFN-γ labeled by “only” seven to eight probe molecules. With about seven pyrene molecules attached to each IFN-γ, the observed excimer emission at 480 nm is caused by intramolecular interaction between pyrene molecules attached to adjacent lysine residues: there are at least 10 loci of two to four adjacent lysine residues in each IFN-γ molecule (Rinderknecht et al., 1984). Py-IFN-γ binding to its membranal receptors has two consequences: (I) specificity and (II) a high degree of pyIFN-γ immobilization. We found that pyrene-labeled IFN-γ specifically binds to its receptor on membranes purified from WISH cells. Competition experiments with excess (500-fold) unlabeled IFN-γ indicated 77.1 ( 5.5% specific binding (Figure 5). Thus, the fluorescent modification of IFN-γ did not alter its ability to recognize specifically its binding site on both membrane preparation as well as intact WISH cells (Figure 6). While additional quantitative binding studies would further characterize this fluorescently labeled IFN-γ, its spectroscopic potential is already evident. Binding of labeled IFN-γ to the receptor is also reflected in its spectroscopic characteristics. Steady-state fluorescence polarization increased 4-fold when compared to free py-IFN-γ (Table 2), indicating reduction in rotational motion. Differential phase studies revealed longer rotational correlation times in bound py-IFN-γ. The enlarged pyrene fluorescence

Falach et al.

lifetime supports the assignment of enhanced fluorescence polarization and rotational correlation times to immobilization. As indicated in Table 2 and already shown by others (Lianos and Georghiou, 1979a), pyrene lifetime depends strongly on the polarity, hydroxylic nature, and viscosity of the solvent. Thus, pyrenesulfonyl chloride in the more viscous (resulting in reduced diffusional quenching), nonhydroxylic, and apolar American white oil has longer lifetimes than in the more polar, less viscous, and hydroxylic ethylene glycol: τ1 ) 30 and 18.5 ns, respectively. When bound to IFN-γ, τ1 rises to 56.4 ns, indicative of the pyrene being buried in the protein in a hydrophobic enclave. The binding of py-IFN-γ to the cell membrane receptors results in a further increase in fluorescence lifetime to τ1 ) 80 ns, presumably reflecting its location in a collision-free environment. The observation of the dramatic rise in steady-state polarization from 0.06 to 0.22, the rise in the rotational correlation time from 8 to 45 ns, and the longer lifetime are all indicative of intimate, tight binding of py-IFN-γ to a hydrophobic enclave of the membrane receptor. The vibronic peak ratio was, however, nearly equal in py-IFN-γ in buffer solution and when bound to membranes. It thus lacks environmental sensitivity, probably because of the heterogeneity in the pyrene-labeled sites. It is possible that the pyrene moieties were in touch with water even after py-IFN-γ was bound to the receptor. Change in the vibronic ratio might occur on sinking from the initial water phospholipids-head-group interface into the hydrophobic core of the lipid bilayer. The preparation of a homogeneous py-IFN-γ with a 1:1 molar ratio of pyrene to IFN-γ would have been advantageous in detecting its immediate environment through the vibronic peak ratio. To detect it at the minute concentrations involved in biological activity, laser excitation would be required and irreversible photobleaching might result. In summary, py-IFN-γ shows specific binding and immobilization and seems to meet the expected requirements essential for examining the dynamics of IFN-γ and its complex with the receptor, for which it was designed. This can be done by monitoring fluorescence polarization and lifetime following the interaction of target cell with IFN-γ. It is still anticipated that receptor recycling, which is associated with its sinking from the initial water phosposlipids-head-group interface into the hydrophobic core of the lipid bilayer, would be detected through the expected change in the vibronic peak ratio. ACKNOWLEDGMENT

We are most indebted to Dr. D. Novick for her gracious donation of recombinant IFN-γ. We thank Prof. D. Gill and Dr. R. Cohen-Luria for critical comments. We are grateful for the technical assistance of Ms. Zipora Zolotov and Ms. Liana Shkolnik. This work was supported in part by the Israel Cancer Research Fund and the Office of Naval Research (A.H.P). LITERATURE CITED Aguet, M. (1980) High affinity binding of 125I-labeled mouse interferon to a specific cell surface receptor. Nature 284, 459461. Bader, T., and Wietzerbin, J. (1994) Nuclear accumulation of interferon gamma. Proc. Natl. Acad. Sci. U.S.A. 91, 1183111835. Branca, A. A., and Baglioni, C. (1981) Evidence that type I and II interferons have different receptors. Nature 294, 768-770. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signalling proteins. Science 264, 1415-1421.

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